WO2024254043A2 - Nmda-r activating peptides in inflammation and neuronal differentiation - Google Patents

Nmda-r activating peptides in inflammation and neuronal differentiation Download PDF

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Publication number
WO2024254043A2
WO2024254043A2 PCT/US2024/032364 US2024032364W WO2024254043A2 WO 2024254043 A2 WO2024254043 A2 WO 2024254043A2 US 2024032364 W US2024032364 W US 2024032364W WO 2024254043 A2 WO2024254043 A2 WO 2024254043A2
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disease
cell
cells
disclosure
prp
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WO2024254043A3 (en
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Steven L. Gonias
Elisabetta MANTUANO
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • BACKGROUND OF THE DISLOSURE Diseases linked to an inflammatory response are difficult to treat for several reasons.
  • the immune system is incredibly complex, involving various cells, proteins, and signaling pathways. Dysregulation of any part of this system can lead to inflammation and disease. Finding therapies that target the specific mechanisms driving inflammation without compromising the body's ability to fight infections or heal wounds is challenging.
  • each inflammatory disease may have different underlying causes, triggers, and manifestations. What works for one patient may not work for another, necessitating personalized treatment approaches.
  • inflammation becomes chronic, persisting over long periods. Chronic inflammation can lead to tissue damage and functional impairment, making it harder to manage and treat the underlying disease.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • corticosteroids corticosteroids
  • biologic therapies targeting specific inflammatory pathways
  • these treatments may not be effective for everyone.
  • some medications may have side effects or lose effectiveness over time.
  • LRP1 Lipoprotein Receptor-related Protein-1
  • LRP1 is a type-1 transmembrane receptor that binds and mediates the endocytosis of diverse ligands, including lipoproteins, proteases, protease inhibitors, growth factors, extracellular matrix proteins, heat shock proteins, and proteins released by injured and dying cells, including microtubule- associated protein Tau and ⁇ -synuclein (1–5).
  • LRP1 may function as an injury detection receptor, as has been most fully defined for Schwann cell LRP1 (6–8).
  • LRP1 Specificity in the function of LRP1 may be manifested in the ability of different ligands to elicit diverse cell-signaling responses by engaging distinct cell-signaling co-receptors, including the N-methyl-D-aspartate receptor (NMDA-R), Trk receptors, and p75 neurotrophic receptor (9–15).
  • NDA-R N-methyl-D-aspartate receptor
  • Trk receptors Trk receptors
  • p75 neurotrophic receptor 9–15.
  • Nonpathogenic cellular prion protein PrP C
  • PrPC which is GPI-anchored to the plasma membrane, laterally associates with LRP1 in the same cell (18–20).
  • PrP C which is released from the cell surface in the form of soluble derivatives by ADAM proteases, binds to LRP1 (21).
  • PrP C that is embedded in exosomes and other extracellular vesicles (EVs) associates with LRP1 in target cells (22, 23).
  • S-PrP a recombinant derivative of PrPC
  • PrPC-bearing EVs isolated from human plasma activate cell-signaling in macrophages and PC12 cells, in an LRP1- and NMDA-R-dependent manner (21–23, 25).
  • PrP C derivatives oppose the activity of Pattern Recognition Receptors, including Toll-like Receptors (TLRs) in macro- phages and promote neurite outgrowth in PC12 cells.
  • TLRs Toll-like Receptors
  • ligands including activated ⁇ 2-macroglobulin ( ⁇ 2M), plasminogen activator inhibitor-1, coagulation Factor -2- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 VIII, and Receptor-associated Protein (RAP) has demonstrated an essential role for ligand- as-sociated Lys residues, typically in tandem (26–31).
  • Applicant screened a series of synthetic peptides, corresponding to the structure of PrP C , and identified a putative LRP1- binding motif just distal to the octarepeat region, in the disordered N-terminal half of PrP C .
  • a 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) replicated all of the cell-signaling activities of full-length S-PrP in a manner that required LRP1 and the NMDA-R.
  • P3 also rescued the increased susceptibility of PrP C gene (Prnp) knock-out mice to lipopolysaccharide (LPS).
  • Lys 100 and Lys 103 from the structure of PrP C , were essential for the cell-signaling activity of P3; when both residues were converted to Ala in P3 (DM) , cell- signaling and biological activity were completely eliminated. Synthetic peptides have been transformed into therapeutics at an increasing rate in recent decades (32). Although PrP C is previously reported to demonstrate anti-inflammatory activity in a variety of contexts, including in experimental autoimmune encephalitis (33–35) and in ischemic brain injury (33, 36–38), incomplete understanding of the responsible molecular mechanism has hindered efforts to exploit this activity of PrP C in therapeutics development. Applicant identified and reports herein that P3 and the LRP1/NMDA-R receptor assembly are members of single system with anti-inflammatory activity.
  • the present disclosure provides a 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) (present on the structure of PrPC and containing amino acids Lys103 and/or Lys105 that replicated all of the cell-signaling activities of full-length soluble-PrP (S-PrP) in a manner that required LRP1 and the NMDA-R.
  • P3 putative LRP1-binding motif
  • S-PrP full-length soluble-PrP
  • the present disclosure also provides a synthetic 4-mer peptide (KPSK) (SEQ ID NO: 33), with the two Lys residues, derived from the structure of P3. Both peptides showed broad anti- inflammatory activity.
  • LPS lipopolysaccharide
  • the present disclosure further provides that both peptides attenuate innate immunity and regulate neuronal differentiation by an NMDA Receptor- dependent mechanism.
  • a treatment based on these peptides is developed for diseases where innate immunity and neuroinflammation play a key role, including Inflammatory Bowel -3- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis.
  • FIGS. 1A – 1C Synthetic peptides and their relation to the structure of PrP C .
  • SEQ ID NOS: 3-32 sequence identifiers relate to sequences from left to right, e.g., SEQ ID NO: 1 is KKRPKPGGWNTGGS-NH 2; SEQ ID NO: 2 is K 23 KRPKPGGWNTGGS 36 and SEQ ID NO: 3 is K 23 KRPKPGGWNTGGS 36.
  • SEQ ID NO: 1 is KKRPKPGGWNTGGS-NH 2; SEQ ID NO: 2 is K 23 KRPKPGGWNTGGS 36 and SEQ ID NO: 3 is K 23 KRPKPGGWNTGGS 36.
  • FIG. 1B SEQ ID NOS: 1 and 2).
  • P1-P4 are located within the primary sequences of human and mouse PrP C .
  • FIG. 1C SEQ ID NOS: 3-32).
  • FIGS. 2A – 2C P3 replicates the effects of S-PrP and EV-associated PrP C in macrophages.
  • FIG. 2A BMDMs from C57BL/6J mice were treated for 6 h with LPS (0.1 ⁇ g/mL) in presence or absence of increasing concentrations (0.1-1.0 ⁇ M) of P1, P2, P3, P3*, or P4, as indicated.
  • FIG. 2B BMDMs were treated for 1 h with LPS (0.1 ⁇ g/mL) in -4- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 presence or absence of different peptides P1, P2, P3, or P4 (each at 0.5 ⁇ M). Immunoblot analysis was performed to detect phospho-I ⁇ B ⁇ , total I ⁇ B ⁇ , and ⁇ -actin. (FIG.
  • FIGS. 3A – 3D P3 activates cell-signaling and promotes neurite outgrowth in PC12 cells.
  • FIG. 3A PC12 cells were treated with P1, P2, P3, P3*, and P4 (each at 0.5 ⁇ M) for 10 min. Cell extracts were subjected to immunoblot analysis to detect phospho-ERK1/2 and total ERK1/2.
  • FIG.3B PC12 cells were stimulated for 10 min with increasing concentrations of P3 (0.1-1 ⁇ M).
  • FIGS. 3C and 3D were determined.
  • PC12 cells were treated for 48 h with S-PrP (40 nM), P1 (0.5 ⁇ M), P3 (0.5 ⁇ M), P4 (0.5 ⁇ M), NGF- ⁇ (50 ng/ml) as a positive control, or vehicle. Neurite outgrowth was examined by phase contrast microscopy. Repre-sentative images are shown (scale bar, 50 ⁇ m).
  • FIG. 3D Neurite length was determined by analyzing all the cells in ⁇ 5 random fields per treatment, in three different experiments (mean ⁇ SEM; one-way ANOVA: ****P ⁇ 0.0001).
  • FIG. 4A Microglia were isolated from C57BL/6J mouse pups and treated with LPS (0.1 ⁇ g/ml) for 6 h, in the presence and absence of S-PrP (40 nM) or P3 (0.5 ⁇ M).
  • S-PrP 40 nM
  • P3 0.5 ⁇ M
  • Conditioned medium CM
  • Representative cytokines that were increased in CM when LPS was added in the absence of S-PrP or P3 are numbered in red boxes.
  • FIG. 4B Microglia were treated for 1 h with LPS (0.1 ⁇ g/mL) in presence or absence of P1, P3, or P4.
  • FIGS. 5A – 5E The NMDA-R is necessary for the response to P3 in macrophages.
  • FIG. 5A BMDMs were pre-treated with MK-801 (1 ⁇ M) or vehicle for 30 min. The cells were then treated with LPS (0.1 ⁇ g/mL), P2 (0.5 ⁇ M), or P3* (0.5 ⁇ M), as indicated for 6 h.
  • FIG. 5C Flow cytometry was -5- 4862-8761-3125.1 Atty. Dkt.
  • 114198-6810 performed to detect cell-surface GluN1 NMDA-R subunit in BMDMs isolated from Grin1fl/fl-LysM-Cre-positive and –negative (WT) mice.
  • WT mice cells from WT mice were incubated with secondary antibody only (grey).
  • FIG. 5D BMDMs from Grin1fl/fl- LysM-Cre-positive mice were treated for 6 h with LPS (0.1 ⁇ g/mL), in presence or absence of increasing concentrations of P3 (1-20 ⁇ M), P4 (1-20 ⁇ M), or vehicle.
  • FIGS. 6A – 6B The anti-inflammatory activity of P3/P3* is strongly facilitated by LRP1.
  • BMDMs from Lrp1 fl/fl- LysM-Cre-positive mice were treated for 6 h with LPS (0.1 ⁇ g/mL) in presence of increasing concentrations (1-20 ⁇ M) of P1, P3, P3*, or vehicle.
  • FIG. 6B BMDMs were treated for 1 h with LPS (0.1 ⁇ g/mL) in the presence of P1, P2, P3, or P3* (each at 0.5 ⁇ M), as indicated.
  • FIGS. 7A – 7C P3 activates ERK1/2 and promotes neurite outgrowth in PC12 cells by a mechanism that requires the NMDA-R and LRP1.
  • FIG. 7A PC12 cells were transfected with Lrp1-specific siRNA, Grin1-specific siRNA, or NTC siRNA and then treated with P3 (0.5 ⁇ M) or vehicle for 10 min. ERK1/2 activation was determined by immunoblotting.
  • FIG. 7B PC12 cells were transfected with Lrp1-specific siRNA, Grin1- specific siRNA, or NTC siRNA, as indicated.
  • FIG. 7C Neurite length was determined in all the cells of ⁇ 5 random fields per treatment, in three different experiments (mean ⁇ SEM; one-way ANOVA: ****P ⁇ 0.0001).
  • FIG. 8 Lys 100 and Lys 103 are required for the function of P3 in PC12 cells. PC12 cells were treated for 10 min with increasing concentrations (0.5-20 ⁇ M) of P3 (K100A) , -6- 4862-8761-3125.1 Atty. Dkt.
  • FIGS. 9A – 9B Lys and Lys are required for the function of P3 in macrophages.
  • FIG.9A BMDMs from wild-type mice were treated for 1 h with LPS (0.1 ⁇ g/mL) and increasing concentrations (2.5-20 ⁇ M) of P3 (K100A) , P3 (K103A) , P3 (K105A) , P3 (K109A) , or P3 (DM) , as indicated above each panel. Immunoblot analysis was performed to detect phospho-I ⁇ B ⁇ , total I ⁇ B ⁇ , and ⁇ -actin.
  • FIG.9B BMDMs from wild-type mice were treated for 6 h with LPS (0.1 ⁇ g/mL) in presence of increasing concentrations of P3 (DM) (2.5-20 ⁇ M).
  • FIG. 10 P3 rescues the increased susceptibility of Prnp mice to LPS.16-20-week old male Prnp -/- mice (orange) and wild-type mice in the same genetic background (black) were challenged with LPS, by IP injection, at 75% of the LD50. A second matched cohort of Prnp -/- mice were treated with LPS and then with P3, 0.5 h later. Toxicity was scored as described in the Methods.
  • FIGS. 11A – 11B A tetrapeptide with two Lys residues mimics the activity of P3.
  • FIGS. 11A BMDMs were treated for 6 h with LPS and the indicated concentrations of KPSK.
  • FIG.11B BMDMs were treated for 1 h with LPS (0.1 ⁇ g/mL) in presence or absence of increasing concentrations of KPSK (0.2-5 ⁇ M). Immunoblot analysis was performed to detect phospho- I ⁇ B ⁇ , total I ⁇ B ⁇ , and ⁇ -actin. DETAILED DESCRIPTION All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
  • each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably.
  • the term -9- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.”
  • the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
  • x to y includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”. -10- 4862-8761-3125.1 Atty. Dkt.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein.
  • PNRP prion protein
  • NMDA-R or N-methyl-D-aspartate (NMDA) receptor is a receptor of glutamate, the primary excitatory neurotransmitter in the human brain.
  • the receptor plays an integral role in synaptic plasticity, which is a neuronal mechanism believed to be a function of memory formation.
  • functional NMDA receptors are heterotetramers comprising different combinations of the GluN1, GluN2 (A-D), and GluN3 (A-B) subunits derived from distinct gene families (Grin1-Grin3).
  • NMDARs contain two of the obligatory GluN1 subunits, which when assembled with GluN2 subunits of the same type, give rise to canonical diheteromeric (d-) NMDARs (e.g., GluN1-2A-1-2A).
  • Triheteromeric NMDARs by contrast, contain three different types of subunits (e.g., GluN1-2A-1-2B), and include receptors that are composed of one or more subunits from each of the three gene families, designated t- NMDARs (e.g., GluN1-2A-3A-2A).
  • t- NMDARs e.g., GluN1-2A-3A-2A
  • Protein and polynucleotide sequences encoding the protein subunits are known in the art, e.g., NMDA type subunit 1 (Homo sapiens, see Accession number Q05586 and https://www.genenames.org/data/gene-symbol-report/#!/hgnc_id/HGNC:4584, last accessed on May 30, 2024); NMDA-A (Accession No. Q12879 and https://www.uniprot.org/uniprotkb/Q12879/entry#sequences, last accessed on May 30, 2024); NMDA 2B, Accession No.
  • NMDA 2C Accession No. Q14957 and https://www.uniprot.org/uniprotkb/Q14957/entry, last accessed on May 30, 2024
  • NMDA 2D Accession No. and O15399 and https://www.uniprot.org/uniprotkb/O15399/entry, last accessed on May 30, 2024
  • NMDA 3A Accession No. Q8TCU5 and https://www.uniprot.org/uniprotkb/Q8TCU5/entry
  • NMDA 3B Accession No.
  • NMDA-R activation intends any activation of the NMDA-R that results in the opening of the ion channel that is nonselective to cations. Activation of postsynaptic NMDA receptors in most hippocampal pathways controls the induction of activity-dependent synaptic modification.
  • LRP1 intends the LDL receptor related protein 1. As noted by the NIH, the LRP1 gene encodes a member of the low-density lipoprotein receptor family of proteins. The encoded preproprotein is proteolytically processed by furin to generate 515 kDa and 85 kDa subunits that form the mature receptor. This receptor is involved in several cellular processes, including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells. In addition, the encoded protein is necessary for the alpha 2-macroglobulin-mediated clearance of secreted amyloid precursor protein and beta-amyloid, the main component of amyloid plaques found in Alzheimer patients.
  • LRP1-binding motif a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111 (peptide P3).
  • the human sequence comprises, or consists essentially of, or yet further consists of amino acids QWNKPSKPKTNMKH.
  • the murine sequence comprises, or consists essentially of, or yet further consists of QWNKPSKPKTNLKH . Variations of the human and murine sequence are disclosed in FIG. 1C.
  • PrPC is a non-pathogenic cellular prion protein.
  • S-PrP soluble cellular prion protein
  • P3 is a synthetic peptide derived from a LRP1-binding motif.
  • P3 peptides are disclosed in FIG. 1C and in the partial sequence below.
  • the term P3 include the disclosed peptides in SEQ ID NOS: 9-14 and 18-32 and equivalents thereof -13- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 that have at least 80% or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% sequence identify to the peptides comprising, consisting essentially of, or consisting of the peptides shown in SEQ ID NOS: 9- 14 and 18-32, with in one aspect, the equivalents retain at least one or both lysines.
  • the equivalent retains one or both lysines and substantial activity as compare to wildtype P3 (SEQ ID Nos 9-11) as determined using an assay as described herein, for example the P3 equivalent replicates cell-signaling and biological activities of full-length shed PrP c .
  • the term “P3” intends 14-mer synthetic peptide corresponding to a putative LRP1-binding motif, wherein the 14-mer synthetic peptide contains one or both Lys 103 and Lys 105 (P3) (murine P3s, or the corresponding lysines in the human P3s), and wherein said P3 replicates cell-signaling and biological activities of full-length shed PrP c .
  • S-PrP Full-length soluble-PrP
  • “Derived from” intends that the protein, polypeptide, or gene is a modification of another protein, polypeptide, or gene, that in one aspect, is a wildtype protein, polypeptide or gene.
