WO2022198073A1 - Compositions and methods for targeting inflammatory or arctivated cells and treating or ameliorating inflammatory conditions and pain - Google Patents

Compositions and methods for targeting inflammatory or arctivated cells and treating or ameliorating inflammatory conditions and pain Download PDF

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WO2022198073A1
WO2022198073A1 PCT/US2022/020991 US2022020991W WO2022198073A1 WO 2022198073 A1 WO2022198073 A1 WO 2022198073A1 US 2022020991 W US2022020991 W US 2022020991W WO 2022198073 A1 WO2022198073 A1 WO 2022198073A1
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Prior art keywords
aibp
optionally
amino acid
virus
acid sequence
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French (fr)
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Yury Miller
Soo-Ho Choi
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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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Priority to AU2022238450A priority Critical patent/AU2022238450A1/en
Priority to CN202280021898.3A priority patent/CN117083381A/zh
Priority to US18/282,749 priority patent/US20240209341A1/en
Priority to KR1020237035521A priority patent/KR20230159847A/ko
Priority to IL305955A priority patent/IL305955A/en
Priority to EP22772305.3A priority patent/EP4308233A4/en
Priority to JP2023557027A priority patent/JP2024510757A/ja
Priority to MX2023010412A priority patent/MX2023010412A/es
Priority to CA3212101A priority patent/CA3212101A1/en
Publication of WO2022198073A1 publication Critical patent/WO2022198073A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/99—Racemaces and epimerases (5.1) acting on other compounds (5.1.99)
    • C12Y501/99006—NAD(P)H-hydrate epimerase (5.1.99.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • This invention generally relates to medicine, inflammation, pain control and cell biology.
  • methods for modification of structure and increasing levels of expression of ApoA-I Binding Protein APOA1BP, AIBP, or AI-BP, also known as NAD(P)HX Epimerase or NAXE
  • APOA1BP, AIBP, or AI-BP also known as NAD(P)HX Epimerase or NAXE
  • a neuropathic pain a CNS inflammation, an allodynia, a post nerve injury pain, a post-surgical pain
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • a neurodegeneration including for example, a neurodegenerative disease or condition such as Alzheimer’s disease, a hyperalgesia, primary headaches such as migraines and cluster headaches, glaucoma, lung inflammation and asthma, HIV infection and its comorbidities, and/or vascular inflammation and cardiovascular disease.
  • APOA1BP polypeptide or protein that is a human or a mammalian APOA1BP, or a peptidomimetic or a synthetic APOAIBP, or a bioisostere thereof, to treat, ameliorate prevent, reverse, decrease the severity of a neuropathic pain, an allodynia, a hyperalgesia, a neurodegenerative disease or condition such as Alzheimer’s disease, a primary headache such as a migraine, glaucoma or other inflammatory diseases of the eye, lung inflammation and asthma, acute respiratory distress syndrome (ARDS), sepsis, viral infection, including influenza, coronavirus (for example, COVID-19) or HIV infection, or its comorbidities, and/or vascular inflammation, atherosclerosis and cardiovascular disease.
  • ARDS acute respiratory distress syndrome
  • Apolipoprotein A- 1 Binding Protein or ApoA-I binding protein (AIBP), also called NAXE, NAD(P)HX epimerase, is a protein discovered in a screen of proteins that physically associate with apoA-I.
  • CIPN Chemotherapy-induced peripheral neuropathy
  • Neuroinflammation mediated by glial cell activation and infiltrating immune cells in the spinal cord and dorsal root ganglia is an important component of CIPN and other neuropathies (Lees et ah, 2017; Makker et ah, 2017).
  • Glial cells express toll-like receptor-4 (TLR4), which mediates secretion of inflammatory cytokines, chemokines, and bioactive lipids (Bruno et ah,
  • TLR4 signaling has been reported in dorsal root ganglion nociceptors (Chen et ah, 2017; Li et ah, 2021).
  • the cell type in which TLR4 activation induces allodynia is unknown.