  • “Innate immunity” is nonspecific immunity that is the immune defense that is present at birth. It protects against all antigens. Examples include the cough reflex, enzymes in tears and skin oils, mucus, skin and stomach acid.
  • Neuroinflammation is an inflammatory response within the brain or spinal cord.
  • Neurodegenerative diseases are chronic conditions that damage and destroy parts of the nervous system, especially the brain.
  • Non-limiting examples include Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy.
  • protein protein
  • peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum -14- 4862-8761-3125.1 Atty. Dkt.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • isolated or recombinant nucleic acids refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides.
  • the term -15- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated or recombinant means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
  • An isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
  • a non- naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In another aspect, an equivalent intends at least about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods.
  • an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70 %, or alternatively at least 75 %, or alternatively at least 80 %, or alternatively at least 85 %, or alternatively at least 90 %, or alternatively at least 95 % affinity or higher affinity to a reference antibody or antigen binding fragment.
  • the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen under a competitive ELISA assay.
  • an equivalent intends at least about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • homologous When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions. "Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. -18- 4862-8761-3125.1 Atty. Dkt.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • prophylaxis or prevention is excluded from the term “treatment.”
  • To prevent intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect. An example of such is preventing the disease from progressing to the state of clinical or diagnostic symptoms.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant or preservative, e.g., DMSO or glycol.
  • pharmaceutical composition is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions of the disclosure.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium -19- 4862-8761-3125.1 Atty. Dkt.
  • No.: 114198-6810 sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
  • administering can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application.
  • An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired effect.
  • the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • the effective amount in some embodiments is -20- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 the amount sufficient to result in a protective response against a pathogen.
  • the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen.
  • the effective amount is the amount required to confer passive immunity on a subject in need thereof.
  • the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject’s immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment.
  • Lipomes are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI phosphatidylinositol
  • SM sphingomyelin
  • DOPE dioleoylphosphatidylethanolamine
  • lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol- amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan- olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), -21- 4862-8761-3125.1 Atty.
  • HSPC hydrogenated soy phosphatidylcholine
  • lecithin phosphatidylethanolamine
  • lysolecithin lysophosphatidylethanol- amine
  • phosphatidylserine phosphat
  • POPC palmitoyloleoylphosphatidylcholine
  • POPE palmitoyloleoylphosphatidylethanolamine
  • DOPE-mal dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carb- oxylate
  • Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol).
  • DDAB DODAC
  • DMRIE DMTAP
  • DOGS DOGS
  • DOTAP DOTMA
  • DOSPA DPTAP
  • DSTAP DC-Chol
  • Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles.
  • liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs).
  • MLVs multi-lamellar vesicles
  • SUVs small uni-lamellar vesicles
  • LUVs large uni-lamellar vesicles
  • a “micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid.
  • a typical micelle in aqueous solution forms an aggregate with the hydrophilic "head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center.
  • This type of micelle is known as a normal phase micelle (oil-in-water micelle).
  • Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle).
  • Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue.
  • pharmaceutically acceptable polymer refers to the group of compounds which can be conjugated to one or more polypeptides described here. It is contemplated that the conjugation of a polymer to the polypeptide is capable of extending the half-life of the polypeptide in vivo and in vitro.
  • Non-limiting examples include polyethylene glycols, polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.
  • the biological active agents can be -22- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 conjugated to a pharmaceutically acceptable polymer for administration in accordance with the methods described herein.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a polynucleotide of this disclosure can be delivered to a cell or tissue using a gene delivery vehicle.
  • Gene delivery “gene transfer,” “transducing,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector- mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • an extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • a “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded.
  • Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally -23- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses.
  • the gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
  • MCS multiple cloning site
  • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.
  • a “yeast artificial chromosome” or “YAC” refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearized by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends.
  • Yeast expression vectors such as YACs, YIps (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than BACs, producing chimeric effects.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors examples include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104).
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, -24- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Schlesinger & Dubensky (1999) Curr.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • “retroviral mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos.
  • Wild-type AAV has high infectivity and specificity integrating into the host cell’s genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art.
  • Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
  • Stratagene La Jolla, CA
  • Promega Biotech Promega Biotech
  • consensus ribosome binding sites can be inserted immediately 5’ of the start codon to enhance expression.
  • Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this disclosure.
  • direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this disclosure are other non-limiting techniques.
  • label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histadine tags (N-His), magnetically active isotopes, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotopes such as 13 C and 15 N, polynucleotide or protein such as an antibody so as to generate a "labeled" composition.
  • N-His N-terminal histadine tags
  • magnetically active isotopes e.g., 115 Sn, 117 Sn and 119 Sn
  • a non-radioactive isotopes such as 13 C and 15 N
  • polynucleotide or protein such as an antibody so as to generate a "labeled” composition.
  • the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences,
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to magnetically active isotopes, non- radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • the label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed
  • a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component -26- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
  • Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal.
  • Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
  • luminescent probes include, but are not limited to, aequorin and luciferases.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue TM , and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton.
  • the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
  • eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian, and human. -27- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called an episome.
  • Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 ⁇ m in diameter and 10 ⁇ m long).
  • Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
  • a “ligand” is a polypeptide.
  • the term "ligand” as used herein refers to any molecule that binds to a specific site on another molecule.
  • the ligand confers the specificity of the protein in a reaction with an immune effector cell or an antibody to a protein or DNA to a protein. In one aspect it is the ligand site within the protein that combines directly with the complementary binding site on the immune effector cell.
  • solid phase support or “solid support”, used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels. As used herein, "solid support” also includes synthetic antigen-presenting matrices, cells, and liposomes.
  • solid phase support may be selected on the basis of desired end use and suitability for various protocols.
  • solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE.RTM. resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel.RTM., Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, Calif.).
  • polystyrene e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.
  • POLYHIPE.RTM. resin obtained from Aminotech, Canada
  • polyamide resin obtained from Peninsula Laboratories
  • polystyrene resin grafted with polyethylene glycol TeentaGel.RTM., Rapp
  • a solid phase support include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble.
  • the support material may have virtually any possible structural configuration so -28- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 long as the coupled molecule is capable of binding to a polynucleotide, polypeptide or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads.
  • modulate an immune response includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response.
  • An immunomodulatory method is one that modulates an immune response in a subject.
  • a “subject” of diagnosis or treatment is a cell or an animal such as a mammal, or a human.
  • Non-human animals subject to diagnosis or treatment are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets.
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non- limiting examples of cytokines which may be used alone or in combination in the practice of the present disclosure include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL- 3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 ⁇ ), interleukin-11 (IL-11), MIP-11, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand.
  • the present disclosure also includes culture conditions in which one or more cytokine is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) and Immunex (Seattle, Wash.). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the disclosure. -29- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere).
  • the present disclosure provides a 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) present on the structure of PrP C and a synthetic 4-mer peptide (KPSK) (SEQ ID NO: 33), with two Lys residues, derived from the structure of P3 that have uniquely broad anti-inflammatory activity.
  • P3 putative LRP1-binding motif
  • KPSK 4-mer peptide
  • the data presented herein show that -30- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 both peptides attenuate innate immunity and regulate neuronal differentiation by an NMDA Receptor-dependent mechanism.
  • the synthetic 14-mer peptide consists essentially of, or yet further consists of an isolated P3, P3*, P3 (K100A) , P3 (K103A) , P3 (K105A) , P3 (K109A) , and P3 (DM) (SEQ ID NOS: 9-32), as shown in the rows of FIG. 1C (from left to right is shown sequence, sequence in human PrPc and sequence in mouse PrPc), fragments, and equivalents of these peptides that retain the mutated amino acids as compared to wildtype P3 (SEQ ID NOS: 9-11).
  • the synthetic 14-mer peptide consists essentially of, or yet further consists of a lysine at one or both of amino acid positions 103 and 105 (Lys 103 and Lys 105 (P3)), fragments, and equivalents of these peptides that retain one or both of the Lys 103 and Lys 105 mutated amino acids as compared to wildtype P3.
  • the amino acid numbering of the variants is relative to the mouse PrP c and the human variants may have different numbering as shown in FIG. 1C.
  • an isolated 4-mer peptide (KPSK) is provided.
  • the 14-mer peptides also comprise N-terminal acetylation and C-terminal amidation.
  • a series of PrP C -derived peptides were screened, and a putative Low Density Lipoprotein Receptor-related Protein-1 (LRP1) recognition motif that includes Lys 100 and Lys 103 was identified.
  • LRP1 Low Density Lipoprotein Receptor-related Protein-1
  • a 14-mer synthetic peptide that contains Lys 103 and Lys 105 (P3) was identified (see FIG. 1C), and it replicated the cell-signaling and biological activities of full-length shed PrP C .
  • P3 inhibited lipo-polysaccharide (LPS)-elicited cytokine expression in macrophages and microglia and induced neurite outgrowth in PC12 cells.
  • LPS lipo-polysaccharide
  • NMDA-R NMDA Receptor
  • Lys 100 and Lys 103 in P3 were converted to Ala the activity of P3 in cell-signaling and in regulating macrophage physiology was entirely blocked.
  • KPSK synthetic 4-mer peptide
  • the tetrapeptide KPSK mimics exactly the activity of P3.
  • new and unique peptides could serve as an excellent template for developing new anti-inflammatory drugs to treat diseases in which innate immunity and neuroinflammation plays an important role, including Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis.
  • the new and unique NMDA-R activating peptides disclosed herein can regulate innate immunity and neuronal differentiation.
  • the data presented herein show for the first time that these novel synthetic peptides replicate the anti-inflammatory activity of the full- length protein of non-pathogenic cellular prion protein (PrP C ) through the NMDA-R receptor and can been transformed into new candidate therapeutics.
  • a peptide linker can be added to the N-terminus or C-terminus of the polypeptide.
  • a “linker” or “peptide linker” refers to a peptide sequence linked to either the N-terminus or the C-terminus of a polypeptide sequence.
  • the linker is from about 1 to about 20 amino acid residues long or alternatively 2 to about 10, about 3 to about 5 amino acid residues long.
  • An example of a peptide linker is Gly-Pro-Ser- Leu-Lys-Leu (SEQ ID NO: 35).
  • the isolated polypeptides of this disclosure are intended to include isolated wildtype and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof. -32- 4862-8761-3125.1 Atty. Dkt.
  • polypeptides are conjugated or linked to a detectable label. Suitable labels are known in the art and described herein.
  • the proteins and polypeptides are obtainable by a number of processes known to those of skill in the art, which include purification, chemical synthesis and recombinant methods.
  • Polypeptides can be isolated from preparations such as host cell systems by methods such as immunoprecipitation with antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography. For such methodology, see for example Deutscher et al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol. 182, Academic Press). Accordingly, this disclosure also provides the processes for obtaining these polypeptides as well as the products obtainable and obtained by these processes.
  • the polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin/ Elmer/Applied Biosystems, Inc., Model 430A or 43lA, Foster City, CA, USA.
  • the synthesized polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this disclosure also provides a process for chemically synthesizing the proteins of this disclosure by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence. Alternatively, the proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook et al. (1989) supra, using a host cell and vector systems described herein. Also provided by this application are the polypeptides described herein conjugated to a detectable agent for use in the diagnostic methods.
  • detectably labeled polypeptides can be bound to a column and used for the detection and purification of -33- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 antibodies. They also are useful as immunogens to produce antibodies.
  • the polypeptides of this disclosure are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes.
  • polypeptides of this disclosure also can be combined with various solid phase carriers, such as an implant, a stent, a paste, a gel, a dental implant, or a medical implant or liquid phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions and emulsions.
  • solid phase carriers such as an implant, a stent, a paste, a gel, a dental implant, or a medical implant or liquid phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions and emulsions.
  • non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one.
  • suitable adjuvants include, but are not limited to Freund’s Complete and Incomplete, mineral salts and polynucleotides.
  • suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat labile enterotoxin of E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene.
  • MPL monophosphoryl lipid A
  • mutant derivatives of the heat labile enterotoxin of E. coli mutant derivatives of cholera toxin
  • CPG oligonucleotides CPG oligonucleotides
  • adjuvants derived from squalene derived from squalene.
  • This disclosure also provides a pharmaceutical composition comprising or alternatively consisting essentially of, or yet further consisting of, any of a polypeptide or an equivalent of this disclosure, alone or in combination with each other or other agents and an acceptable carrier or solid support. These composition
  • Polynucleotides This disclosure also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides and their respective complementary strands.
  • Vectors comprising the isolated or recombinant polynucleotides are further provided examples of which are known in the art and briefly described herein.
  • the isolated or recombinant polynucleotides can be contained within a polycistronic vector.
  • the polynucleotides can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.
  • operatively linked means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule.
  • promoters are SP6, T4 and T7.
  • cell-specific promoters are used for cell-specific expression of the inserted polynucleotide.
  • Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are known in the art and commercially available. For general methodology and cloning strategies, see Gene Expression Technology (Goeddel ed., Academic Press, Inc.
  • polynucleotides derived from the polynucleotides of the disclosure encode polypeptides or proteins having diagnostic and therapeutic utilities as described herein as well as probes to identify transcripts of the protein that may or may not be present.
  • These nucleic acid fragments can by prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker.
  • random fragments can be generated using nick translation of the molecule.
  • Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA.
  • suitable expression vectors include plasmids, yeast vectors, viral vectors and liposomes.
  • Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient -35- 4862-8761-3125.1 Atty. Dkt.
  • a nucleic acid When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced.
  • suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989) supra.
  • the nucleic acid can be inserted into the host cell by methods known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See, Sambrook et al. (1989) supra, for methodology.
  • a host cell e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules.
  • any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a pharmaceutically acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector.
  • Pharmaceutically acceptable vectors containing the nucleic acids of this disclosure can be further modified for transient or stable expression of the inserted polynucleotide.
  • the term “pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
  • a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
  • An example of such a vector is a “replication-incompetent” vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell.
  • An example of a replication-incompetent retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7:980-990). The methodology of using replication-incompetent -36- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 retroviruses for retroviral-mediated gene transfer of gene markers has been established.
  • This disclosure also provides genetically modified cells that contain and/or express the polynucleotides of this disclosure.
  • the genetically modified cells can be produced by insertion of upstream regulatory sequences such as promoters or gene activators (see, U.S. Patent No. 5,733,761).
  • the polynucleotides can be conjugated to a detectable marker, e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
  • detectable markers include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
  • enzyme tags calorimetric indicator substrates can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • this disclosure further provides a method for detecting a single-stranded polynucleotide or its complement, by contacting target single-stranded polynucleotide with a labeled, single-stranded polynucleotide (a probe) which is a portion of the polynucleotide of this disclosure under conditions permitting hybridization (preferably moderately stringent hybridization conditions) of complementary single-stranded polynucleotides, or more preferably, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from un-hybridized, single-stranded polynucleotides.
  • the hybridized polynucleotide pairs are detected using methods known to those of skill in the art and set forth, for example, in Sambrook et al. (1989) supra.
  • the polynucleotide embodied in this disclosure can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein.
  • One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service. -37- 4862-8761-3125.1 Atty. Dkt.
  • the polynucleotides of this disclosure can be isolated or replicated using PCR.
  • the PCR technology is the subject matter of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston (1994)), and references cited therein.
  • one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA.
  • this disclosure also provides a process for obtaining the polynucleotides by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
  • these polynucleotides are further isolated.
  • one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
  • the DNA so amplified can be isolated from the cell by methods known to those of skill in the art.
  • RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
  • the DNA can be delivered by any appropriate method, e.g., using an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation.
  • an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
  • electroporation e.g., electroporation
  • mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al.
  • polynucleotides exhibiting sequence complementarity or homology to a polynucleotide of this disclosure are useful as hybridization probes or as an equivalent of the specific polynucleotides identified herein. Since the full coding sequence of the transcript is known, any portion of this sequence or homologous sequences, can be used in the methods of this disclosure. It is known in the art that a “perfectly matched” probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or -38- 4862-8761-3125.1 Atty. Dkt.
  • a probe useful for detecting the mRNA is at least about 80% identical to the homologous region. More preferably, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region; even more preferably, it exhibits 90% identity.
  • These probes can be used in radioassays (e.g., Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various cells or tissues containing these cells.
  • the probes also can be attached to a solid support or an array such as a chip for use in high throughput screening assays for the detection of expression of the gene corresponding a polynucleotide of this disclosure. Accordingly, this disclosure also provides a probe comprising or corresponding to a polynucleotide of this disclosure, or its equivalent, or its complement, or a fragment thereof, attached to a solid support for use in high throughput screens.
  • the total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the nucleic acid segment.
  • Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between at least 5 to 10 to about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect.
  • Nucleotide probes having complementary sequences over stretches greater than 5 to 10 nucleotides in length are generally preferred, to increase stability and selectivity of the hybrid, and thereby improving the specificity of hybrid molecules obtained. More preferably, one can design polynucleotides having gene-complementary stretches of 10 or more or more than 50 nucleotides in length, or even longer where desired.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology with two priming oligonucleotides as described in U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production.
  • a probe is about 50-75 or more alternatively, 50-100, nucleotides in length.
  • the polynucleotides of the present disclosure can serve as primers for the detection of -39- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 genes or gene transcripts that are expressed in cells described herein.
  • amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity.
  • Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.
  • a primer is the same length as that identified for probes.
  • One method to amplify polynucleotides is PCR and kits for PCR amplification are commercially available. After amplification, the resulting DNA fragments can be detected by any appropriate method known in the art, e.g., by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • Methods for administering an effective amount of a gene delivery vector or vehicle to a cell have been developed and are known to those skilled in the art and described herein.
  • Methods for detecting gene expression in a cell include techniques such as in hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays and Northern blot analysis. Such methods are useful to detect and quantify expression of the gene in a cell.
  • expression of the encoded polypeptide can be detected by various methods. It is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with the target polypeptide. Such antibodies are useful for visualizing cells that express the polypeptide using techniques such as immunohistology, ELISA, and Western blotting.
  • compositions are further provided.
  • the compositions comprise a carrier and one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure.
  • the carriers can be one or more of a solid support or a pharmaceutically acceptable carrier.
  • the compositions can further comprise an adjuvant or other components suitable for administration.
  • the compositions are formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants.
  • embodiments of the compositions of the present disclosure include one or more of an isolated -40- 4862-8761-3125.1 Atty.
  • any one or more of an isolated or recombinant polypeptide as described herein, an isolated or recombinant polynucleotide as described herein, a vector as described herein, an isolated host cell as described herein can be used alone or in pharmaceutical formulations of the disclosure comprising, or consisting essentially of, the compound in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as
  • compositions can 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.
  • 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
  • compositions and unit dose forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infections, and therapies in which the patient self-administers the drug.
  • the formulation is specific for pediatric administration.
  • the disclosure provides pharmaceutical formulations in which the one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure can be formulated into preparations for injection in accordance with the disclosure by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, -41- 4862-8761-3125.1 Atty.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. Aerosol formulations provided by the disclosure can be administered via inhalation and can be propellant or non-propellant based.
  • embodiments of the pharmaceutical formulations of the disclosure comprise a compound of the disclosure formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a non-limiting example of a non-propellant is a pump spray that is ejected from a closed container by means of mechanical force (i.e., pushing down a piston with one's finger or by compression of the container, such as by a compressive force applied to the container wall or an elastic force exerted by the wall itself (e.g. by an elastic bladder)).
  • Suppositories of the disclosure can be prepared by mixing a compound of the disclosure with any of a variety of bases such as emulsifying bases or water-soluble bases.
  • Embodiments of this pharmaceutical formulation of a compound of the disclosure can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the disclosure.
  • unit dosage forms for injection or intravenous administration may comprise a compound of the disclosure in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Embodiments of the pharmaceutical formulations of the disclosure include those in -42- 4862-8761-3125.1 Atty. Dkt.
  • injectable pharmaceutical formulations of the disclosure are prepared as liquid solutions or suspensions, or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection.
  • the preparation may also be emulsified, or the active ingredient encapsulated in liposome vehicles in accordance with other embodiments of the pharmaceutical formulations of the disclosure.
  • one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure is formulated for delivery by a continuous delivery system.
  • continuous delivery system is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
  • Continuous delivery system is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
  • Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos.
  • a compound of the disclosure is in a liquid formulation in a drug-impermeable reservoir and is delivered in a continuous fashion to the individual.
  • the drug delivery system is an at least partially implantable device.
  • the implantable device can be implanted at any suitable implantation site using methods and devices well known in the art.
  • An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned.
  • Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.
  • Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.
  • Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation.
  • the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system).
  • the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material).
  • the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc. Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure.
  • a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396).
  • Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.
  • a further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).
  • the drug delivery device is an implantable device.
  • the drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art.
  • an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. -44- 4862-8761-3125.1 Atty. Dkt.
  • Suitable excipient vehicles for a compound of the disclosure are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the compound adequate to achieve the desired state in the subject being treated.
  • compositions of the present disclosure include those that comprise a sustained-release or controlled release matrix.
  • a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
  • a sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), poly
  • Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.
  • the interfering agent (as well as combination compositions) is delivered in a controlled release system.
  • a compound of the disclosure may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al.
  • a controlled release system is placed in proximity of the therapeutic target, i.e., the liver, thus requiring only a fraction of the systemic dose.
  • a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic.
  • Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.
  • compositions of the present disclosure include those formed by impregnation of an inhibiting agent described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions.
  • absorptive materials such as sutures, bandages, and gauze
  • solid phase materials such as surgical staples, zippers and catheters to deliver the compositions.
  • Screening Assays The present disclosure provides methods for screening for equivalent agents, such as equivalent peptides and small molecules that modulate the activity of the active agents and pharmaceutical compositions of the disclosure or the function of a polypeptide or peptide product encoded by the polynucleotide of this disclosure.
  • an “agent” is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a ribozyme.
  • a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a ribozyme.
  • a vast array of compounds can be synthesized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term “agent.”
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like.
  • composition comprising, or consisting essentially of, or consisting of, the 14-mer synthetic peptide or a polynucleotide encoding the 14-mer synthetic peptide (e.g., SEQ ID NOS: 9-11, 12-14, or 18-29) and/or the synthetic 4-mer peptide or a polynucleotide encoding the 4-mer (KPSK) (e.g., SEQ ID NO: 34) as disclosed herein for treating a disease in which innate immunity and neuroinflammation plays an important role.
  • KPSK 4-mer
  • the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle.
  • the disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis.
  • the 14-mer Also provided are methods for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising, or consisting essentially of, or yet further consisting of administering to a subject-in-need an effective amount of the 14-mer synthetic peptide (e.g., SEQ ID NOS: 9-11, 12-14, or 18-29) or synthetic 4-mer peptide (e.g., SEQ ID NO: 33) as disclosed herein.
  • the 14-mer synthetic peptide e.g., SEQ ID NOS: 9-11, 12-14, or 18-29
  • synthetic 4-mer peptide e.g., SEQ ID NO: 33
  • the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle.
  • the disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis.
  • the 14-mer and 4-mer peptides can be combined with other therapies as appropriate.
  • the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle and are combined with other therapies as appropriate.
  • a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle.
  • routes of administration include by one or more method comprising transdermally, urethrally, sublingually, rectally, vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally, intranasally, by inhalation or orally.
  • routes of administration applicable to the methods of the disclosure include intranasal, intramuscular, urethrally, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted -47- 4862-8761-3125.1 Atty. Dkt.
  • routes of administration suitable for the methods of the disclosure include, but are not limited to, direct injection, enteral, parenteral, or inhalational routes.
  • Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal.
  • Parenteral administration can be conducted to effect systemic or local delivery of the inhibiting agent.
  • administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
  • the peptides and compositions of the disclosure can also be delivered to the subject by enteral administration.
  • Enteral routes of administration include, but are not limited to, oral and rectal (e.g., using a suppository) delivery.
  • Methods of administration of the active through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transcutaneous transmission, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • Iontophoretic transmission may be accomplished using commercially available "patches” that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more. Dosing of can be accomplished in accordance with the methods of the disclosure using capsules, tablets, oral suspension, suspension for intra-muscular injection, suspension for intravenous infusion, gel or cream for topical application, or suspension for intra-articular injection. Dosage, toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic -48- 4862-8761-3125.1 Atty. Dkt.
  • compositions which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount of a composition sufficient for achieving a therapeutic or prophylactic effect ranges from about 0.000001 mg per kilogram body weight per administration to about 10,000 mg per kilogram body weight per administration.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per administration to about 100 mg per kilogram body weight per administration.
  • Administration can be provided as an initial dose, followed by one or more “booster” doses.
  • Booster doses can be provided a day, two days, three days, a week, two weeks, three weeks, one, two, three, six or twelve months after an initial dose.
  • a booster dose is administered after an evaluation of the subject’s response to prior administrations.
  • certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • S-PrP (residues 23-231 from mouse PrP C ) was expressed and purified as previously described (21).
  • Peptides P1, P2, P3, P3*, P4, P3 (K100A) , P3 (K103A) were provided from AnaSpec. All peptides had N-terminal acetylation and C-terminal amidation.
  • coli was from Sigma- Aldrich.
  • Recombinant human NGF- ⁇ was from R&D Systems. Animals. WT C57BL/6J mice were obtained from Jackson Laboratory. To generate mice in which BMDMs are LRP1 deficient (Lrp1 ⁇ / ⁇ mice), Lrp1 flox/flox mice were bred with mice that express Cre recombinase under the control of the lysozyme-M promoter (LysM- Cre), in the C57BL/6J background, as previously described (44).
  • mice For experiments with macrophages harvested from Lrp1 ⁇ / ⁇ mice, control cells were harvested from littermates that were Lrp1 flox/flox but LysM-Cre-negative (Lrp1 +/+ mice). To generate mice in which macrophages are deficient in the essential NMDA-R GluN1 subunit (Grin1 ⁇ / ⁇ mice), Grin1 flox/flox mice were bred with mice that express Cre recombinase under the control of the LysM-Cre promoter in the C57BL/6J background. When BMDMs were harvested from Grin1 -/- mice, control cells were harvested from littermates that were Grin1f lox/flox but LysM-Cre-negative.
  • Prnp ⁇ / ⁇ mice were generously provided by Dr. Adriano Aguzzi (University Hospital of Zurich, Zurich, Switzerland). Cell culture model systems. BMDMs were harvested from 16-week-old wild-type male mice, as previously described (40, 44). Briefly, bone marrow cells were flushed from mouse femurs, plated in non-tissue culture-treated dishes, and cultured in DMEM/F-12 medium containing 10% fetal bovine serum (FBS) and 20 nM mouse macrophage colony- stimulating factor (BioLegend) for 7 days. Non-adherent cells were eliminated. Adherent cells included >95% BMDMs, as determined by F4/80 and CD11b immunoreactivity.
  • FBS fetal bovine serum
  • BioLegend mouse macrophage colony- stimulating factor
  • Rat PC12 cells were from the ATCC (CRL-1721) and subjected to quality control tests by the ATCC.
  • PC12 cells were cultured in Dulbecco's modified Eagle's medium -50- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 (DMEM, high glucose; Thermo Fisher Scientific) containing 10% heat-inactivated FBS, 5% heat-inactivated horse serum (Thermo Fisher Scientific), in plates that were pre-coated with 0.01 mg/mL type IV collagen (Sigma-Aldrich). Cells were passaged no more than eight times.
  • DMEM high glucose
  • Thermo Fisher Scientific heat-inactivated FBS
  • Thermo Fisher Scientific heat-inactivated horse serum
  • Microglia were isolated from C57BL/6J mouse pups, as described previously (60). In brief, brains were harvested from postnatal day 1–6 mice. The cortices were dissected from the forebrain, and the surrounding meninges were removed. Intact cortices were mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec). Mixed glial cultures were established in DMEM/F-12 supplemented with GlutaMAX (Thermo Fisher Scientific), 10% FBS, and 1 ⁇ Gibco Antibiotic-Antimycotic (Thermo Fisher Scientific). After culturing for 10–14 days, microglia were harvested by shaking the mixed cultures at 200 rpm for 30 min at 37 °C.
  • the floating cells were collected by centrifugation (5 min, 1500 rpm) and re-plated at 3 ⁇ 10 5 cells/well. Culture purity was >96% as determined by immunofluorescence microscopy for Iba1 (positive), glial fibrillary acidic protein (negative), ⁇ -III tubulin (negative), and OLIG1 (negative). Experiments were performed within 24 h of completing cell isolations. Gene silencing. Rat-specific ON-TARGETplus SMARTpool siRNA, targeting Lrp1 or Grin1, and pooled NTC siRNA were from Horizon Discovery. PC12 cells (2 ⁇ 10 6 ) were transfected with siRNA by electroporation using the Cell Line Nucleofector Kit V (Lonza), following the manufacturer's instructions.
  • BMDMs were transferred to serum-free medium (SFM) for 30 min and treated for 6 h with various proteins and reagents, alone or simultaneously as noted, including: LPS (0.1 ⁇ g/ml); various synthetic peptides at different concentrations; MK-801 (1 ⁇ M); or vehicle (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, PBS).
  • RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad). qPCR was performed using TaqMan gene -51- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 expression products (Thermo Fisher Scientific).
  • Primer-probe sets were as follows: GAPDH (Mm99999915_g1); TNF ⁇ (Mm00443258_m1); IL-6 (Mm00446190_m1). The relative change in mRNA expression was calculated using the 2 ⁇ Ct method with GAPDH mRNA as a normalizer.
  • Flow Cytometry Loss of the NMDA-R from the surfaces of purified BMDMs from Grin1 ⁇ / ⁇ mice was demonstrated by flow cytometry. Non-permeabilized cells were labeled with NMDA-R Glun1 subunit-specific antibody (Invitrogen #PA3-102, Thermo Fisher Scientific).
  • Cell-associated PA3-102 was detected with A647-conjugated secondary antibody (Thermo Fisher Scientific). Control cells were treated with secondary antibody only. All data were analyzed using FlowJo Software version 10.7.1 (BD Biosciences). Cell-signaling. BMDMs were transferred to SFM for 30 min and treated for 1 h with various proteins and reagents, alone or simultaneously as noted. PC12 cells were cultured in serum-containing medium until ⁇ 70% confluent. The cells were then transferred into SFM for 2 h before treatment with various reagents. Some cultures were pretreated with MK-801 (1 ⁇ M), as noted.
  • Microglia were cultured in SFM for 30 min and then treated with LPS (0.1 ⁇ g/ml) for 1 h in the presence and absence of S-PrP (40 nM) or various synthetic peptides. Extracts of BMDMs, PC12 cells, and microglia were prepared in RIPA buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts of protein were subjected to SDS-PAGE and electro- transferred to poly-vinylidene fluoride membranes.
  • the membranes were blocked with 5% nonfat dried milk and then incubated with primary antibodies from Cell Signaling Technology that recognize: phospho-ERK1/2, total ERK1/2, phospho-I ⁇ B ⁇ , total I ⁇ B ⁇ , and ⁇ -actin.
  • the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch). Immunoblots were developed using Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and imaged using the Azure Biosystems c300 digital system. Images were processed with Adobe Photoshop 23.3.2. The presented results are representative of at least three independent experiments. -52- 4862-8761-3125.1 Atty.
  • Prnp -/- mice Male Prnp -/- mice and wild-type mice in the same genetic background (16-20-week-old, 26-28 g) were injected intraperitoneally with 9 mg/kg LPS.
  • the LD 50 for the specific LPS lot was predetermined in Applicant’s laboratory, as previously described (44), and was 12 mg/kg.
  • the mice were treated by intravenous injection with P3 (2.5 ⁇ g/g body weight) or PBS, 30 min after LPS administration. Animals were monitored and scored for signs of toxicity at 1 h intervals using the murine sepsis scoring system (47).
  • FIG. 1A Three of the peptides corresponded to sequences in the disordered N-terminal region of PrPC (FIG. 1A). Differences in the sequences of human and mouse PrP C in the regions corresponding to the synthetic peptides were conservative (FIG. 1B). Because P3 emerged as important for the activities studied here, Applicant synthesized two variants of this peptide (P3 and P3*) to completely replicate the mouse and human sequences.
  • FIG. 1C summarizes the sequences of the first five peptides, in relation to the structure of human and mouse PrP C , and a secondary set of peptides designed to explore the molecular requirements for engaging the LRP1/NMDA-R receptor assembly.
  • BMDMs bone marrow-derived macrophages
  • S- PrP and EV-associated PrP C 22, 25.
  • BMDMs were harvested as previously described (39, 40) and treated with 0.1 ⁇ g/mL LPS in the presence of increasing concentrations of each peptide for 6 h.
  • FIG. 2A shows that, in the absence of peptides, LPS significantly increased TNF ⁇ mRNA expression in the BMDMs as determined by RT-qPCR.
  • P1, P2, and P4 had no effect on LPS-induced TNF ⁇ expression.
  • P3 and P3* at concentrations of 0.2 ⁇ M or higher, blocked LPS-induced TNF ⁇ expression.
  • P3 and P3* also blocked LPS-induced interleukin-6 (IL-6) mRNA expression, whereas P1 and P4 were inactive.
  • Increased expression of pro-inflammatory cytokines in response to LPS requires NF ⁇ B activation, which may be monitored by examining I ⁇ B ⁇ phosphorylation and the -54- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 accompanying decrease in total abundance of I ⁇ B ⁇ (41).
  • FIG. 2B shows that in the absence of PrPC-derived peptides, BMDMs treated with LPS (0.1 ⁇ g/mL) for 1 h demonstrated increased phospho-I ⁇ B ⁇ and decreased total I ⁇ B ⁇ , as anticipated. P1, P2, and P4 (each at 0.5 ⁇ M) had no effect on this response. By contrast, 0.5 ⁇ M P3 blocked LPS-induced I ⁇ B ⁇ phosphory-lation and the associated decrease in total cellular I ⁇ B ⁇ .
  • FIG. 2C shows that the effects of P3 on I ⁇ B ⁇ phosphorylation were concentration-dependent; P3 at concentrations ⁇ 0.5 ⁇ M completely blocked the cell-signaling event whereas 0.2 ⁇ M P3 typically generated an inter-mediate effect.
  • P3 replicates the activity of S-PrP and EV-associated PrP C as an inhibitor of LPS-induced NF ⁇ B activation.
  • P3 is bioactive in PC12 cell and microglial cell culture model systems.
  • S-PrP and EV- associated PrP C activate ERK1/2 and promote neurite outgrowth in PC12 cells (21, 23).
  • Applicant treated PC12 cells with P1, P2, P3, P3*, and P4 (each at 0.5 ⁇ M) for 10 min.
  • FIG. 3A shows that P3 and P3* activated ERK1/2. The other peptides were inactive. ERK1/2 activation by P3 was evident throughout the P3 concentration studied (0.1-1.0 ⁇ M) (FIG. 3B).
  • CM conditioned medium
  • FIG. 4B shows that S-PrP (40 nM) and P3 (0.5 ⁇ M) were -55- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 effective, completely blocking I ⁇ B ⁇ phosphorylation and the associated decrease in total abundance of I ⁇ B ⁇ . P1 and P4 were without effect.