  • polypeptides or chimeric polypeptide
  • the polypeptide is comprised of (or comprises) a ApoA-I Binding Protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence
  • AIBP ApoA-I Binding Protein
  • the amino acid sequence N-terminal to the AIBP amino acid sequence is comprised of at least eight amino acids, or the amino acid sequence N-terminal to the AIBP amino acid sequence is 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, or 16 or more amino acids in length
  • the amino acid sequence N-terminal to the AIBP amino acid sequence is capable of inducing unfolding, exposing or otherwise making accessible the cryptic domain in the AIBP amino acid sequence for binding of the polypeptide to TLR4 under relevant physiological conditions
  • relevant physiological conditions refer to those conditions to be experienced by the polypeptide compound in vivo upon providing it to a subject in need thereof by administration, with the proviso that the amino acid sequence N-terminal to the
  • the amino acid sequence N-terminal to the AIBP amino acid sequence is comprised of between about 8 and about 40 contiguous amino acid residues (or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19 or 20 or more contiguous amino acid residues) of which between about 3 and about 12, or between about 8 and 20, or between about 10 and 40, amino acid residues are independently selected from the group consisting of arginine (R), histidine (H) and lysine (K);
  • the N-terminus of the amino acid sequence N-terminal to the AIBP amino acid sequence is or comprises a secretion signal amino acid sequence
  • the secretion signal amino acid sequence is (or comprises) a fibronectin secretion signal domain, an immunoglobulin heavy chain secretion signal domain, an immunoglobulin kappa light chain secretion signal domain, or an interleukin-2 signal peptide secretion signal domain
  • the fibronectin secretion signal domain is MLRGPGPGRLLLL AVLCLGT S VRCTET GKSKR (SEQ ID: NO:24):
  • AIBP sequence is hAIBP (SEQ ID NO:6, or is encoded by SEQ ID NO:5) or d24hAIBP (SEQ ID NO:21, or encoded by SEQ ID NO:20);
  • the amino acid sequence N-terminal to the AIBP amino acid sequence is comprised of about 6, or between about 5 and 40, consecutive histidine amino acid residues (for example, HHHHHH (SEQ ID NO: 1)), N-terminal to the TLR4 binding domain of the AIBP amino acid sequence;
  • the polypeptide has (or comprises) a thrombin cleavage domain intervening between the N-terminus of the TLR4 binding domain of the ApoA-I Binding Protein sequence, wherein the thrombin cleavage domain has one or more amino acid deletions and/or mutations within this domain so as to render it functionally inoperable;
  • the amino acid sequence N-terminal to the AIBP amino acid sequence is: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO:2) or MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), each having an amino acid mutation of its thrombin cleavage domain so as to render it functionally inoperative;
  • amino acid sequence N-terminal to the AIBP amino acid sequence is selected from the group consisting of: TETGKSKR (SEQ ID NO:26);
  • MD YKDHDGD YKDHDID YKDDDDKL A AAN S (SEQ ID NO:33), or MSPIDPMGHHHHHHGRRRASVAAGILVPAASPGLDGICSR (SEQ ID NO: 7);
  • the AIBP amino acid sequence is that of (or is derived from) a mammalian AIBP amino acid sequence, and optionally the mammalian AIBP amino acid sequence is that of (or is derived from) a human AIBP amino acid sequence; and/or
  • the human AIBP amino acid sequence is (or comprises the full-length amino acid sequence of 288 amino acid residues with NCBI Reference Sequence: NP 658985.2, or optionally the human AIBP amino acid sequence is the human AIBP amino acid sequence with NCBI Reference Sequence: NP 658985.2 having deletion of amino acids 1-24 from said AIBP amino acid sequence.
  • compositions or formulations comprised of (or comprising) a polypeptide compound as provided herein and at least one excipient suitable for (of formulated for) parenteral administration.
  • parenteral administration is by intrathecal injection or intrathecal implant, or by intravenous or intracular injection.
  • nucleic acids wherein the nucleic acid compound is comprised of (or comprises) a nucleic acid sequence that encodes for the polypeptide as provided herein.
  • expression vectors comprised of (or comprising, or having contained therein) a nucleic acid sequence that encodes for a polypeptide as provided herein.
  • the expression vector can be a recombinant virus such as a recombinant adenovirus or a recombinant lentivirus.
  • inflammation-induced neuropathic pain wherein optionally the inflammation-induced neuropathic pain comprises a Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain,
  • TLR4 Toll-like receptor 4
  • nerve or CNS inflammation comprises a TLR4- mediated nerve or CNS inflammation
  • the allodynia comprises a TLR4-mediated allodynia
  • post nerve or tissue injury pain or neuropathic pain wherein optionally the post nerve or tissue injury pain or neuropathic pain is generated or caused by, or is a sequelae to, trauma, chemotherapy, arthritis, diabetes, or viral infection,
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • a neurodegenerative disease or condition optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post-traumatic stress syndrome (PTSS),
  • CTE Chronic Traumatic Encephalopathy
  • TBI traumatic brain injury
  • PTSS posttraumatic stress disorder
  • PTSS post-traumatic stress syndrome
  • a primary headache optionally a migraine or a cluster headache
  • ARDS acute respiratory distress syndrome
  • the virus comprises an influenza or a coronavirus
  • the coronavirus is COVID-19 or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally an influenza A, B or C), or a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus , a viral meningitis, a rhinovirus, a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EB V), an Adenovirus , a Hantavirus , a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection, or its comorbidities, and
  • AOAIBP ApoA-I Binding Protein