  • P3 activity in macrophages requires the NMDA-R and LRP1. LRP1 and the NMDA- R collaborate to mediate cell-signaling in response to S-PrP (21, 22, 25). The requirement for the NMDA-R appears to be absolute.
  • BMDMs express the NMDA-R (44).
  • LPS 0.1 ⁇ g/mL
  • P2 or P3* the non-competitive NMDA-R antagonist, MK-801
  • FIG. 5A the absence of MK-801, P3* neutralized the effects of LPS on TNF ⁇ mRNA expression.
  • mice were bred in which the gene encoding the essential NMDA-R GluN1 subunit was floxed (Grin1 fl/fl ) with mice that express Cre recombinase under the control of the LysM promoter.
  • BMDMs were harvest-ed from Grin1 fl/fl -LysM-Cre mice and GluN1 mRNA expression was compared with that detected in wild-type BMDMs isolated from Grin1 fl/fl mice that did not carry LysM-Cre.
  • Loss of the NMDA-R protein from the surfaces of BMDMs was demonstrated by flow cytometry (FIG. 5C). The degree of loss was ⁇ 70%, as determined by comparing mean fluorescence intensity.
  • LPS induced TNF ⁇ mRNA expression, as anticipated (FIG. 5D).
  • FIG. 7A shows that in cells transfected with non-targeting control (NTC) siRNA, P3 caused ERK1/2 activation. By contrast, silencing Lrp1 or Grin1 in PC12 cells blocked ERK1/2 activation in response to P3.
  • NTC non-targeting control
  • FIG. 7C summarizes image analysis studies examining individual cells in ⁇ 5 randomly selected fields from three separate experiments with each agonist and gene-silencing reagent. Statistically significant neurite outgrowth was observed only in cells transfected with NTC siRNA and stimulated with P3 or S-PrP. Lys 100 and Lys 103 are essential for the function of P3 as an agonist for the LRP1/ NMDA-R cell-signaling receptor assembly. Given the documented role of Lys residues in -57- 4862-8761-3125.1 Atty. Dkt.
  • FIG. 8 shows that although all four modified peptides demonstrated decreased potency compared with P3, peptides in which either Lys 100 or Lys 103 was modified to Ala demonstrated the most substantial change and were 5-fold decreased in potency compared with P3 variants in which either Lys 105 or Lys 109 was modified.
  • P3 (DM) A P3 derivative in which both Lys 100 and Lys 103 were modified to Ala (P3 (DM) ) failed to activate ERK1/2 at concentrations up to 20 ⁇ M.
  • Applicant examined the ability of modified P3 peptides to inhibit LPS-induced NF ⁇ B activation in BMDMs. Cells were treated with 0.1 ⁇ g/mL LPS and with the indicated concentrations of peptide for 1 h (FIG. 9A). P3 (K015A) and P3 (K109A) were only slightly less active than unmodified P3.
  • P3 (K100A) and P3 (K103A) demonstrated substantially decreased potency compared with P3 and completely inhibited LPS-induced I ⁇ B ⁇ phosphorylation only when present at 20 ⁇ M.
  • P3 (DM) was ineffective throughout the concentration range studied. These results support those obtained examining PC12 cells.
  • TNF ⁇ mRNA expression in cells treated for 6 h with LPS and with the indicated concentrations of P3 (DM) (FIG. 9B).
  • P3 (DM) failed to inhibit LPS-induced TNF ⁇ mRNA ex- pression throughout the studied P3 (DM) concentration range.
  • P3 failed to inhibit LPS-induced IL-6 mRNA expression.
  • Prnp -/- mice demonstrated significantly increased sensitivity to LPS, compared with wild-type mice.
  • Prnp -/- mice -58- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 were injected intravenously with a single dose of P3 (2.5 ⁇ g/g body weight), 30 min after LPS administration, toxicity was significantly decreased.
  • a tetrapeptide with two Lys residues mimics the activity of P3.
  • Applicant also examined the ability of a synthetic 4-mer peptide (KPSK), with two Lys residues, derived from the structure of P3, to inhibit LPS-induced NF ⁇ B activation in BMDMs.
  • KPSK synthetic 4-mer peptide
  • FIG. 11A shows that in the absence of KPSK, BMDMs treated with LPS (0.1 ⁇ g/mL) for 1 h demonstrated increased phospho-I ⁇ B ⁇ and decreased total I ⁇ B ⁇ , as anticipated.
  • PrPC has been identified as a gene product capable of attenuating inflammation in a variety of contexts (33–38, 45, 48–52), including experimental autoimmune encephalitis (33– 35) and ischemic brain injury (33, 36–38).
  • Previously reported work identified PrP C derivatives released by cells, including soluble fragments of PrP C and EV-associated PrP C , as candidate mediators of the known anti-inflammatory activity of PrP C (22, 25).
  • LRP1 and the NMDA-R as cell-signaling receptors for soluble- and EV-associated PrP C derivatives.
  • PrP C that localizes to lipid rafts, within the original cell of synthesis also may express LRP1-dependent anti-inflammatory activity by laterally associating with LRP1 within the plasma membrane; this interaction facilitates the anti-inflammatory activity of LRP1, when it is presented with ligands other than S-PrP, such as tPA (25).
  • tPA tPA
  • LRP1 ligands that activate anti- inflammatory cell-signaling pathways share a common mechanism of receptor engagement, in which at least two receptors, LRP1 and the NMDA-R, play an instrumental role (22, 25, 44).
  • the NMDA-R appears to be essential.
  • LRP1 substantially decreases the concentration of ligand required to trigger cell-signaling.
  • LRP1 captures soluble ligands, like S-PrP, and then delivers them to the NMDA-R to trigger calcium influx and activation of cell-signaling factors such as Src family kinases and PI3K.
  • the NMDA-R is reported to bind tPA and PrP C (53–55). If LRP1 transfers anti-inflammatory ligands to the NMDA-R, the ligand would most likely form a transient ternary complex in which different regions of the ligand engage LRP1 and the NMDA-R simultaneously.
  • Tandem Lys residues in a number of full-length proteins also have been implicated in LRP1-binding (26–31), although the activity of the Lys residues in LRP1/ NMDA-R-dependent cell-signaling has not been formally addressed in previous studies. Although it is unlikely that P3 bridges LRP1 to the NMDA-R, both receptors were necessary to elicit potent P3 biological activities. In addition to its activity in cell culture model systems, P3 rescued the known increased susceptibility of Prnp -/- mice to LPS challenge. This result has a number of implications. First, these studies suggest that overly exuberant pro-inflammatory responses in Prn p-/- mice may be rescued entirely by soluble derivatives of PrP C .
  • P2 peptide is italicized.
  • P3 peptide is bolded.
  • SEQ ID NO: 2 Prnp Mus musculus 1 MANLGYWLLA LFVTMWTDVG LCKKRPKPGG 31 WNTGGSRYPG QGSPGGNRYP PQGGTWGQPH 61 GGGWGQPHGG SWGQPHGGSW GQPHGGGWGQ 91 GGGTHNQWNK PSKPKTNLKH VAGAAAAGAV 121 VGGLGGYMLG SAMSRPMIHF GNDWEDRYYR 151 ENMYRYPNQV YYRPVDQYSN QNNFVHDCVN 181 ITIKQHTVTT TTKGENFTET DVKMMERVVE 211 QMCVTQYQKE SQAYYDGRRS SSTVLFSSPP 241 VILLISFLIF LIVG P1 peptide is underlined.
  • P3 peptide is bolded.
  • P4 peptide is bolded and underlined. -63- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 SEQ ID NOS: 3-32 SEQ ID Peptide Sequence Sequence in human PrP Sequence in mouse PrP Nos: 3-5 P1 Ac-KKRPKPGGWNTGGS-NH K KRPKPGGWNTGGS K KRPKPGGWNTGGS 6-8 P2 Ac-GGWGQPHGGGWGQP-NH G GWGQPHGGGWGQP G TWGQPHGGGWGQP 9-11 P3 Ac-QWNKPSKPKTNLKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 12-14 P3* Ac-QWNKPSKPKTNMKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 15-17 P4 Ac-DVKMMERVVEQMCV-NH D VKMMERVVEQMC
  • LDL Low Density Lipoprotein
  • CD91 Basu S, et al. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 2001;14(3):303–313.
  • Rauch JN, et al. LRP1 is a master regulator of tau uptake and spread. Nature 2020;580(7803):381–385. 5. Chen K, et al.
  • LRP1 is a neuronal receptor for ⁇ -synuclein uptake and spread. Molecular Neurodegeneration 2022;17(1):57. 6. Campana WM, et al. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells: Possible implications in peripheral nerve injury. Journal of Neuroscience 2006;26(43):11197–11207. 7. Mantuano E, et al. The Unfolded Protein Response Is a Major Mechanism by Which LRP1 Regulates Schwann Cell Survival after Injury. Journal of Neuroscience 2011;31(38):13376– 13385. 8. remplitsch A, et al.
  • LDL receptor-related protein 1 acts as an early injury detection receptor and activates c-Jun in Schwann cells. Neuroreport 2016;27(18):1305– 1311.
  • Bacskai BJ et al.
  • the endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A 2000;97(21):11551– 11556.
  • Martin AM et al. The functional role of the second NPXY motif of the LRP1 ⁇ -chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors. Journal of Biological Chemistry 2008;283(18):12004–12013. 11.
  • LDL receptor-related protein-1 is a sialic-acid-independent receptor for myelin-associated glycoprotein that functions in neurite outgrowth inhibition by MAG and CNS myelin. Journal of Cell Science 2013;126(1):209–220. -65- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 13. Mantuano E, Lam MS, Gonias SL.
  • LRP1 assembles unique co-receptor systems to initiate cell signaling in response to tissue-type plasminogen activator and myelin-associated glycoprotein. Journal of Biological Chemistry 2013;288(47):34009–34018. 14. May P, et al. Neuronal LRP1 Functionally Associates with Postsynaptic Proteins and Is Required for Normal Motor Function in Mice. Molecular and Cellular Biology 2004;24(20):8872–8883. 15. Nakajima C, et al.
  • LRP1 Low Density Lipoprotein Receptor-related Protein 1
  • NMDA N -Methyl-d-aspartate
  • LRP1 low-density lipoprotein receptor-related protein 1
  • Mattei V, et al. A multimolecular signaling complex including PrPC and LRP1 is strictly dependent on lipid rafts and is essential for the function of tissue plasminogen activator. Journal of Neurochemistry 2020;152(4):468–481. 21. Mantuano E, et al.
  • Watanabe T et al. Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells. Neurosci Lett 2011;496(2):121–124. 37. Shao J, et al. Cellular Prion Protein Attenuates OGD/R-Induced Damage by Skewing Microglia toward an Anti-inflammatory State via Enhanced and Prolonged Activation of Autophagy. Mol Neurobiol 2023;60(3):1297–1316. -67- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 38. Steele AD, et al. Context dependent neuroprotective properties of prion protein (PrP).
  • Salvesen ⁇ , et al. LPS-induced systemic inflammation reveals an immunomodulatory role for the prion protein at the blood-brain interface. J Neuroinflammation 2017;14(1):106. 60. Chida J, et al. Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice. PLOS Pathogens 2020;16(8):e1008823. 61. Salvesen ⁇ , et al. Goats without Prion Protein Display Enhanced Proinflammatory Pulmonary Signaling and Extracellular Matrix Remodeling upon Systemic Lipopolysaccharide Challenge. Front Immunol 2017;8:1722. 62. Bakkeb ⁇ MK, et al.
  • the Cellular Prion Protein A Player in Immunological Quiescence. Front Immunol 2015;6:450.
  • a ⁇ neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A 2012;109(5):1737– 1742. 66. Gonias SL. Plasminogen activator receptor assemblies in cell-signaling, innate immunity, and inflammation. American Journal of Physiology-Cell Physiology [published online ahead of print: August 18, 2021]; doi:10.1152/ajpcell.00269.2021 -69- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 67. Van Leuven F. Human ⁇ 2-macroglobulin: structure and function. Trends in Biochemical Sciences 1982;7(5):185–187.

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Abstract

Provided herein are synthetic peptides that replicate the cell-signaling and biological activities of full-length shed PrPc, and a synthetic 4-mer peptide (KPSK), with two Lys residues, derived from the structure of P3 have uniquely broad anti-inflammatory activity.

Description

Atty. Dkt. No.: 114198-6810 NMDA-R ACTIVATING PEPTIDES IN INFLAMMATION AND NEURONAL DIFFERENTIATION CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Serial No. 63/506,609, filed June 7, 2023, the contents of which are hereby incorporated by reference into the present disclosure in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under HL136395 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE DISLOSURE Diseases linked to an inflammatory response are difficult to treat for several reasons. For example, the immune system is incredibly complex, involving various cells, proteins, and signaling pathways. Dysregulation of any part of this system can lead to inflammation and disease. Finding therapies that target the specific mechanisms driving inflammation without compromising the body's ability to fight infections or heal wounds is challenging. In addition, each inflammatory disease may have different underlying causes, triggers, and manifestations. What works for one patient may not work for another, necessitating personalized treatment approaches. Moreover, in many cases, inflammation becomes chronic, persisting over long periods. Chronic inflammation can lead to tissue damage and functional impairment, making it harder to manage and treat the underlying disease. While there are various medications available to manage inflammation, such as nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and biologic therapies targeting specific inflammatory pathways, these treatments may not be effective for everyone. Moreover, some medications may have side effects or lose effectiveness over time. -1- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Thus, a need exists in the art to find safe and effective treatment for diseases and conditions that involve inflammation. This disclosure satisfies this need and provides related advantages as well. SUMMARY OF THE DISCLOSURE Low Density Lipoprotein Receptor-related Protein-1 (LRP1) is a type-1 transmembrane receptor that binds and mediates the endocytosis of diverse ligands, including lipoproteins, proteases, protease inhibitors, growth factors, extracellular matrix proteins, heat shock proteins, and proteins released by injured and dying cells, including microtubule- associated protein Tau and α-synuclein (1–5). The evolutionary foundation for a receptor with such a broad scope of ligands remains unclear; however, LRP1 may function as an injury detection receptor, as has been most fully defined for Schwann cell LRP1 (6–8). Specificity in the function of LRP1 may be manifested in the ability of different ligands to elicit diverse cell-signaling responses by engaging distinct cell-signaling co-receptors, including the N-methyl-D-aspartate receptor (NMDA-R), Trk receptors, and p75 neurotrophic receptor (9–15). Nonpathogenic cellular prion protein (PrPC) is expressed by numerous cells inside and outside the nervous system (16, 17) and interacts with LRP1 in three different states. First, PrPC, which is GPI-anchored to the plasma membrane, laterally associates with LRP1 in the same cell (18–20). Second, PrPC, which is released from the cell surface in the form of soluble derivatives by ADAM proteases, binds to LRP1 (21). Finally, PrPC that is embedded in exosomes and other extracellular vesicles (EVs) associates with LRP1 in target cells (22, 23). It has been demonstrated that a recombinant derivative of PrPC (S-PrP), corresponding closely to the product released from cells by ADAM10 (24), and PrPC-bearing EVs isolated from human plasma activate cell-signaling in macrophages and PC12 cells, in an LRP1- and NMDA-R-dependent manner (21–23, 25). As a result, these PrPC derivatives oppose the activity of Pattern Recognition Receptors, including Toll-like Receptors (TLRs) in macro- phages and promote neurite outgrowth in PC12 cells. Molecular analysis of the interaction of LRP1 with a number of ligands, including activated α2-macroglobulin (α2M), plasminogen activator inhibitor-1, coagulation Factor -2- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 VIII, and Receptor-associated Protein (RAP), has demonstrated an essential role for ligand- as-sociated Lys residues, typically in tandem (26–31). Applicant screened a series of synthetic peptides, corresponding to the structure of PrPC, and identified a putative LRP1- binding motif just distal to the octarepeat region, in the disordered N-terminal half of PrPC. A 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) replicated all of the cell-signaling activities of full-length S-PrP in a manner that required LRP1 and the NMDA-R. P3 also rescued the increased susceptibility of PrPC gene (Prnp) knock-out mice to lipopolysaccharide (LPS). Lys100 and Lys103, from the structure of PrPC, were essential for the cell-signaling activity of P3; when both residues were converted to Ala in P3(DM), cell- signaling and biological activity were completely eliminated. Synthetic peptides have been transformed into therapeutics at an increasing rate in recent decades (32). Although PrPC is previously reported to demonstrate anti-inflammatory activity in a variety of contexts, including in experimental autoimmune encephalitis (33–35) and in ischemic brain injury (33, 36–38), incomplete understanding of the responsible molecular mechanism has hindered efforts to exploit this activity of PrPC in therapeutics development. Applicant identified and reports herein that P3 and the LRP1/NMDA-R receptor assembly are members of single system with anti-inflammatory activity. Applicant also reports new studies exploring the efficacy of PrPC derivatives as candidate therapeutics in a variety of disease states in which inflammation plays a role. The present disclosure provides a 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) (present on the structure of PrPC and containing amino acids Lys103 and/or Lys105 that replicated all of the cell-signaling activities of full-length soluble-PrP (S-PrP) in a manner that required LRP1 and the NMDA-R. The present disclosure also provides a synthetic 4-mer peptide (KPSK) (SEQ ID NO: 33), with the two Lys residues, derived from the structure of P3. Both peptides showed broad anti- inflammatory activity. P3 inhibited lipopolysaccharide (LPS)-elicited cytokine expression in macrophages and microglia. The present disclosure further provides that both peptides attenuate innate immunity and regulate neuronal differentiation by an NMDA Receptor- dependent mechanism. A treatment based on these peptides is developed for diseases where innate immunity and neuroinflammation play a key role, including Inflammatory Bowel -3- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A – 1C: Synthetic peptides and their relation to the structure of PrPC. (FIG. 1A) (SEQ ID NOS: 3-32, sequence identifiers relate to sequences from left to right, e.g., SEQ ID NO: 1 is KKRPKPGGWNTGGS-NH2; SEQ ID NO: 2 is K23KRPKPGGWNTGGS36 and SEQ ID NO: 3 is K23KRPKPGGWNTGGS36.) Location of the primary set of four synthetic peptides in relation to the structure of PrPC. (FIG. 1B) (SEQ ID NOS: 1 and 2). , P1-P4 are located within the primary sequences of human and mouse PrPC. (FIG. 1C) (SEQ ID NOS: 3-32). The sequences of all studied synthetic peptides, including variants of P3/P3* are shown. Lys residues and Lys residues that were converted to Ala in second generation peptides are underlined and bolded. Conservative sequence differences between the synthetic peptides and the structure of human and mouse PrPC are italicized. FIGS. 2A – 2C: P3 replicates the effects of S-PrP and EV-associated PrPC in macrophages. (FIG. 2A) BMDMs from C57BL/6J mice were treated for 6 h with LPS (0.1 μg/mL) in presence or absence of increasing concentrations (0.1-1.0 μM) of P1, P2, P3, P3*, or P4, as indicated. RT-qPCR was performed to determine mRNA levels of TNFα and IL-6. (n = 3-9, individual points are shown). Data are expressed as the mean ± SEM (one-way ANOVA: ****P<0.0001). (FIG. 2B) BMDMs were treated for 1 h with LPS (0.1 μg/mL) in -4- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 presence or absence of different peptides P1, P2, P3, or P4 (each at 0.5 μM). Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. (FIG. 2C) BMDMs were treated for 1 h with increasing concentrations of P3 (0.1-1 μM). Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. FIGS. 3A – 3D: P3 activates cell-signaling and promotes neurite outgrowth in PC12 cells. (FIG. 3A) PC12 cells were treated with P1, P2, P3, P3*, and P4 (each at 0.5 μM) for 10 min. Cell extracts were subjected to immunoblot analysis to detect phospho-ERK1/2 and total ERK1/2. (FIG.3B) PC12 cells were stimulated for 10 min with increasing concentrations of P3 (0.1-1 μM). Phospho-ERK1/2 and total ERK1/2 were determined. (FIG. 3C) PC12 cells were treated for 48 h with S-PrP (40 nM), P1 (0.5 μM), P3 (0.5 μM), P4 (0.5 μM), NGF-β (50 ng/ml) as a positive control, or vehicle. Neurite outgrowth was examined by phase contrast microscopy. Repre-sentative images are shown (scale bar, 50 μm). (FIG. 3D) Neurite length was determined by analyzing all the cells in ≥5 random fields per treatment, in three different experiments (mean ± SEM; one-way ANOVA: ****P<0.0001). FIGS. 4A – 4B: P3 inhibits the pro-inflammatory activity of LPS in microglia. (FIG. 4A) Microglia were isolated from C57BL/6J mouse pups and treated with LPS (0.1 μg/ml) for 6 h, in the presence and absence of S-PrP (40 nM) or P3 (0.5 μM). Conditioned medium (CM) was collected and analyzed using Proteome Profiler Mouse Cytokine Array. Representative cytokines that were increased in CM when LPS was added in the absence of S-PrP or P3 are numbered in red boxes. (FIG. 4B) Microglia were treated for 1 h with LPS (0.1 μg/mL) in presence or absence of P1, P3, or P4. Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. FIGS. 5A – 5E: The NMDA-R is necessary for the response to P3 in macrophages. (FIG. 5A) BMDMs were pre-treated with MK-801 (1 μM) or vehicle for 30 min. The cells were then treated with LPS (0.1 μg/mL), P2 (0.5 μM), or P3* (0.5 μM), as indicated for 6 h. RT-qPCR was performed to compare mRNA levels for TNFα and IL-6 (n=3-7, individual points are shown). (FIG. 5B) BMDMs were harvested from Grin1fl/fl- LysM-Cre-positive mice. GluN1 mRNA expression was determined by RT-qPCR and compared with that detected in BMDMs isolated from Grin1fl/fl-LysM-Cre-negative mice (n=3; mean ± SEM; one-way ANOVA: ****P<0.0001). (FIG. 5C) Flow cytometry was -5- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 performed to detect cell-surface GluN1 NMDA-R subunit in BMDMs isolated from Grin1fl/fl-LysM-Cre-positive and –negative (WT) mice. As a control, cells fromWT mice were incubated with secondary antibody only (grey). (FIG. 5D) BMDMs from Grin1fl/fl- LysM-Cre-positive mice were treated for 6 h with LPS (0.1 μg/mL), in presence or absence of increasing concentrations of P3 (1-20 μM), P4 (1-20 μM), or vehicle. RT-qPCR was performed to determine TNFα mRNA (n=3). Data are expressed as the mean ± SEM (one- way ANOVA: ****P<0.0001). (FIG. 5E) BMDMs from Grin1fl/fl-LysM-Cre-positive mice were treated for 1 h with LPS (0.1 μg/mL), in presence of P1, P2, P3, P3*, and P4, as indicated (each at 0.5 μM). Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. FIGS. 6A – 6B: The anti-inflammatory activity of P3/P3* is strongly facilitated by LRP1. (FIG. 6A) BMDMs from Lrp1fl/fl-LysM-Cre-positive mice were treated for 6 h with LPS (0.1 μg/mL) in presence of increasing concentrations (1-20 μM) of P1, P3, P3*, or vehicle. RT-qPCR was performed to determine mRNA levels for TNFα and IL-6 (n=3-4). Data are expressed as the mean ± SEM (one-way ANOVA: *P<0.05, **P<0.01, ****P<0.0001). (FIG. 6B) BMDMs were treated for 1 h with LPS (0.1 μg/mL) in the presence of P1, P2, P3, or P3* (each at 0.5 μM), as indicated. Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. FIGS. 7A – 7C: P3 activates ERK1/2 and promotes neurite outgrowth in PC12 cells by a mechanism that requires the NMDA-R and LRP1. (FIG. 7A) PC12 cells were transfected with Lrp1-specific siRNA, Grin1-specific siRNA, or NTC siRNA and then treated with P3 (0.5 μM) or vehicle for 10 min. ERK1/2 activation was determined by immunoblotting. (FIG. 7B) PC12 cells were transfected with Lrp1-specific siRNA, Grin1- specific siRNA, or NTC siRNA, as indicated. The cells were then treated with S-PrP (40 nM), P3 (0.5 μM), or P4 (0.5 μM) for 48 h. Neurite outgrowth was detected by phase contrast microscopy. Representative images are shown (scale bar, 50 μm). (FIG. 7C) Neurite length was determined in all the cells of ≥5 random fields per treatment, in three different experiments (mean ± SEM; one-way ANOVA: ****P<0.0001). FIG. 8: Lys100 and Lys103 are required for the function of P3 in PC12 cells. PC12 cells were treated for 10 min with increasing concentrations (0.5-20 μM) of P3(K100A), -6- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 P3(K103A), P3(K105A), P3(K109A), or P3(DM), as indicated above each panel. Immunoblot analysis was per-formed to determine activation of ERK1/2. FIGS. 9A – 9B: Lys and Lys are required for the function of P3 in macrophages. (FIG.9A) BMDMs from wild-type mice were treated for 1 h with LPS (0.1 μg/mL) and increasing concentrations (2.5-20 μM) of P3(K100A), P3(K103A), P3(K105A), P3(K109A), or P3(DM), as indicated above each panel. Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. (FIG.9B) BMDMs from wild-type mice were treated for 6 h with LPS (0.1 μg/mL) in presence of increasing concentrations of P3(DM) (2.5-20 μM). RT-qPCR was performed to determine mRNA levels for TNFα and IL-6 (n=3). Data are expressed as the mean ± SEM (one-way ANOVA: **P<0.01, ***P<0.001). FIG. 10: P3 rescues the increased susceptibility of Prnp mice to LPS.16-20-week old male Prnp-/- mice (orange) and wild-type mice in the same genetic background (black) were challenged with LPS, by IP injection, at 75% of the LD50. A second matched cohort of Prnp-/- mice were treated with LPS and then with P3, 0.5 h later. Toxicity was scored as described in the Methods. Prnp mice demonstrated significantly more toxicity compared with wild-type mice (n=4; mean ± SEM; two-way ANOVA: *P<0.05; ***P<0.001; ****P<0.0001). P3 significantly reversed the toxicity of LPS in Prnp-/- mice (n=4; mean ± SEM; two-way ANOVA: †P<0.05; †††P<0.001; ††††P<0.0001). FIGS. 11A – 11B: A tetrapeptide with two Lys residues mimics the activity of P3. (FIG. 11A) BMDMs were treated for 6 h with LPS and the indicated concentrations of KPSK. RT-qPCR was performed to determine mRNA levels for TNFα and IL-6 (n=3). Data are expressed as the mean ± SEM (one-way ANOVA: *P<0.05, ***P<0.001). (FIG.11B) BMDMs were treated for 1 h with LPS (0.1 μg/mL) in presence or absence of increasing concentrations of KPSK (0.2-5 μM). Immunoblot analysis was performed to detect phospho- IκBα, total IκBα, and β-actin. DETAILED DESCRIPTION All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing -7- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. -8- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate or alternatively by a variation of +/- 15 %, or alternatively 10% or alternatively 5% or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term -9- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a catalyst,” “a metal,” or “a substrate,” includes, but are not limited to, mixtures or combinations of two or more such catalysts, metals, or substrates, and the like. It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”. -10- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. “A synthetic peptide intends” a peptide or protein that does not exist in nature and has been made by human intervention. -11- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 The term “PNRP” intends a gene that encodes a protein called prion protein (PrP) which is active in the brain and several other tissues. The human and mouse sequences of the proteins are disclosed in FIG. 1B (SEQ ID NOS: 1 and 2). NMDA-R or N-methyl-D-aspartate (NMDA) receptor is a receptor of glutamate, the primary excitatory neurotransmitter in the human brain. The receptor plays an integral role in synaptic plasticity, which is a neuronal mechanism believed to be a function of memory formation. Structurally, functional NMDA receptors are heterotetramers comprising different combinations of the GluN1, GluN2 (A-D), and GluN3 (A-B) subunits derived from distinct gene families (Grin1-Grin3). All NMDARs contain two of the obligatory GluN1 subunits, which when assembled with GluN2 subunits of the same type, give rise to canonical diheteromeric (d-) NMDARs (e.g., GluN1-2A-1-2A). Triheteromeric NMDARs, by contrast, contain three different types of subunits (e.g., GluN1-2A-1-2B), and include receptors that are composed of one or more subunits from each of the three gene families, designated t- NMDARs (e.g., GluN1-2A-3A-2A). There is one GluN1, four GluN2, and two GluN3 subunit encoding genes, and each gene may produce more than one splice variant. Protein and polynucleotide sequences encoding the protein subunits are known in the art, e.g., NMDA type subunit 1 (Homo sapiens, see Accession number Q05586 and https://www.genenames.org/data/gene-symbol-report/#!/hgnc_id/HGNC:4584, last accessed on May 30, 2024); NMDA-A (Accession No. Q12879 and https://www.uniprot.org/uniprotkb/Q12879/entry#sequences, last accessed on May 30, 2024); NMDA 2B, Accession No. Q13224 and https://www.uniprot.org/uniprotkb/Q13224/entry, last accessed on May 30, 2024; NMDA 2C, Accession No. Q14957 and https://www.uniprot.org/uniprotkb/Q14957/entry, last accessed on May 30, 2024; and NMDA 2D, Accession No. and O15399 and https://www.uniprot.org/uniprotkb/O15399/entry, last accessed on May 30, 2024); NMDA 3A, Accession No. Q8TCU5 and https://www.uniprot.org/uniprotkb/Q8TCU5/entry; and NMDA 3B, Accession No. O15399 and https://www.uniprot.org/uniprotkb/O15399/entry, last accessed on May 30, 2024 NMDA 3B, Accession No. and O60391 and https://www.uniprot.org/uniprotkb/O60391/entry, last accessed on May 30, 2024. -12- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “NMDA-R activation” intends any activation of the NMDA-R that results in the opening of the ion channel that is nonselective to cations. Activation of postsynaptic NMDA receptors in most hippocampal pathways controls the induction of activity-dependent synaptic modification. “LRP1” intends the LDL receptor related protein 1. As noted by the NIH, the LRP1 gene encodes a member of the low-density lipoprotein receptor family of proteins. The encoded preproprotein is proteolytically processed by furin to generate 515 kDa and 85 kDa subunits that form the mature receptor. This receptor is involved in several cellular processes, including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells. In addition, the encoded protein is necessary for the alpha 2-macroglobulin-mediated clearance of secreted amyloid precursor protein and beta-amyloid, the main component of amyloid plaques found in Alzheimer patients. Expression of this gene decreases with age and has been found to be lower than controls in brain tissue from Alzheimer's disease patients. See https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=4035, last accessed on May 30, 2024. The human protein is reported at Accession No. Q07954 and https://www.uniprot.org/uniprotkb/Q07954/entry, last accessed on May 30, 2024. “LRP1-binding motif” a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111 (peptide P3). The human sequence comprises, or consists essentially of, or yet further consists of amino acids QWNKPSKPKTNMKH. The murine sequence comprises, or consists essentially of, or yet further consists of QWNKPSKPKTNLKH. Variations of the human and murine sequence are disclosed in FIG. 1C. PrPC is a non-pathogenic cellular prion protein. S-PrP (soluble cellular prion protein) is a recombinant derivative of PrPC that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin and Metalloprotease (ADAM) family. “P3” is a synthetic peptide derived from a LRP1-binding motif. Examples of P3 peptides are disclosed in FIG. 1C and in the partial sequence below. In one aspect, the term P3 include the disclosed peptides in SEQ ID NOS: 9-14 and 18-32 and equivalents thereof -13- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 that have at least 80% or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% sequence identify to the peptides comprising, consisting essentially of, or consisting of the peptides shown in SEQ ID NOS: 9- 14 and 18-32, with in one aspect, the equivalents retain at least one or both lysines. In a further aspect, the equivalent retains one or both lysines and substantial activity as compare to wildtype P3 (SEQ ID Nos 9-11) as determined using an assay as described herein, for example the P3 equivalent replicates cell-signaling and biological activities of full-length shed PrPc. In a further aspect, the term “P3” intends 14-mer synthetic peptide corresponding to a putative LRP1-binding motif, wherein the 14-mer synthetic peptide contains one or both Lys 103 and Lys 105 (P3) (murine P3s, or the corresponding lysines in the human P3s), and wherein said P3 replicates cell-signaling and biological activities of full-length shed PrPc. Full-length soluble-PrP (S-PrP) is the soluble form of PrP. “Derived from” intends that the protein, polypeptide, or gene is a modification of another protein, polypeptide, or gene, that in one aspect, is a wildtype protein, polypeptide or gene. “Innate immunity” is nonspecific immunity that is the immune defense that is present at birth. It protects against all antigens. Examples include the cough reflex, enzymes in tears and skin oils, mucus, skin and stomach acid. “Neuroinflammation” is an inflammatory response within the brain or spinal cord. “Neurodegenerative diseases” are chronic conditions that damage and destroy parts of the nervous system, especially the brain. Non-limiting examples include Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy. The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum -14- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 number of amino acids which may comprise a protein’s or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides. The term -15- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non- naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In another aspect, an equivalent intends at least about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Where the polypeptide has been modified (mutated) from a parent sequence such as the 14-mer P3 peptides, the equivalent thereof or biological equivalent thereof has retained the mutated or modified amino acids but the remaining amino acids can be modified without significant loss of function. Thus, an equivalent of P3* can have at least 80% percent identity to the parent P3* peptide but retains the mutated amino acids. -16- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 In one aspect, an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods. In another aspect, an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70 %, or alternatively at least 75 %, or alternatively at least 80 %, or alternatively at least 85 %, or alternatively at least 90 %, or alternatively at least 95 % affinity or higher affinity to a reference antibody or antigen binding fragment. In another aspect, the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen under a competitive ELISA assay. In another aspect, an equivalent intends at least about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address://align.genome.jp/, last accessed on March 7, 2011. “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing -17- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. -18- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell. The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. As used herein, the terms "treating," "treatment" and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder. In one aspect, prophylaxis or prevention is excluded from the term “treatment.” To prevent intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect. An example of such is preventing the disease from progressing to the state of clinical or diagnostic symptoms. A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant or preservative, e.g., DMSO or glycol. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions of the disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium -19- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application. An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. The term “effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. In the context of an immunogenic composition, in some embodiments the effective amount is -20- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject’s immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment. “Liposomes” are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol- amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan- olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), -21- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carb- oxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs). The biological active agents can be encapsulated in such for administration in accordance with the methods described herein. A “micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle). Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue. The phrase “pharmaceutically acceptable polymer” refers to the group of compounds which can be conjugated to one or more polypeptides described here. It is contemplated that the conjugation of a polymer to the polypeptide is capable of extending the half-life of the polypeptide in vivo and in vitro. Non-limiting examples include polyethylene glycols, polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof. The biological active agents can be -22- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 conjugated to a pharmaceutically acceptable polymer for administration in accordance with the methods described herein. A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. A polynucleotide of this disclosure can be delivered to a cell or tissue using a gene delivery vehicle. “Gene delivery,” “gene transfer,” “transducing,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector- mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein. A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally -23- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for. A “yeast artificial chromosome” or “YAC” refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearized by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. Yeast expression vectors, such as YACs, YIps (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than BACs, producing chimeric effects. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, -24- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, “retroviral mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell’s genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in -25- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 vitro transcription, it may be necessary to remove, add or alter 5’ and/or 3’ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5’ of the start codon to enhance expression. Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this disclosure. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this disclosure are other non-limiting techniques. As used herein, the term “label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histadine tags (N-His), magnetically active isotopes, e.g., 115Sn, 117Sn and 119Sn, a non-radioactive isotopes such as 13C and 15N, polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non- radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component -26- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent. “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian, and human. -27- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called an episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2µm in diameter and 10 µm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium. In the context of this disclosure, a “ligand” is a polypeptide. In one aspect, the term "ligand" as used herein refers to any molecule that binds to a specific site on another molecule. In other words, the ligand confers the specificity of the protein in a reaction with an immune effector cell or an antibody to a protein or DNA to a protein. In one aspect it is the ligand site within the protein that combines directly with the complementary binding site on the immune effector cell. As used herein, "solid phase support" or "solid support", used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels. As used herein, "solid support" also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE.RTM. resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel.RTM., Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, Calif.). An example of a solid phase support include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble. The support material may have virtually any possible structural configuration so -28- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 long as the coupled molecule is capable of binding to a polynucleotide, polypeptide or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen or will be able to ascertain the same by use of routine experimentation. The term "modulate an immune response" includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response. An immunomodulatory method (or protocol) is one that modulates an immune response in a subject. A “subject” of diagnosis or treatment is a cell or an animal such as a mammal, or a human. Non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets. As used herein, the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. Non- limiting examples of cytokines which may be used alone or in combination in the practice of the present disclosure include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL- 3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1α), interleukin-11 (IL-11), MIP-11, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present disclosure also includes culture conditions in which one or more cytokine is specifically excluded from the medium. Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) and Immunex (Seattle, Wash.). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the disclosure. -29- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere). Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. It should be emphasized that the described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above- described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims. Modes for Carrying Out the Disclosure Polypeptides and Compositions The present disclosure provides a 14-mer synthetic peptide corresponding to the putative LRP1-binding motif (P3) present on the structure of PrPC and a synthetic 4-mer peptide (KPSK) (SEQ ID NO: 33), with two Lys residues, derived from the structure of P3 that have uniquely broad anti-inflammatory activity. The data presented herein show that -30- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 both peptides attenuate innate immunity and regulate neuronal differentiation by an NMDA Receptor-dependent mechanism. In one aspect, the synthetic 14-mer peptide consists essentially of, or yet further consists of an isolated P3, P3*, P3(K100A), P3(K103A), P3(K105A), P3(K109A), and P3(DM) (SEQ ID NOS: 9-32), as shown in the rows of FIG. 1C (from left to right is shown sequence, sequence in human PrPc and sequence in mouse PrPc), fragments, and equivalents of these peptides that retain the mutated amino acids as compared to wildtype P3 (SEQ ID NOS: 9-11). In one aspect, the synthetic 14-mer peptide consists essentially of, or yet further consists of a lysine at one or both of amino acid positions 103 and 105 (Lys103 and Lys105 (P3)), fragments, and equivalents of these peptides that retain one or both of the Lys103 and Lys105mutated amino acids as compared to wildtype P3. The amino acid numbering of the variants is relative to the mouse PrPc and the human variants may have different numbering as shown in FIG. 1C. In another aspect, an isolated 4-mer peptide (KPSK) is provided. In a further aspect, the 14-mer peptides also comprise N-terminal acetylation and C-terminal amidation. In certain embodiments, a series of PrPC-derived peptides were screened, and a putative Low Density Lipoprotein Receptor-related Protein-1 (LRP1) recognition motif that includes Lys100 and Lys103 was identified. A 14-mer synthetic peptide that contains Lys103 and Lys105 (P3) was identified (see FIG. 1C), and it replicated the cell-signaling and biological activities of full-length shed PrPC. P3 inhibited lipo-polysaccharide (LPS)-elicited cytokine expression in macrophages and microglia and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA Receptor (NMDA-R), which is associated to form a cell-signaling receptor assembly. P3 rescued the heightened sensitivity to LPS in mice in which the gene encoding PrPC (Prnp) is deleted. When Lys100 and Lys103 in P3 were converted to Ala, the activity of P3 in cell-signaling and in regulating macrophage physiology was entirely blocked. In certain embodiments, the ability of a synthetic 4-mer peptide (KPSK), with two Lys residues, derived from the structure of P3, was also examined to inhibit LPS-induced NFκB activation in BMDMs. The tetrapeptide KPSK mimics exactly the activity of P3. These results suggest that the anti-inflammatory activity of PrPC and the LRP1/NMDA-R -31- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 receptor assembly may be harnessed in new candidate therapeutics, such as the 14-mer synthetic peptide P3 and the derived synthetic 4-mer peptide (KPSK), disclosed herein. Synthetic peptides have been transformed into therapeutics at an increasing rate in recent decades. Here, for the first time, the present disclosure provides that NMDA-R activating peptides can regulate inflammation and neuronal differentiation. The scientific discovery disclosed herein opens up opportunities to develop unique chemistries with anti- inflammatory activity. These new and unique peptides could serve as an excellent template for developing new anti-inflammatory drugs to treat diseases in which innate immunity and neuroinflammation plays an important role, including Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. The new and unique NMDA-R activating peptides disclosed herein can regulate innate immunity and neuronal differentiation. The data presented herein show for the first time that these novel synthetic peptides replicate the anti-inflammatory activity of the full- length protein of non-pathogenic cellular prion protein (PrPC) through the NMDA-R receptor and can been transformed into new candidate therapeutics. These data give the foundation to develop unique chemistries with anti-inflammatory activity and regulation of neuronal differentiation. Novel future therapies can be identified using this preparatory information. In any of the above embodiments, a peptide linker can be added to the N-terminus or C-terminus of the polypeptide. A “linker” or “peptide linker” refers to a peptide sequence linked to either the N-terminus or the C-terminus of a polypeptide sequence. In one aspect, the linker is from about 1 to about 20 amino acid residues long or alternatively 2 to about 10, about 3 to about 5 amino acid residues long. An example of a peptide linker is Gly-Pro-Ser- Leu-Lys-Leu (SEQ ID NO: 35). Other examples include Gly-Gly-Gly; Gly-Pro-Ser-Leu (SEQ ID NO: 36); Gly-Pro-Ser; Pro-Ser-Leu-Lys (SEQ ID NO: 37); Gly-Pro-Ser-Leu-Lys (SEQ ID NO: 38); and Ser-Leu-Lys-Leu (SEQ ID NO: 39). The isolated polypeptides of this disclosure are intended to include isolated wildtype and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof. -32- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 It is understood that functional equivalents or variants of the wildtype polypeptide or protein also are within the scope of this disclosure, for example, those having conservative amino acid substitutions of the amino acids, see for example, FIG. 1C that retain the synthetic mutations as compared to wildtype. In a further aspect, the polypeptides are conjugated or linked to a detectable label. Suitable labels are known in the art and described herein. The proteins and polypeptides are obtainable by a number of processes known to those of skill in the art, which include purification, chemical synthesis and recombinant methods. Polypeptides can be isolated from preparations such as host cell systems by methods such as immunoprecipitation with antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography. For such methodology, see for example Deutscher et al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol. 182, Academic Press). Accordingly, this disclosure also provides the processes for obtaining these polypeptides as well as the products obtainable and obtained by these processes. The polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin/ Elmer/Applied Biosystems, Inc., Model 430A or 43lA, Foster City, CA, USA. The synthesized polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this disclosure also provides a process for chemically synthesizing the proteins of this disclosure by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence. Alternatively, the proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook et al. (1989) supra, using a host cell and vector systems described herein. Also provided by this application are the polypeptides described herein conjugated to a detectable agent for use in the diagnostic methods. For example, detectably labeled polypeptides can be bound to a column and used for the detection and purification of -33- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 antibodies. They also are useful as immunogens to produce antibodies. The polypeptides of this disclosure are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes. The polypeptides of this disclosure also can be combined with various solid phase carriers, such as an implant, a stent, a paste, a gel, a dental implant, or a medical implant or liquid phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one. However, for the purpose of illustration only, suitable adjuvants include, but are not limited to Freund’s Complete and Incomplete, mineral salts and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat labile enterotoxin of E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene. This disclosure also provides a pharmaceutical composition comprising or alternatively consisting essentially of, or yet further consisting of, any of a polypeptide or an equivalent of this disclosure, alone or in combination with each other or other agents and an acceptable carrier or solid support. These compositions are useful for various diagnostic and therapeutic methods as described herein. Polynucleotides This disclosure also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides and their respective complementary strands. Vectors comprising the isolated or recombinant polynucleotides are further provided examples of which are known in the art and briefly described herein. In one aspect where more than one isolated or recombinant polynucleotide is to be expressed as a single unit, the isolated or recombinant polynucleotides can be contained within a polycistronic vector. The polynucleotides can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA. -34- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 One of skill in the art can make such polynucleotides using the information provided herein and knowledge of those of skill in the art. See Goodman and Kay (1999) J. Biological Chem. 274(52):37004-37011 and Kamashev and Rouviere-Yaniv (2000) EMBO J. 19(23):6527-6535. The disclosure further provides the isolated or recombinant polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA. As used herein, the term “operatively linked” means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, cell-specific promoters are used for cell-specific expression of the inserted polynucleotide. Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are known in the art and commercially available. For general methodology and cloning strategies, see Gene Expression Technology (Goeddel ed., Academic Press, Inc. (1991)) and references cited therein and Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, N.Y. (1994)) which contains maps, functional properties, commercial suppliers and a reference to GenEMBL accession numbers for various suitable vectors. In one embodiment, polynucleotides derived from the polynucleotides of the disclosure encode polypeptides or proteins having diagnostic and therapeutic utilities as described herein as well as probes to identify transcripts of the protein that may or may not be present. These nucleic acid fragments can by prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker. Alternatively, random fragments can be generated using nick translation of the molecule. For methodology for the preparation and labeling of such fragments, see Sambrook, et al. (1989) supra. Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Non-limiting examples of suitable expression vectors include plasmids, yeast vectors, viral vectors and liposomes. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient -35- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989) supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See, Sambrook et al. (1989) supra, for methodology. Thus, this disclosure also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. When the vectors are used for gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector. Pharmaceutically acceptable vectors containing the nucleic acids of this disclosure can be further modified for transient or stable expression of the inserted polynucleotide. As used herein, the term “pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells. An example of such a vector is a “replication-incompetent” vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell. An example of a replication-incompetent retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7:980-990). The methodology of using replication-incompetent -36- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 retroviruses for retroviral-mediated gene transfer of gene markers has been established. (Bordignon (1989) PNAS USA 86:8912-8952; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood 79(10):2694-2700). This disclosure also provides genetically modified cells that contain and/or express the polynucleotides of this disclosure. The genetically modified cells can be produced by insertion of upstream regulatory sequences such as promoters or gene activators (see, U.S. Patent No. 5,733,761). The polynucleotides can be conjugated to a detectable marker, e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell. A wide variety of appropriate detectable markers are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In one aspect, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. Thus, this disclosure further provides a method for detecting a single-stranded polynucleotide or its complement, by contacting target single-stranded polynucleotide with a labeled, single-stranded polynucleotide (a probe) which is a portion of the polynucleotide of this disclosure under conditions permitting hybridization (preferably moderately stringent hybridization conditions) of complementary single-stranded polynucleotides, or more preferably, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from un-hybridized, single-stranded polynucleotides. The hybridized polynucleotide pairs are detected using methods known to those of skill in the art and set forth, for example, in Sambrook et al. (1989) supra. The polynucleotide embodied in this disclosure can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service. -37- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 The polynucleotides of this disclosure can be isolated or replicated using PCR. The PCR technology is the subject matter of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston (1994)), and references cited therein. Alternatively, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA. Accordingly, this disclosure also provides a process for obtaining the polynucleotides by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods known to those of skill in the art. A process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained. RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any appropriate method, e.g., using an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) supra. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989) supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures. Polynucleotides exhibiting sequence complementarity or homology to a polynucleotide of this disclosure are useful as hybridization probes or as an equivalent of the specific polynucleotides identified herein. Since the full coding sequence of the transcript is known, any portion of this sequence or homologous sequences, can be used in the methods of this disclosure. It is known in the art that a “perfectly matched” probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or -38- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 insertion of a small number of bases do not affect the hybridization specificity. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated. Preferably, a probe useful for detecting the mRNA is at least about 80% identical to the homologous region. More preferably, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region; even more preferably, it exhibits 90% identity. These probes can be used in radioassays (e.g., Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various cells or tissues containing these cells. The probes also can be attached to a solid support or an array such as a chip for use in high throughput screening assays for the detection of expression of the gene corresponding a polynucleotide of this disclosure. Accordingly, this disclosure also provides a probe comprising or corresponding to a polynucleotide of this disclosure, or its equivalent, or its complement, or a fragment thereof, attached to a solid support for use in high throughput screens. The total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between at least 5 to 10 to about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect. Nucleotide probes having complementary sequences over stretches greater than 5 to 10 nucleotides in length are generally preferred, to increase stability and selectivity of the hybrid, and thereby improving the specificity of hybrid molecules obtained. More preferably, one can design polynucleotides having gene-complementary stretches of 10 or more or more than 50 nucleotides in length, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology with two priming oligonucleotides as described in U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production. In one aspect, a probe is about 50-75 or more alternatively, 50-100, nucleotides in length. The polynucleotides of the present disclosure can serve as primers for the detection of -39- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 genes or gene transcripts that are expressed in cells described herein. In this context, amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. For illustration purposes only, a primer is the same length as that identified for probes. One method to amplify polynucleotides is PCR and kits for PCR amplification are commercially available. After amplification, the resulting DNA fragments can be detected by any appropriate method known in the art, e.g., by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination. Methods for administering an effective amount of a gene delivery vector or vehicle to a cell have been developed and are known to those skilled in the art and described herein. Methods for detecting gene expression in a cell are known in the art and include techniques such as in hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays and Northern blot analysis. Such methods are useful to detect and quantify expression of the gene in a cell. Alternatively, expression of the encoded polypeptide can be detected by various methods. It is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with the target polypeptide. Such antibodies are useful for visualizing cells that express the polypeptide using techniques such as immunohistology, ELISA, and Western blotting. These techniques can be used to determine expression level of the expressed polynucleotide. Compositions Compositions are further provided. The compositions comprise a carrier and one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure. The carriers can be one or more of a solid support or a pharmaceutically acceptable carrier. The compositions can further comprise an adjuvant or other components suitable for administration. In one aspect, the compositions are formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants. In addition, embodiments of the compositions of the present disclosure include one or more of an isolated -40- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or a host cell formulated with one or more pharmaceutically acceptable auxiliary substances. For oral preparations, any one or more of an isolated or recombinant polypeptide as described herein, an isolated or recombinant polynucleotide as described herein, a vector as described herein, an isolated host cell as described herein, can be used alone or in pharmaceutical formulations of the disclosure comprising, or consisting essentially of, the compound in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can 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. Pharmaceutical formulations and unit dose forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infections, and therapies in which the patient self-administers the drug. In one aspect, the formulation is specific for pediatric administration. The disclosure provides pharmaceutical formulations in which the one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure can be formulated into preparations for injection in accordance with the disclosure by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, -41- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 emulsifying agents, stabilizers and preservatives or other antimicrobial agents. 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 for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. Aerosol formulations provided by the disclosure can be administered via inhalation and can be propellant or non-propellant based. For example, embodiments of the pharmaceutical formulations of the disclosure comprise a compound of the disclosure formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. A non-limiting example of a non-propellant is a pump spray that is ejected from a closed container by means of mechanical force (i.e., pushing down a piston with one's finger or by compression of the container, such as by a compressive force applied to the container wall or an elastic force exerted by the wall itself (e.g. by an elastic bladder)). Suppositories of the disclosure can be prepared by mixing a compound of the disclosure with any of a variety of bases such as emulsifying bases or water-soluble bases. Embodiments of this pharmaceutical formulation of a compound of the disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the disclosure. Similarly, unit dosage forms for injection or intravenous administration may comprise a compound of the disclosure in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Embodiments of the pharmaceutical formulations of the disclosure include those in -42- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 which one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure formulated in an injectable composition. Injectable pharmaceutical formulations of the disclosure are prepared as liquid solutions or suspensions, or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. The preparation may also be emulsified, or the active ingredient encapsulated in liposome vehicles in accordance with other embodiments of the pharmaceutical formulations of the disclosure. In an embodiment, one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure is formulated for delivery by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art. Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery of a compound of the disclosure can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. In some embodiments, a compound of the disclosure is in a liquid formulation in a drug-impermeable reservoir and is delivered in a continuous fashion to the individual. In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device. -43- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc. Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic). In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted herein, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. -44- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Suitable excipient vehicles for a compound of the disclosure are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the compound adequate to achieve the desired state in the subject being treated. Compositions of the present disclosure include those that comprise a sustained-release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix. In another embodiment, the interfering agent (as well as combination compositions) is delivered in a controlled release system. For example, a compound of the disclosure may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, polymeric materials are used. In yet another embodiment a controlled release system is placed in proximity of the therapeutic target, i.e., the liver, thus requiring only a fraction of the systemic dose. In yet -45- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 another embodiment, a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic. Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533. In another embodiment, the compositions of the present disclosure (as well as combination compositions separately or together) include those formed by impregnation of an inhibiting agent described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the instant disclosure. Screening Assays The present disclosure provides methods for screening for equivalent agents, such as equivalent peptides and small molecules that modulate the activity of the active agents and pharmaceutical compositions of the disclosure or the function of a polypeptide or peptide product encoded by the polynucleotide of this disclosure. For the purposes of this disclosure, an “agent” is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a ribozyme. A vast array of compounds can be synthesized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term “agent.” In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen. Therapeutic Methods Provided herein is a composition comprising, or consisting essentially of, or consisting of, the 14-mer synthetic peptide or a polynucleotide encoding the 14-mer synthetic peptide (e.g., SEQ ID NOS: 9-11, 12-14, or 18-29) and/or the synthetic 4-mer peptide or a polynucleotide encoding the 4-mer (KPSK) (e.g., SEQ ID NO: 34) as disclosed herein for treating a disease in which innate immunity and neuroinflammation plays an important role. -46- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 In one aspect, the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle. In one aspect, the disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. In one aspect, the 14-mer Also provided are methods for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising, or consisting essentially of, or yet further consisting of administering to a subject-in-need an effective amount of the 14-mer synthetic peptide (e.g., SEQ ID NOS: 9-11, 12-14, or 18-29) or synthetic 4-mer peptide (e.g., SEQ ID NO: 33) as disclosed herein. In one aspect, the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle. In one aspect, the disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. The 14-mer and 4-mer peptides can be combined with other therapies as appropriate. In one aspect, the 14-mers or the 4-mer peptides and/or polynucleotides encoding them are combined with a linker peptide and/or a carrier such as a vector in the case of a polynucleotide or a pharmaceutically acceptable carrier such as a micelle or lipid nanoparticle and are combined with other therapies as appropriate. The agents and compositions of this disclosure can be concurrently or sequentially administered. In one aspect, administration is locally to the site of the injury or disease or by inhalation for example. Other non-limiting examples of administration include by one or more method comprising transdermally, urethrally, sublingually, rectally, vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally, intranasally, by inhalation or orally. Thus, routes of administration applicable to the methods of the disclosure include intranasal, intramuscular, urethrally, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted -47- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 depending upon the agent and/or the desired effect. An active agent can be administered in a single dose or in multiple doses. Embodiments of these methods and routes suitable for delivery, include systemic or localized routes. In general, routes of administration suitable for the methods of the disclosure include, but are not limited to, direct injection, enteral, parenteral, or inhalational routes. Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be conducted to effect systemic or local delivery of the inhibiting agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations. The peptides and compositions of the disclosure can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal (e.g., using a suppository) delivery. Methods of administration of the active through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transcutaneous transmission, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. Iontophoretic transmission may be accomplished using commercially available "patches" that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more. Dosing of can be accomplished in accordance with the methods of the disclosure using capsules, tablets, oral suspension, suspension for intra-muscular injection, suspension for intravenous infusion, gel or cream for topical application, or suspension for intra-articular injection. Dosage, toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic -48- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, an effective amount of a composition sufficient for achieving a therapeutic or prophylactic effect, ranges from about 0.000001 mg per kilogram body weight per administration to about 10,000 mg per kilogram body weight per administration. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per administration to about 100 mg per kilogram body weight per administration. Administration can be provided as an initial dose, followed by one or more “booster” doses. Booster doses can be provided a day, two days, three days, a week, two weeks, three weeks, one, two, three, six or twelve months after an initial dose. In some embodiments, a booster dose is administered after an evaluation of the subject’s response to prior administrations. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments. -49- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Experimental Methods Proteins and reagents. S-PrP (residues 23-231 from mouse PrPC) was expressed and purified as previously described (21). Peptides P1, P2, P3, P3*, P4, P3(K100A), P3(K103A),
Figure imgf000051_0001
were provided from AnaSpec. All peptides had N-terminal acetylation and C-terminal amidation. LPS serotype 055:B5 from E. coli was from Sigma- Aldrich. The uncompetitive NMDA-R antagonist, dizocilpine (MK-801), was from Cayman Chemicals. Recombinant human NGF-β was from R&D Systems. Animals. WT C57BL/6J mice were obtained from Jackson Laboratory. To generate mice in which BMDMs are LRP1 deficient (Lrp1−/− mice), Lrp1flox/flox mice were bred with mice that express Cre recombinase under the control of the lysozyme-M promoter (LysM- Cre), in the C57BL/6J background, as previously described (44). For experiments with macrophages harvested from Lrp1−/− mice, control cells were harvested from littermates that were Lrp1flox/flox but LysM-Cre-negative (Lrp1+/+ mice). To generate mice in which macrophages are deficient in the essential NMDA-R GluN1 subunit (Grin1−/− mice), Grin1flox/flox mice were bred with mice that express Cre recombinase under the control of the LysM-Cre promoter in the C57BL/6J background. When BMDMs were harvested from Grin1-/- mice, control cells were harvested from littermates that were Grin1flox/flox but LysM-Cre-negative. Prnp−/− mice were generously provided by Dr. Adriano Aguzzi (University Hospital of Zurich, Zurich, Switzerland). Cell culture model systems. BMDMs were harvested from 16-week-old wild-type male mice, as previously described (40, 44). Briefly, bone marrow cells were flushed from mouse femurs, plated in non-tissue culture-treated dishes, and cultured in DMEM/F-12 medium containing 10% fetal bovine serum (FBS) and 20 nM mouse macrophage colony- stimulating factor (BioLegend) for 7 days. Non-adherent cells were eliminated. Adherent cells included >95% BMDMs, as determined by F4/80 and CD11b immunoreactivity. This method was approved by the Institutional Animal Care and Use Committee of University of California San Diego. Rat PC12 cells were from the ATCC (CRL-1721) and subjected to quality control tests by the ATCC. PC12 cells were cultured in Dulbecco's modified Eagle's medium -50- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 (DMEM, high glucose; Thermo Fisher Scientific) containing 10% heat-inactivated FBS, 5% heat-inactivated horse serum (Thermo Fisher Scientific), in plates that were pre-coated with 0.01 mg/mL type IV collagen (Sigma-Aldrich). Cells were passaged no more than eight times. Microglia were isolated from C57BL/6J mouse pups, as described previously (60). In brief, brains were harvested from postnatal day 1–6 mice. The cortices were dissected from the forebrain, and the surrounding meninges were removed. Intact cortices were mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec). Mixed glial cultures were established in DMEM/F-12 supplemented with GlutaMAX (Thermo Fisher Scientific), 10% FBS, and 1× Gibco Antibiotic-Antimycotic (Thermo Fisher Scientific). After culturing for 10–14 days, microglia were harvested by shaking the mixed cultures at 200 rpm for 30 min at 37 °C. The floating cells were collected by centrifugation (5 min, 1500 rpm) and re-plated at 3 × 105 cells/well. Culture purity was >96% as determined by immunofluorescence microscopy for Iba1 (positive), glial fibrillary acidic protein (negative), β-III tubulin (negative), and OLIG1 (negative). Experiments were performed within 24 h of completing cell isolations. Gene silencing. Rat-specific ON-TARGETplus SMARTpool siRNA, targeting Lrp1 or Grin1, and pooled NTC siRNA were from Horizon Discovery. PC12 cells (2 × 106) were transfected with siRNA by electroporation using the Cell Line Nucleofector Kit V (Lonza), following the manufacturer's instructions. Briefly, cell suspensions were treated with 300 nM Lrp1-specific siRNA, Grin1-specific siRNA, or NTC siRNA, and electroporated with the PC12-specific program in a Lonza Nucleofector 2b device. Gene silencing was determined 48 h after transfection by RT-qPCR as previously described (23). Experiments were performed 48 h after transfection. Gene expression studies. BMDMs were transferred to serum-free medium (SFM) for 30 min and treated for 6 h with various proteins and reagents, alone or simultaneously as noted, including: LPS (0.1 μg/ml); various synthetic peptides at different concentrations; MK-801 (1 μM); or vehicle (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, PBS). RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad). qPCR was performed using TaqMan gene -51- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 expression products (Thermo Fisher Scientific). Primer-probe sets were as follows: GAPDH (Mm99999915_g1); TNFα (Mm00443258_m1); IL-6 (Mm00446190_m1). The relative change in mRNA expression was calculated using the 2ΔΔCt method with GAPDH mRNA as a normalizer. Flow Cytometry. Loss of the NMDA-R from the surfaces of purified BMDMs from Grin1−/− mice was demonstrated by flow cytometry. Non-permeabilized cells were labeled with NMDA-R Glun1 subunit-specific antibody (Invitrogen #PA3-102, Thermo Fisher Scientific). Cell-associated PA3-102 was detected with A647-conjugated secondary antibody (Thermo Fisher Scientific). Control cells were treated with secondary antibody only. All data were analyzed using FlowJo Software version 10.7.1 (BD Biosciences). Cell-signaling. BMDMs were transferred to SFM for 30 min and treated for 1 h with various proteins and reagents, alone or simultaneously as noted. PC12 cells were cultured in serum-containing medium until ∼70% confluent. The cells were then transferred into SFM for 2 h before treatment with various reagents. Some cultures were pretreated with MK-801 (1 μM), as noted. Microglia were cultured in SFM for 30 min and then treated with LPS (0.1 μg/ml) for 1 h in the presence and absence of S-PrP (40 nM) or various synthetic peptides. Extracts of BMDMs, PC12 cells, and microglia were prepared in RIPA buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts of protein were subjected to SDS-PAGE and electro- transferred to poly-vinylidene fluoride membranes. The membranes were blocked with 5% nonfat dried milk and then incubated with primary antibodies from Cell Signaling Technology that recognize: phospho-ERK1/2, total ERK1/2, phospho-IκBα, total IκBα, and β-actin. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch). Immunoblots were developed using Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and imaged using the Azure Biosystems c300 digital system. Images were processed with Adobe Photoshop 23.3.2. The presented results are representative of at least three independent experiments. -52- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 PC12 cell neurite outgrowth. Wild-type PC12 cells and cells that were transfected to silence Lrp1 or Grin1 and cells transfected with NTC siRNA were plated at 1 × 105 cells/well and maintained in serum-containing medium for 24 h. The medium was then replaced with SFM supplemented with S-PrP (40 nM) or various synthetic peptides (each at 0.2 μM). Incubations were conducted for 48 h. The cells were imaged by phase contrast microscopy, using a Leica DMi8 microscope (Leica Microsystems) equipped with a Leica DFC3000 G digital camera and Leica Application Suite X software. Neurite length was determined in all of the cells imaged in ≥5 representative fields in three separate experiments using the NeuronJ plugin of ImageJ software (National Institutes of Health). Proteome Profiler Mouse Cytokine Array. Microglia were transferred to SFM for 30 min and treated with LPS (0.1 μg/ml) in the presence and absence of S-PrP (40 nM) or P3 (0.5 μM) for 6 h. Conditioned medium (CM) was collected and particulates were removed by centrifugation. An equivalent amount of CM (1 ml for each condition) was incubated with the nitrocellulose membranes provided in the Proteome Profiler Mouse Cytokine Array Kit (R&D Systems). Membrane were developed following the instructions of the manufacturer. LPS challenge experiments in Prnp-/- mice. Male Prnp-/- mice and wild-type mice in the same genetic background (16-20-week-old, 26-28 g) were injected intraperitoneally with 9 mg/kg LPS. The LD50 for the specific LPS lot was predetermined in Applicant’s laboratory, as previously described (44), and was 12 mg/kg. The mice were treated by intravenous injection with P3 (2.5 μg/g body weight) or PBS, 30 min after LPS administration. Animals were monitored and scored for signs of toxicity at 1 h intervals using the murine sepsis scoring system (47). In brief, the following variables were scored from 0 to 4: appearance, level of consciousness, activity, responses to auditory stimuli, eye function, respiration rate, and respiration quality. Mice were considered moribund and euthanized if the murine sepsis score was ≥21. Investigators were blinded to treatment groups. Statistics. Statistical analysis was performed using GraphPad Prism 9.4. All results are expressed as the mean±SEM. Each replicate was performed using a different BMDM or PC12 cell preparation. Comparisons between two groups were performed using two-tailed unpaired t tests. When more than two groups were compared, we performed one-way ANOVA followed by post-hoc Dunnett’s multiple comparison test. LPS challenge -53- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 experiments were analyzed by two-way ANOVA followed by Šidák’s multiple comparison test. P-values of *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 were considered statistically significant. Results*** A synthetic peptide corresponding to a sequence in the unstructured N-terminal region of PrPC replicates the effects of S-PrP on macrophage physiology. A series of peptides corresponding to a series of peptides corresponding to sequences in the structure of PrPC, including two with clusters of Lys residues (P1 and P3) and two without sequence elements suggestive of LRP1 binding (P2 and P4). Three of the peptides corresponded to sequences in the disordered N-terminal region of PrPC (FIG. 1A). Differences in the sequences of human and mouse PrPC in the regions corresponding to the synthetic peptides were conservative (FIG. 1B). Because P3 emerged as important for the activities studied here, Applicant synthesized two variants of this peptide (P3 and P3*) to completely replicate the mouse and human sequences. FIG. 1C summarizes the sequences of the first five peptides, in relation to the structure of human and mouse PrPC, and a secondary set of peptides designed to explore the molecular requirements for engaging the LRP1/NMDA-R receptor assembly. Applicant initially screened for the ability of PrPC-derived peptides to inhibit expression of tumor necrosis factor alpha (TNFα) mRNA in response to lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and thus, replicate the activity of S- PrP and EV-associated PrPC (22, 25). BMDMs were harvested as previously described (39, 40) and treated with 0.1 μg/mL LPS in the presence of increasing concentrations of each peptide for 6 h. FIG. 2A shows that, in the absence of peptides, LPS significantly increased TNFα mRNA expression in the BMDMs as determined by RT-qPCR. P1, P2, and P4 had no effect on LPS-induced TNFα expression. By contrast, P3 and P3*, at concentrations of 0.2 μM or higher, blocked LPS-induced TNFα expression. P3 and P3* also blocked LPS-induced interleukin-6 (IL-6) mRNA expression, whereas P1 and P4 were inactive. Increased expression of pro-inflammatory cytokines in response to LPS requires NFκB activation, which may be monitored by examining IκBα phosphorylation and the -54- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 accompanying decrease in total abundance of IκBα (41). FIG. 2B shows that in the absence of PrPC-derived peptides, BMDMs treated with LPS (0.1 μg/mL) for 1 h demonstrated increased phospho-IκBα and decreased total IκBα, as anticipated. P1, P2, and P4 (each at 0.5 μM) had no effect on this response. By contrast, 0.5 μM P3 blocked LPS-induced IκBα phosphory-lation and the associated decrease in total cellular IκBα. FIG. 2C shows that the effects of P3 on IκBα phosphorylation were concentration-dependent; P3 at concentrations ≥0.5 μM completely blocked the cell-signaling event whereas 0.2 μM P3 typically generated an inter-mediate effect. These results demonstrated that P3 replicates the activity of S-PrP and EV-associated PrPC as an inhibitor of LPS-induced NFκB activation. P3 is bioactive in PC12 cell and microglial cell culture model systems. S-PrP and EV- associated PrPC activate ERK1/2 and promote neurite outgrowth in PC12 cells (21, 23). Applicant treated PC12 cells with P1, P2, P3, P3*, and P4 (each at 0.5 μM) for 10 min. FIG. 3A shows that P3 and P3* activated ERK1/2. The other peptides were inactive. ERK1/2 activation by P3 was evident throughout the P3 concentration studied (0.1-1.0 μM) (FIG. 3B). Furthermore, P3 (0.5 μM) induced PC12 cell neurite outgrowth after 48 h, replicating the activity of S-PrP (40 nM), as shown in the representative images in FIG. 3C. The other PrPC-derived peptides were inactive. Image analysis of individual cells in ≥5 randomly selected fields in three separate experiments confirmed that the effects of P3 and S-PrP on neurite outgrowth were highly significant (FIG.3D). Microglia are macrophage-like cells and the principal cell type responsible for innate immune responses in the CNS (42, 43). Applicant isolated microglia from mouse pups and established primary cultures. The microglia were treated with LPS (0.1 μg/mL) for 6 h in the presence or absence of S-PrP (40 nM) or P3 (0.5 μM). To examine cytokine production in an unbiased manner, conditioned medium (CM) was recovered and subjected to cytokine array analysis. In the absence of other reagents, LPS induced microglial production of multiple pro- inflammatory cytokines and chemokines, including but not limited to TNFα, IL-6, CCL3/MIP-1α, CXCL2/MIP-2, and CCL5/RANTES (FIG. 4A). S-PrP inhibited cytokine expression in response to LPS, as did P3. Applicant also examined the ability of S-PrP and P3 to block LPS-induced IκBα phosphorylation in microglia. FIG. 4B shows that S-PrP (40 nM) and P3 (0.5 μM) were -55- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 effective, completely blocking IκBα phosphorylation and the associated decrease in total abundance of IκBα. P1 and P4 were without effect. P3 activity in macrophages requires the NMDA-R and LRP1. LRP1 and the NMDA- R collaborate to mediate cell-signaling in response to S-PrP (21, 22, 25). The requirement for the NMDA-R appears to be absolute. In the absence of LRP1, signaling is still observed; however, the concentration of S-PrP required to elicit responses increases substantially, leading us to propose that LRP1 “captures” the ligand and then delivers it to the NMDA-R. BMDMs express the NMDA-R (44). To test whether the NMDA-R is necessary for the response to P3 in macrophages, first BMDMs were treated for 6 h with LPS (0.1 μg/mL) and P2 or P3*, in the presence or absence of the non-competitive NMDA-R antagonist, MK-801 (FIG. 5A). In the absence of MK-801, P3* neutralized the effects of LPS on TNFα mRNA expression. P2 was ineffective, as anticipated. In the presence of MK-801, the activity of P3* was blocked and TNFα mRNA expression was restored to the level observed in the absence of P3*. Similarly, MK-801 re- versed the ability of P3* to neutralize IL-6 mRNA expression in response to LPS. To confirm a role for macrophage NMDA-R in the response to P3, mice were bred in which the gene encoding the essential NMDA-R GluN1 subunit was floxed (Grin1fl/fl) with mice that express Cre recombinase under the control of the LysM promoter. BMDMs were harvest-ed from Grin1fl/fl-LysM-Cre mice and GluN1 mRNA expression was compared with that detected in wild-type BMDMs isolated from Grin1fl/fl mice that did not carry LysM-Cre. GluN1 mRNA was decreased by 63.8 ± 0.4% (n=3) (FIG. 5B). Loss of the NMDA-R protein from the surfaces of BMDMs was demonstrated by flow cytometry (FIG. 5C). The degree of loss was ~70%, as determined by comparing mean fluorescence intensity. In GluN1-deficient BMDMs, LPS induced TNFα mRNA expression, as anticipated (FIG. 5D). P3, at concentrations up to 20 μM, was ineffective at inhibiting LPS-stimulated TNFα expression. Similarly, none of the PrPC-derived peptides (0.5 μM), including P3 and P3*, inhibited LPS-induced IκBα phosphorylation (FIG. 5E). Next, Applicant isolated LRP1-deficient BMDMs from Lrp1fl/fl-LysM-Cre mice, which are previously described (40). LPS induced expression of TNFα mRNA in these -56- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 BMDMs, as anticipated, and P3 blocked the effects of LPS on TNFα expression; however, the minimum con-centration of P3 required to inhibit LPS was increased about 100-fold to 20 μM (FIG. 6A). Equivalent results were obtained with P3*. P1 was inactive as inhibitor of LPS-induced TNFα expression in LRP1-deficient BMDMs throughout this expanded peptide concentration range, as anticipated. In experiments examining IL-6 mRNA expression, once again P3 and P3* blocked the activity of LPS and once again, the minimum concentration of P3 or P3* required to observe activity was increased about 100-fold compared with that observed in wild-type BMDMs. These results mimic those observed with S-PrP and demonstrate a robust but non- essential role for LRP1 as a facilitator of the activity of P3/P3*. In IκBα phosphorylation experiments using LRP1-deficient BMDMs from Lrp1fl/fl-LysM-Cre mice, P3 and P3* (0.5 μM) failed to counteract the activity of LPS (FIG. 6B), confirming the results of our cytokine mRNA experiments. To confirm that LRP1 and the NMDA-R mediate the effects of P3 on cell-signaling and cell physiology in a second model system, Applicant silenced expression of Lrp1 and Grin1 in PC12 cells. FIG. 7A shows that in cells transfected with non-targeting control (NTC) siRNA, P3 caused ERK1/2 activation. By contrast, silencing Lrp1 or Grin1 in PC12 cells blocked ERK1/2 activation in response to P3. Applicant next studied neurite outgrowth. Representative images showing PC12 cells, transfected with Lrp1, Grin1, or NTC siRNA and treated with P3 (0.5 μM), P4 (0.5 μM), S- PrP (40 nM), or vehicle are shown in FIG. 7B. In cells transfected with NTC siRNA, neurite outgrowth was observed in response to S-PrP and P3, but not in response to P4. In cells in which Lrp1 or Grin1 was silenced, the response to P3 and S-PrP was eliminated. P4 also was without effect, as anticipated. FIG. 7C summarizes image analysis studies examining individual cells in ≥5 randomly selected fields from three separate experiments with each agonist and gene-silencing reagent. Statistically significant neurite outgrowth was observed only in cells transfected with NTC siRNA and stimulated with P3 or S-PrP. Lys100 and Lys103 are essential for the function of P3 as an agonist for the LRP1/ NMDA-R cell-signaling receptor assembly. Given the documented role of Lys residues in -57- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 LRP1-binding motifs (26–31), Applicant modified the four Lys residues in P3 to Ala, one at a time. To test the activity of the resulting set of new synthetic peptides, Applicant began by examining ERK1/2 activation in PC12 cells, which is measured in the absence of a secondary reagent (LPS). FIG. 8 shows that although all four modified peptides demonstrated decreased potency compared with P3, peptides in which either Lys100 or Lys103 was modified to Ala demonstrated the most substantial change and were 5-fold decreased in potency compared with P3 variants in which either Lys105 or Lys109 was modified. A P3 derivative in which both Lys100 and Lys103 were modified to Ala (P3(DM)) failed to activate ERK1/2 at concentrations up to 20 μM. Next, Applicant examined the ability of modified P3 peptides to inhibit LPS-induced NFκB activation in BMDMs. Cells were treated with 0.1 μg/mL LPS and with the indicated concentrations of peptide for 1 h (FIG. 9A). P3(K015A) and P3(K109A) were only slightly less active than unmodified P3. By contrast, P3(K100A) and P3(K103A) demonstrated substantially decreased potency compared with P3 and completely inhibited LPS-induced IκBα phosphorylation only when present at 20 μM. P3(DM) was ineffective throughout the concentration range studied. These results support those obtained examining PC12 cells. To confirm that P3(DM) is ineffective at opposing the response to LPS in BMDMs, we examined TNFα mRNA expression in cells treated for 6 h with LPS and with the indicated concentrations of P3(DM) (FIG. 9B). P3(DM) failed to inhibit LPS-induced TNFα mRNA ex- pression throughout the studied P3(DM) concentration range. Similarly, P3(DM) failed to inhibit LPS-induced IL-6 mRNA expression. P3 rescues the phenotype of Prnp-/- mice in LPS challenge experiments. Applicant performed experiments to test whether it could replicate the reported increase in sensitivity of Prnp-/- mice to LPS challenge (45). These experiments were performed as previously described (22, 44), using the PrnpZH3/ZH3 strain (46). Male Prnp-/- mice and wild-type mice in the same genetic background (26-28 g) were challenged with LPS at 75% of the LD50 calculated for wild-type mice. Animals were monitored and scored for signs of toxicity using the murine sepsis scoring system (47). FIG. 10 shows that Prnp-/- mice demonstrated significantly increased sensitivity to LPS, compared with wild-type mice. When Prnp-/- mice -58- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 were injected intravenously with a single dose of P3 (2.5 μg/g body weight), 30 min after LPS administration, toxicity was significantly decreased. A tetrapeptide with two Lys residues mimics the activity of P3. Applicant also examined the ability of a synthetic 4-mer peptide (KPSK), with two Lys residues, derived from the structure of P3, to inhibit LPS-induced NFκB activation in BMDMs. Cells were treated with 0.1 μg/mL LPS and with the indicated concentrations of KPSK for 1 h (FIG. 11A). In the absence of KPSK, LPS significantly increased TNFα expression in the BMDMs as determined by RT-qPCR. The KPSK tetrapeptide at concentrations of 1 μM or higher, blocked LPS-induced TNFα mRNA expression. Similarly, KPSK neutralizes IL-6 mRNA expression in response to LPS. FIG. 11B shows that in the absence of KPSK, BMDMs treated with LPS (0.1 μg/mL) for 1 h demonstrated increased phospho-IκBα and decreased total IκBα, as anticipated. By contrast, at concentration of 1 μM and higher KPSK blocked LPS-induced IκBα phosphorylation and the associated decrease in total cellular IκBα. Discussion PrPC has been identified as a gene product capable of attenuating inflammation in a variety of contexts (33–38, 45, 48–52), including experimental autoimmune encephalitis (33– 35) and ischemic brain injury (33, 36–38). Previously reported work identified PrPC derivatives released by cells, including soluble fragments of PrPC and EV-associated PrPC, as candidate mediators of the known anti-inflammatory activity of PrPC (22, 25). Applicant also implicated LRP1 and the NMDA-R as cell-signaling receptors for soluble- and EV-associated PrPC derivatives. PrPC that localizes to lipid rafts, within the original cell of synthesis, also may express LRP1-dependent anti-inflammatory activity by laterally associating with LRP1 within the plasma membrane; this interaction facilitates the anti-inflammatory activity of LRP1, when it is presented with ligands other than S-PrP, such as tPA (25). The studies presented here further support Applicant’s model in which PrPC derivatives released from cells function as LRP1-dependent cell-signaling agonists and anti- inflammatory agents. For the first time, Applicant demonstrated that S-PrP blocks inflammatory responses in microglia, supporting the hypothesis that the PrPC/LRP1 interaction may be responsible for the documented anti-inflammatory activity of PrPC in the -59- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 CNS (33–38). Applicant also harnessed the cell-signaling and anti-inflammatory activity of PrPC within a single 14-mer peptide, derived from the structure of PrPC. This advance suggests that it is feasible to translate the known anti-inflammatory activities of PrPC into novel small molecule candidate therapeutics. The ability of a small peptide to mimic the cell-signaling and anti-inflammatory activities of full-length S-PrP was not anticipated. LRP1 ligands that activate anti- inflammatory cell-signaling pathways share a common mechanism of receptor engagement, in which at least two receptors, LRP1 and the NMDA-R, play an instrumental role (22, 25, 44). The NMDA-R appears to be essential. LRP1 substantially decreases the concentration of ligand required to trigger cell-signaling. Without being bound by theory, it is reasonable to propose that LRP1 captures soluble ligands, like S-PrP, and then delivers them to the NMDA-R to trigger calcium influx and activation of cell-signaling factors such as Src family kinases and PI3K. Notably, the NMDA-R is reported to bind tPA and PrPC (53–55). If LRP1 transfers anti-inflammatory ligands to the NMDA-R, the ligand would most likely form a transient ternary complex in which different regions of the ligand engage LRP1 and the NMDA-R simultaneously. Such a model seems highly feasible for tPA, which has multiple domains (56), and for α2M, which is a large tetramer of four identical subunits (57). The size of P3, a synthetic 14-amino acid peptide, argues against the bridged receptor model. Tandem Lys residues in the structure of P3, including Lys100 and Lys103, were essential for activation of the LRP1/NMDA-R receptor assembly. Replacement of both Lys residues with Ala in P3(DM) eliminated activity. Tandem Lys residues in a number of full-length proteins also have been implicated in LRP1-binding (26–31), although the activity of the Lys residues in LRP1/ NMDA-R-dependent cell-signaling has not been formally addressed in previous studies. Although it is unlikely that P3 bridges LRP1 to the NMDA-R, both receptors were necessary to elicit potent P3 biological activities. In addition to its activity in cell culture model systems, P3 rescued the known increased susceptibility of Prnp-/- mice to LPS challenge. This result has a number of implications. First, these studies suggest that overly exuberant pro-inflammatory responses in Prnp-/- mice may be rescued entirely by soluble derivatives of PrPC. Second, although Applicant did not study the pharmacokinetics of P3, synthetic peptides typically have a short -60- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 circulating half-life (32). Assuming an initial distribution volume corresponding to the plasma volume in a mouse (1.5 mL) and the molecular mass of P3 of 1,743, the maximum concentration of P3 in the plasma following injection was estimated at ~50 μM. Without being bound by theory, Applicant hypothesize that P3 rapidly engages cellular receptor targets and stimulates changes in cell physiology that are long-lasting in vivo, despite clearance of the peptide. In support of this hypothesis, Applicant previously demonstrated that a single intravenously-administered injection of enzymatically-inactive tPA not only neutralizes LPS toxicity but also significantly reverses inflammation and disease progression in the dextran sodium sulfate model of inflammatory bowel disease (44, 58). These results are observed despite the fact that the circulating half-life of inactive tPA in mice is only 3 min (59). Equivalents Additional advantages of the present disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the present disclosure. The advantages of the present disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein. -61- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly -62- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Partial Listing of Sequences SEQ ID NO: 1 Prnp Homo sapiens 1 MANLGCWMLV LFVATWSDLG LCKKRPKPGG 31 WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP 61 HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG 91 QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA 121 VVGGLGGYML GSAMSRPIIH FGSDYEDRYY 151 RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV 181 NITIKQHTVT TTTKGENFTE TDVKMMERVV 211 EQMCITQYER ESQAYYQRGS SMVLFSSPPV 241 ILLISFLIFL IVG P1 peptide is underlined. P2 peptide is italicized. P3 peptide is bolded. SEQ ID NO: 2 Prnp Mus musculus 1 MANLGYWLLA LFVTMWTDVG LCKKRPKPGG 31 WNTGGSRYPG QGSPGGNRYP PQGGTWGQPH 61 GGGWGQPHGG SWGQPHGGSW GQPHGGGWGQ 91 GGGTHNQWNK PSKPKTNLKH VAGAAAAGAV 121 VGGLGGYMLG SAMSRPMIHF GNDWEDRYYR 151 ENMYRYPNQV YYRPVDQYSN QNNFVHDCVN 181 ITIKQHTVTT TTKGENFTET DVKMMERVVE 211 QMCVTQYQKE SQAYYDGRRS SSTVLFSSPP 241 VILLISFLIF LIVG P1 peptide is underlined. P3 peptide is bolded. P4 peptide is bolded and underlined. -63- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 SEQ ID NOS: 3-32 SEQ ID Peptide Sequence Sequence in human PrP Sequence in mouse PrP Nos: 3-5 P1 Ac-KKRPKPGGWNTGGS-NH K KRPKPGGWNTGGS K KRPKPGGWNTGGS 6-8 P2 Ac-GGWGQPHGGGWGQP-NH G GWGQPHGGGWGQP G TWGQPHGGGWGQP 9-11 P3 Ac-QWNKPSKPKTNLKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 12-14 P3* Ac-QWNKPSKPKTNMKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 15-17 P4 Ac-DVKMMERVVEQMCV-NH D VKMMERVVEQMCI D VKMMERVVEQMCV18-20 P3 Ac-QWNAPSKPKTNLKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 21-23 P3 Ac-QWNKPSAPKTNLKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH24-26 P3 Ac-QWNKPSKPKTNLAH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH27-29 P3 Ac-QWNKPSKPKTNLAH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH 30-32 P3 Ac-QWNAPSAPKTNLKH-NH Q WNKPSKPKTNMKH Q WNKPSKPKTNLKH Given their pivotal role in LRP1-binding, Lys residues are shown in bold and underlined. Amino acids that are conservatively modified compared with experimental peptides are shown in italics. † The amino acid numbering of peptide variants is relative to the sequence of mouse PrPC. P3(DM) is a double amino acid substitution at K100 and K103. -64- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 REFERENCES 1. Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. Journal of Clinical Investigation 2001;108(6):779–784. 2. Fernandez-Castaneda A, et al. Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris*. Journal of Biological Chemistry 2013;288(7):4538– 4548. 3. Basu S, et al. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 2001;14(3):303–313. 4. Rauch JN, et al. LRP1 is a master regulator of tau uptake and spread. Nature 2020;580(7803):381–385. 5. Chen K, et al. 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American Journal of Physiology-Cell Physiology [published online ahead of print: August 18, 2021]; doi:10.1152/ajpcell.00269.2021 -69- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 67. Van Leuven F. Human α2-macroglobulin: structure and function. Trends in Biochemical Sciences 1982;7(5):185–187. 68. Das L, et al. Enzymatically Inactive Tissue-Type Plasminogen Activator Reverses Disease Progression in the Dextran Sulfate Sodium Mouse Model of Inflammatory Bowel Disease. American Journal of Pathology 2021;191(4):590–601. 69. Fuchs HE, Berger H, Pizzo SV. Catabolism of human tissue plasminogen activator in mice. Blood 1985;65(3):539–544. 70. Ni M, Aschner M. Neonatal rat primary microglia: Isolation, culturing, and selected applications. Current Protocols in Toxicology 2010;(SUPPL.43). doi:10.1002/0471140856.tx1217s43 -70- 4862-8761-3125.1

Claims

Atty. Dkt. No.: 114198-6810 WHAT IS CLAIMED IS: 1. An isolated 14-mer synthetic P3 peptide corresponding to a putative LRP1-binding motif, and wherein said P3 replicates cell-signaling and biological activities of full-length shed PrPc. 2. An isolated synthetic 4-mer peptide (KPSK), with two Lys residues, derived from the structure of P3 of claim 1. 3. A composition comprising the isolated 14-mer synthetic peptide of claim 1 for treating a disease in which innate immunity and neuroinflammation plays an important role. 4. The composition of claim 3, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. 5. A composition comprising the synthetic isolated 4-mer peptide (KPSK) of claim 2 for treating a disease in which innate immunity and neuroinflammation plays an important role. 6. The composition of claim 5, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. 7. A method for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising administering to a subject-in-need an effective amount of the 14-mer synthetic peptide of claim 1. 8. The method of claim 7, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. 9. A method for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising administering to a subject-in-need an effective amount of the composition of claim 3. -71- 4862-8761-3125.1 Atty. Dkt. No.: 114198-6810 10. The method of claim 9, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. 11. A method for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising administering to a subject-in-need an effective amount of the synthetic 4-mer peptide of claim 2. 12. The method of claim 11, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. 13. A method for treating a disease in which innate immunity and neuroinflammation plays an important role, said method comprising administering to a subject-in-need an effective amount of the composition of claim 5. 14. The method of claim 13, wherein said disease is selected from the group consisting of Inflammatory Bowel Disease, Rheumatoid Arthritis, Psoriasis, Chronic Pain Disorders, Spinal Cord Injury, Diabetes, Neurodegenerative Diseases, and Multiple Sclerosis. -72- 4862-8761-3125.1
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AU2006204705C1 (en) * 2005-01-13 2012-07-05 Novartis Vaccines And Diagnostics, Inc. ELISA assays using prion-specific peptide reagents
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