  • a recombinant or synthetic ApoA-I Binding Protein (APOAIBP, AIBP, or AI BP) polypeptide compound or composition having a heterologous (or non-native, or non- AIBP, or non-wild type (wt), or any sequence not present in wild type (wt) AIBP) amino terminus amino acid sequence of at least about ten amino acids, or between about 5 to 20 amino acids, or between about 10 to 100 amino acids, or between about 20 to 80 amino acids, or between about 30 to 50 amino acids, or having on the AIBP amino terminus 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more amino acid residues that are not present in wt AIBP or are non-native (to AIBP) amino acid residues or peptides (also called AIBP variants as provided herein), and optionally with the proviso that the amino acid sequence N-terminal to the AIBP amino acid sequence is not comprised of a His-tag and
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO:2), wherein all these AIBP variants as provided herein (or, all the AIBP amino acids that also comprise an amino terminal sequence not present in wt AIPB, or comprise a heterologous amino terminal peptide or amino acid residues) are capable of, or serve the purpose of, in physiologic conditions, cause unfolding or exposing or making accessible a cryptic domain in the AIBP molecule that comprises of amino acids 25-51, which mediates AIBP binding to a toll-like receptor-4 (TLR4) polypeptide (in other words, the AIBP variants as provided herein have a TLR4 binding domain exposed to the extracellular milieu such that the AIBP variants as provided herein can bind to TLR4 polypeptide under physiologic conditions);
  • TLR4 toll-like receptor-4
  • a recombinant nucleic acid encoding the APOAIBP polypeptide of (i), and optionally the nucleic acid that expresses or encodes a APOAIBP polypeptide or a polypeptide having a APOAIBP polypeptide activity is contained in an expression vehicle, vector, recombinant virus, or equivalent, and optionally the vector or virus is or comprises an adenovirus vector or an adeno-associated virus (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector, and optionally the AAV vector comprises or is: an adeno-associated virus (AAV), or an adenovirus vector, an AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV- DJ/8TM (Cell Biolabs, Inc., San Diego, CA), a rhesus-derived AAV
  • a formulation or pharmaceutical composition comprising a recombinant or synthetic ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP) polypeptide or protein of (i), or a recombinant nucleic acid of (ii), wherein optionally the recombinant or synthetic ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP) polypeptide or protein is or comprises all or part of a human or a mammalian APOAIBP, or a AIBP1 or a AIBP2 sequence;
  • APIBP ApoA-I Binding Protein
  • (v) a formulation or pharmaceutical composition of any of (iii) to (iv), formulated for as a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant (for example, an intrathecal implant); and
  • APIBP ApoA-I Binding Protein
  • inflammation-induced neuropathic pain wherein optionally the inflammation-induced neuropathic pain comprises a Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain,
  • TLR4 Toll-like receptor 4
  • nerve or CNS inflammation comprises a TLR4- mediated nerve or CNS inflammation
  • the allodynia comprises a TLR4-mediated allodynia
  • post nerve or tissue injury pain or neuropathic pain wherein optionally the post nerve or tissue injury pain or neuropathic pain is generated or caused by, or is a sequela to, trauma, chemotherapy, arthritis, diabetes, or viral infection,
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • a neurodegenerative disease or condition optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post-traumatic stress syndrome (PTSS),
  • CTE Chronic Traumatic Encephalopathy
  • TBI traumatic brain injury
  • PTSS posttraumatic stress disorder
  • PTSS post-traumatic stress syndrome
  • a primary headache optionally a migraine or a cluster headache
  • the virus comprises an influenza or a coronavirus (optionally the coronavirus is COVID-19) or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally an influenza A, B or C), or a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps vims, a Herpes simplex vims (HS V), a Cytomegalovirus (CMV), a Rubivirus or rubella vims, an Enterovirus , a viral meningitis, a rhinovims, a varicella-zoster or chickenpox vims, an Orthopoxvirus or variola or smallpox vims, an Epstein-Barr vims (EB V), an Adenovirus , a Hantavirus , a Flavivirida
  • kits comprising: a recombinant or synthetic ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP) polypeptide or protein; a recombinant nucleic acid; and/or a formulation or a pharmaceutical composition as used in a method as provided herein, and optionally comprising instmctions on practicing a method as provided herein.
  • APIBP ApoA-I Binding Protein
  • AIBP ApoA-I Binding Protein
  • a formulation or a pharmaceutical composition as provided herein in the manufacture of a medicament for treating, ameliorating, preventing, reversing or decreasing the severity or duration of, or decreasing the severity of symptoms of:
  • inflammation-induced neuropathic pain wherein optionally the inflammation-induced neuropathic pain comprises a Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain,
  • TLR4 Toll-like receptor 4
  • nerve or CNS inflammation comprises a TLR4- mediated nerve or CNS inflammation
  • the allodynia comprises a TLR4-mediated allodynia
  • post nerve or tissue injury pain or neuropathic pain wherein optionally the post nerve or tissue injury pain or neuropathic pain is generated or caused by, or is a sequelae to, trauma, chemotherapy, arthritis, diabetes, or viral infection,
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • a neurodegenerative disease or condition optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post-traumatic stress syndrome (PTSS),
  • TBI Traumatic Encephalopathy
  • TBI traumatic brain injury
  • PTSS posttraumatic stress disorder
  • PTSS post-traumatic stress syndrome
  • a primary headache optionally a migraine or a cluster headache
  • ARDS acute respiratory distress syndrome
  • the coronavirus is COVID-19 or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally an influenza A, B or C), or a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus , a viral meningitis, a rhinovirus, a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EB V), an Adenovirus , a Hantavirus , a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection, or its comorbidities, and
  • a formulation, a pharmaceutical composition or a therapeutic combination for use in a method for treating, ameliorating, preventing, reversing or decreasing the severity or duration of, or decreasing the severity of symptoms of:
  • inflammation-induced neuropathic pain wherein optionally the inflammation-induced neuropathic pain comprises a Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain,
  • TLR4 Toll-like receptor 4
  • post nerve or tissue injury pain or neuropathic pain wherein optionally the post nerve or tissue injury pain or neuropathic pain is generated or caused by, or is a sequelae to, trauma, chemotherapy, arthritis, diabetes, or viral infection,
  • CIPN chemotherapeutic-induced peripheral neuropathy
  • a neurodegenerative disease or condition optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post-traumatic stress syndrome (PTSS),
  • CTE Chronic Traumatic Encephalopathy
  • TBI traumatic brain injury
  • PTSS posttraumatic stress disorder
  • PTSS post-traumatic stress syndrome
  • a primary headache optionally a migraine or a cluster headache
  • ARDS acute respiratory distress syndrome
  • the virus comprises an influenza or a coronavirus
  • the coronavirus is COVID-19 or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally an influenza A, B or C), or a hepatitis virus, a rous sarcoma virus (RSV), a Paramyxoviridae or measles virus, a Paramyxovirus or mumps virus, a Herpes simplex virus (HSV), a Cytomegalovirus (CMV), a Rubivirus or rubella virus, an Enterovirus , a viral meningitis, a rhinovirus, a varicella-zoster or chickenpox virus, an Orthopoxvirus or variola or smallpox virus, an Epstein-Barr virus (EB V), an Adenovirus , a Hantavirus , a Flaviviridae or Dengue virus, a Zika virus, or a chikungunya virus infection, or its comorbidities, and
  • the formulation or the therapeutic combination comprises a formulation or a therapeutic combination as provided herein, and wherein the formulation or a therapeutic combination is administered to an individual or patient in need thereof.
  • an ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP) polypeptide, comprising adding to a native (or wild type) AIBP polypeptide a heterologous (or non-native, or non-wild type) amino terminus amino acid sequence of at least about ten amino acid, or between about 5 to 50 amino acids, or between about 10 to 100 amino acids, or between about 20 to 80 amino acids, or between about 30 to 50 amino acids, or adding to the AIBP amino terminus 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more amino acid residues that are not present in wt AIBP or that are non-native (non- AIBP) amino acid residues or peptides, and optionally the heterologous amino terminus amino acid sequence comprises a peptide tag, and optionally the
  • polypeptide compounds wherein a polypeptide compound is comprised of a ApoA-I Binding Protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence is comprised of at least eight amino acids, or between 4 and 12 amino acids, or between 5 and 10 amino acids, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence is capable of inducing unfolding, exposing or otherwise making accessible the cryptic domain in the AIBP amino acid sequence for binding of the polypeptide to TLR4 under relevant physiological conditions, with the proviso that the amino acid sequence N- terminal to the AIBP amino acid sequence is not comprised of a His-tag and a proteolytic cleavage site that when acted upon under said physiological conditions results in loss of the His-tag.
  • AIBP ApoA-I Binding Protein
  • a pharmaceutically acceptable composition comprising a polypeptide compound or a nucleic acid compound as provided herein, wherein the nucleic acid sequence of the nucleic acid compound encodes the amino acid sequence of said polypeptide
  • the TLR4-mediated diseases or conditions include, but are not limited to, inflammation-induced pain, CNS inflammatory diseases and conditions, arthritis, neurodegenerative diseases and conditions, allodynia, hyperalgesia, lung inflammatory diseases or conditions, ocular inflammatory diseases and conditions, sepsis, vascular inflammatory diseases and conditions, diseases and conditions generated or caused by, or sequela to posttraumatic stress disorder, traumatic war neuroses, post-traumatic stress syndromes (PTSS), and viral infections.
  • FIG. 1 A-F illustrate data showing that chemotherapy-induced peripheral neuropathy alters TLR4 dimerization and lipid rafts in spinal microglia, and reversal by AIBP:
  • FIG. 1 A graphically illustrates data showing withdrawal thresholds in wild type (WT) mice in response to intraperitoneal (i.p.) cisplatin (2 injections of 2.3 mg/kg/day), followed by a single dose of intrathecal (i.t.) saline (5m1) or AIBP (0.5pg/5pl); naive mice received no injections;
  • FIG. 1B-C graphically illustrate data showing an analysis of CD1 lbVTMEMl 19 + spinal microglia cells showing TLR4 dimerization (FIG. IB) and lipid raft content measured by CTxB staining (FIG. 1C) 24 hours after i.t. saline or AIBP, i.e. at day 8 of the time course shown in FIG. 1 A;
  • FIG. 1C illustrates images of BV-2 microglia cells (left panels) incubated for 30 min with AIBP (0.2pg/mL) or vehicle in complete media, followed by a 5 min incubation with LPS (lOOng/mL), and graphically illustrates (right panel) data showing the Manders’ coefficients (a colocalization analysis) with or without LPS and/or AIBP; and
  • FIG. 1E-F graphically illustrate data showing AIBP levels over time in CSF (FIG. IE) and lumbar spinal cord (FIG. IF), as discussed in detail in Example 1, below.
  • FIG. 2A-C illustrate data showing gene expression in spinal microglia of CIPN mice:
  • FIG. 2A-B illustrate data from studies where microglia (CD1 lb + TEMEMl 19 + ) were FACS-sorted from 3 groups shown in Fig. 1 A,
  • FIG. 1 A illustrates images of a heatmap plot of DEGs across all samples
  • FIG. IB graphically illustrates data showing that Groups of significant DEGs were clustered based on expression profile patterns in different treatment conditions
  • FIG. 1C graphically illustrates data showing a pathway and GO enrichment analysis of upregulated (group 1 in right panel) and downregulated (group 2 in left panel) genes induced by cisplatin treatment, upregulated pathways are shown in in right panel “group 1” (red) and downregulated pathways in left panel “group 2” (in blue), as discussed in detail in Example 1, below.
  • FIG. 3 A-H illustrate data showing disease associated microglia (DAM) and lipid related gene expression and lipid droplets in spinal microglia of CIPN mice:
  • FIG. 3 A-C illustrate the same groups as in FIG 2:
  • FIG. 3 A illustrates an image of a volcano plot of upregulated and downregulated genes in spinal microglia of cisplatin- treated vs. naive mice;
  • FIG. 3B illustrates an image of a heatmap depicting disease associated microglia (DAM) signature genes;
  • FIG. 3B illustrates an image of a heatmap of log2 normalized gene counts scaled by row showing lipid related gene sets; and
  • FIG. 3D-H graphically illustrates data showing lipid droplet accumulation in spinal microglia measured by PLIN2 immunostaining in spinal cord sections co-stained with IBA1 and DAPI, with FIG. 3D showing ;
  • FIG. 3E graphically illustrating IBA1+/ PLIN2+ cells of total IBA1+ cells per field, with or without cisplatin and/or AIBP;
  • FIG. 3F graphically illustrating average LD numbers/cell with or without AIBP;
  • FIG. 3G graphically illustrating average LD size with or without cisplatin and/or AIBP;
  • FIG. 3H graphically illustrating normalized Plin2 gene counts with or without AIPB, as discussed in detail in Example 1, below.
  • FIG. 4A-H illustrate data showing gene expression in spinal microglia of CIPN mice, and the effect of AIBP:
  • FIG. 4A illustrates a pathway and Gene Ontology (GO) enrichment analysis of CIPN-upregulated genes that were downregulated by AIBP (see group 3 in FIG. 2B)) and CIPN-downregulated genes that were upregulated by AIBP (group 4);
  • GO Gene Ontology
  • FIG. 4B illustrates differentially expressed genes (DEGs) in spinal microglia induced by i.t. AIBP, a volcano plot of up and down regulated genes in cisplatin/ AIBP versus (vs.) cisplatin/saline treated mice;
  • DEGs differentially expressed genes
  • FIG. 4C illustrates a heatmap of inflammatory genes in group 3 upregulated in CIPN and downregulated by AIBP;
  • FIG. 4D graphically illustrates data showing cytokine protein expression in spinal tissue from WT naive, cisplatin/saline and cisplatin/ AIBP groups;
  • FIG. 4E illustrates a heatmap of inflammatory genes not induced by cisplatin but downregulated by AIBP
  • FIG. 4F graphically illustrates a pathway and GO enrichment analysis of all genes downregulated by AIBP
  • FIG. 4G illustrates a heatmap of non-inflammatory genes downregulated by AIBP included in the most enriched pathway: peptidase inhibitor activity pathway; and
  • FIG. 4H illustrates a heatmap of genes whose downregulation in CIPN was reversed by AIBP, as discussed in detail in Example 1, below.
  • FIG. 5A-J illustrate data showing that ABCA1 and ABCG1 expression in microglia controls nociception and is required for AIBP-mediated reversal of allodynia in a mouse model of CIPN:
  • FIG. 5A-B illustrates data showing data from BV-2 cells incubated for 30 min with AIBP (0.2 pg/mL) or vehicle in complete media, followed by a 5 min incubation with LPS (100 ng/mL), showing colocalization of accessible cholesterol with ABCAl (FIG. 5A) and APOAl (FIG. 5B) in lipid rafts;
  • FIG. 5C schematically illustrates an exemplary experimental design and timeline of tamoxifen, cisplatin, AIBP or saline injection in mice;
  • FIG. 5D graphically illustrates data showing baseline (day 0) withdrawal thresholds before the start of cisplatin intervention
  • FIG. 5F graphically illustrates data showing withdrawal thresholds after i.t. saline or AIBP (0.5pg/5pl), followed by i.t. LPS (0.1pg/5pl) in TAM-induced ABC-imKO mice;
  • FIG. 5G-H graphically illustrate data showing withdrawal thresholds following i.p. cisplatin and i.t. saline or AIBP (0.5pg/5pl) injections in TAM-induced ABC-imKO (FIG. 5G) and non-induced (vehicle) ABC-imKO (FIG. 5H) mice;
  • FIG. 5I-J graphically illustrate data showing TLR4 dimerization (FIG. 51) and lipid rafts (FIG. 5J) in CD1 lb + TEMEMl 19 + spinal microglia at day 8 in the groups shown in panels FIG. 5G and FIG. 5H, as discussed in detail in Example 1, below.
  • FIG. 6A-G illustrate data showing expression in spinal microglia of ABC-imKO mice:
  • FIG. 6A top image schematically illustrates overlapping genes and pathways induced in naive ABC-imKO microglia and shared with WT microglia in mice treated with cisplatin, showed in purple (darker, upper) lines connecting overlapping genes and in blue (lighter, lower) lines connecting the overlapping enriched pathways
  • FIG. 6A bottom image is a Venn diagram of upregulated genes in spinal microglia from WT cisplatin and ABC-imKO naive mice;
  • FIG. 6B illustrates an enrichment pathway analysis of up and down regulated genes induced by ABCA1 and ABCG1 knockdown in microglia
  • FIG. 6C illustrates DEGs in naive spinal microglia of TAM-induced ABC-imKO mice
  • FIG. 6D schematically illustrates overlapping genes and pathways induced by cisplatin treatment in ABC-imKO microglia and shared with WT microglia in mice treated with cisplatin;
  • FIG. 6E illustrates DEGs in spinal microglia of cisplatin-treated, TAM-induced ABC-imKO mice compared to cisplatin-treated WT mice.
  • FIG. 6F-G illustrate a heatmap of DEGs upregulated genes (FIG. 6F) or downregulated genes (FIG. 6G) in ABC-imKO microglia either in naive or cisplatin condition, as discussed in detail in Example 1, below.
  • FIG. 7A-F illustrate data showing that microglial reprogramming by AIBP is dependent on ABCAl/ABCGl expression:
  • FIG. 7A schematically illustrates a Venn diagram comparing the effect of AIBP treatment on gene expression in WT and ABC-imKO mice in which CIPN was induced by cisplatin;
  • FIG. 7B schematically illustrates a Volcano plot representation of up and down regulated genes by AIBP treatment in CIPN comparing AIBP effect on ABC-imKO vs. WT mice;
  • FIG. 7C schematically illustrates a heatmap of log2 normalized gene counts of inflammatory genes altered by AIBP in an ABC-dependent manner (downregulated by AIBP in WT microglia but upregulated by AIBP in ABC-imKO;
  • FIG. 7D schematically illustrates a heatmap of cholesterol synthesis and LXR related genes comparing cisplatin and AIBP effect in wild type and ABC-imKO;
  • FIG. 7E schematically illustrates a heatmap of non-inflammatory genes regulated by AIBP in an ABC-dependent manner
  • FIG. 7F schematically illustrates an enrichment pathway analysis of upregulated genes by AIBP in ABC-imKO microglia, as discussed in detail in Example 1, below.
  • FIG. 8A-G illustrate that endogenous AIBP and TLR4 in microglia are important in nociception:
  • FIG. 8 A schematically illustrates an exemplary experimental design and timeline: Tamoxifen; cisplatin; AIBP; and/or saline are injected;
  • FIG. 8B graphically illustrates baseline (day 0 in FIG. 8A) withdrawal thresholds before the start of cisplatin intervention
  • FIG. 8C graphically illustrates WT and Cx3crl-Cre ERT2 (no floxed genes) mice were tested for withdrawal threshold before (naive, day -7 in panel A timeline) and after (TAM, day 0) tamoxifen injection regimen;
  • FIG. 8D-F graphically illustrate withdrawal thresholds following i.p. cisplatin and i.t. saline or AIBP injections in: (FIG. 8D) TAM-induced AIBP-imKO mice; non-induced (vehicle) AIBP-imKO mice (FIG. 8E); and bred in-house whole body AIBP knockout mice (FIG. 8F); and
  • FIG. 8G graphically illustrates withdrawal thresholds in WT and tamoxifen- induced TLR4-imKO mice following cisplatin injections, as discussed in detail in Example 1, below.
  • FIG. 9A-H illustrates data showing the identification of the domain in the AIBP molecule responsible for TLR4 binding:
  • FIG. 9A schematically illustrates human AIBP with signal peptide, amino acids (aa) 1-24, previously uncharacterized N-terminal domain (aa 25-51), and YjeF_N domain (aa 52-288);
  • FIG. 9B illustrates an image of a PAGE separation of flag-tagged deletion mutants of human AIBP, which were co-expressed in HEK293 cells with the Flag-tagged TLR4 ectodomain (eTLR4); cell lysates were immunoprecipitated (IP) with an anti-TLR4 antibody and immunoblotted (IB) with an anti-Flag antibody;
  • eTLR4 Flag-tagged TLR4 ectodomain
  • FIG. 9C illustrates an image of a PAGE separation of his-tagged human (hu), mouse (mo) and zebrafish (zf) AIBP, all lacking the signal peptide, expressed in a baculovirus/insect cell system, and were combined in a test-tube with eTLR4-His, followed by IP with an anti-TLR4 antibody and IB with an anti-His antibody;
  • FIG. 9D-H illustrate data showing binding of His-tagged wild type (wt, 25-288 aa) and the deletion mutant (mut, 52-288 aa) human AIBP to eTLR4, APOAl and microglia, and immunoprecipitation (IP) of eTLR4 and wtAIBP or mutAIBP in a test tube with an anti- AIBP antibody, blot and quantification from 3 independent experiments:
  • FIG. 9D illustrates (left image) a PAGE separation, where ELISA were done with plates coated with eTLR4 and incubated with wtAIBP or mutAIBP, the right image graphically shows the amounts of TLR4/AIBP for wt and mu AIPB;
  • FIG. 9E graphically illustrates AIBP binding on immobilized eTLR4 with wt or mut AIBP (or no AIBP), where ELISA wasre done with plates coated with BSA, wtAIBP or mutAIBP and incubated with APOAl;
  • FIG. 9F graphically illustrates APOAl bound to AIBP
  • FIG. 9G graphically illustrates number of cells with APOAl bound to AIBP using flow cytometry (upper graphs), and (lower graph) AIBP binding (fold change) to wt and mut AIBP in non-stimulated LPS stimulated cells;
  • FIG. 9H illustrates APOAl bound to AIBP using confocal imaging, showing binding of wtAIBP and mutAIBP to BV-2 microglia cells, unstimulated or treated for 15 min with LPS, as discussed in detail in Example 1, below.
  • FIG. 10A-G illustrate data showing that intrathecal delivery of AIBP lacking the TLR4 binding domain cannot alleviate CIPN allodynia:
  • FIG. 10A-B graphically illustrate TLR4 dimerization (FIG. 10 A) and lipid rafts (FIG. 10B) in BV-2 cells pre-treated with wt AIBP or mut AIBP and stimulated with LPS;
  • FIG. IOC graphically illustrates withdrawal thresholds in WT mice that received i.t. AIBP (0.5pg/5pL) or saline (5pL), followed by i.t. LPS;
  • FIG. 10D graphically illustrates withdrawal thresholds in WT mice in response to i.p. cisplatin, followed by i.t. wtAIBP, mutAIBP or saline;
  • FIG. 10E-F graphically illustrate TLR4 dimerization (FIG. 10E) and lipid rafts (FIG. 10F) in CD1 lb + /TMEMl 19 + microglia from lumbar spinal cord of mice in experimental groups shown in panel FIG. 10D, at day 21;
  • FIG. 10G schematically illustrates a diagram illustrating the effect of chemotherapeutic-induced peripheral neuropathy (CIPN) using cisplatin-induced tissue damage (damage-associated molecular patterns (DAMPs)) and AIBP treatment on microglia gene expression and lipid droplet accumulation, black dots in the plasma membrane and the ER depict cholesterol, as discussed in detail in Example 1, below.
  • FIG. 11 schematically illustrates a model of unfolding or exposing a cryptic N- terminal domain in the AIBP molecule; the diagram summarizes and illustrates results of experiments shown in FIGs.
  • FIG. 12 illustrates an exemplary amino acid sequence of an engineered AIBP, as provided herein (SEQ ID NO:35): the amino acid sequence of an extended AIBP molecule depicted in the bottom panel of FIG. 11, blue letters, amino acids from the native AIBP sequence; green box, the TLR4-binding sequence (amino acids 25-51 of the human AIBP sequence); black letters and (red) box, added amino acids.
  • SEQ ID NO:35 the amino acid sequence of an extended AIBP molecule depicted in the bottom panel of FIG. 11, blue letters, amino acids from the native AIBP sequence; green box, the TLR4-binding sequence (amino acids 25-51 of the human AIBP sequence); black letters and (red) box, added amino acids.
  • FIG 13 schematically illustrates TLR4 binding of various exemplary engineered forms of AIBP: all proteins were expressed and purified from a baculovirus/insect cell system:
  • His-d24AIBP corresponds to the amino acid sequence shown in FIG. 12, the amino acid sequence shows the sequence of the orange box “cleavable His tag”, all other drawings show different modifications and corresponding changes in the amino acid sequence introduced to the AIBP molecule, the green “N-terminal domain” box depicts the amino acid 25-51 sequence of native AIBP, the column on the right shows the results of co-immunoprecipitation experiments of the AIBP variants with a recombinant ectodomain of TLR4, for His-24 AIBP:
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR for “cleaved His-d24 AIBP”
  • GSPGLDGICSR for “5xD mut His-d24 AIPB”:
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), for “cleaved 5xD His-d24 AIPB”
  • GSDGDDGDDDR (SEQ ID NO: 10), for “2xD mut His-d24 AIBP
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGICSR (SEQ ID NO: 11), and for “cleaved His-d24 AIBP” GSPGLDGICSR (SEQ ID NO:9).
  • FIG. 14 schematically illustrates TLR4 binding of various engineered forms of AIBP: all proteins were co-expressed with the full-length TLR4 in a mammalian system: SS, secretion signal, corresponding to the amino acids 1-24 in the human AIBP sequence; the column on the right shows the results of co-immunoprecipitation from cell lysates of the AIBP variants with TLR4,
  • FIG. 15 schematically illustrates various AIBP constructs to optimize the structure for TLR4 affinity: baculovirus/insect cell expression system:
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR for “PKA site + Thrombin cleavage site”: MGRRRAS VAAGILVPRGSPGLDGIC SR (SEQ ID NO: 17) for “thrombin cleavage site”
  • GSDGDDGDDDR (SEQ ID NO: 11), for “5XD”.
  • FIG. 16 schematically illustrates TLR4 binding of various engineered forms of AIBP: all proteins were expressed and purified from an E.coli , the column on the right shows the results of co-immunoprecipitation experiments of the AIBP variants with a recombinant ectodomain of TLR4.
  • FIG. 17A-D provides validation of the specificity of TLR4 antibodies used for flow cytometry and microscopy, and also shows TLR4 dimerization and lipid rafts measured in dorsal root ganglia macrophages:
  • FIG. 17A graphically illustrates flow cytometry of single cell suspensions from spinal cords of WT (left images) and Tlr4-/- mice (right images) showing TLR4-APC and TLR4/MD2-PE antibodies staining of CDllb+(PercP-Cy5.5)/TMEM199+(Pe-Cy7) microglia;
  • FIG. 17B illustrates confocal images of peritoneal elicited macrophages from WT and Tlr4-/ ⁇ mice co-stained with F4/80-FITC and TLR4-647 antibodies; Scale bar, 5 pm; and
  • FIG. 17C-D graphically illustrate flow cytometry analysis of CD1 lb+ DRG macrophages cells showing TLR4 dimerization (FIG. 17C) and lipid raft content measured by CTxB staining (FIG. 17D) 24 hours after i.t. saline or AIBP, as discussed in Example 1, below.
  • FIG. 18A-E shows FACS sorting strategy for spinal microglia, quality controls and phenotypic controls for RNA-seq:
  • FIG. 18A illustrates sorting strategy for lumbar CD1 lb+TMEMl 19+ spinal microglia, including: SSC-A and FSC-A, SSC-W and SSC-H, UVE/DEAD (APC-Cy7-A) and SSC-A, GLAST1 and CD24, and, CDllb and TMEM119;
  • FIG. 18B illustrates flow cytometry analysis of sorted microglia measuring purity of sorted cells and absence of GLAST1+ astrocytes or CD24+ neurons, including TMEM119 and CDllb, SSC-A and GLAST1, and SSC-1 and CD24;
  • FIG. 18C illustrates microglial linage analysis with a heatmap of microglia specific genes
  • FIG. 18D-E illustrate heatmaps of CIPN-repressed genes that were up-regulated by AIBP (group 4) (FIG. 18D) and CIPN-induced genes that were downregulated by AIBP (group 3) in wildtype mice (FIG. 18E); as discussed in Example 1, below.
  • FIG. 19A-D provides immunohistochemical validation of conditional knockout of ABCAl and ABCGl in spinal microglia of tamoxifen-induced ABC-imKO mice:
  • FIG. 19A illustrates DAPI, IBA1, ABCAl, MERGE, and COLOC MASK, with and without tamixifen
  • FIG. 19B illustrates DAPI, IBA1, ABCGl, MERGE, and COLOC MASK, with and without tamixifen,
  • FIG. 19C illustrates DAPI, NeuN, ABCAl, MERGE, and COLOC MASK, with and without tamixifen, and
  • FIG. 19D illustrates DAPI, GFAP, ABCAl, MERGE, and COLOC MASK, with and without tamixifen, as discussed in further detail in Example 1, below.
  • FIG. 20A-E show tactile allodynia data for tamoxifen-treated WT mice in i.t. LPS and CIPN experiments, and provide additional RNA-seq data for ABC-imKO dependent genes and the cisplatin effect on ABC-imKO vs. WT mice:
  • FIG. 20A-B graphically illustrate data where, as a control for ABC-imKO mice, inhouse bred WT littermate mice were subjected to the tamoxifen regimen (TAM, 200pL/day, lOmg/mL, 5 consecutive days), followed by (FIG. 20A) i.t. injection of AIBP (0.5pg/5pL) or saline (5pL) and i.t. LPS(0.1pg/5pL) 2 hours later; and (FIG. 20B) i.p. injections of cisplatin (2.3 mg/Kg) on day 1 and day 3 followed by i.t. injection of AIBP (0.5pg/5pL) or saline (5pL) on day 7;
  • TAM tamoxifen regimen
  • FIG. 20C graphically illustrate data where ABC-imKO mice were injected with TAM and then cisplatin as above, followed by i.t. saline (5pL), AIBP (0.5pg/5pL) or hp- b-CD (0.25mg/5pL) on day 7;
  • FIG. 20D illustrates a heatmap of differentially regulated genes across all conditions (naive, induced by cisplatin/saline or cisplatin/ AIBP) regulated in an ABC- imKO manner;
  • FIG. 20D illustrates all significant genes from likelihood ratio test using a reduced model without interaction term (condition: genotype);
  • FIG. 20E illustrates a heatmap of pathway enrichment of cisplatin upregulated genes in WT and ABC-imKO microglia using cutoff ⁇ 0.05, enrichment >1.5 and a minimum overlap of 3 genes in the pathway, as discussed in further detail in Example 1, below.
  • FIG. 21 A-B provides immunohistochemical validation of AIBP knockout in spinal microglia of tamoxifen-induced AIBP-imKO mice, and demonstrates that the BE-1 monoclonal antibody has similar affinity to wtAIBP and mutAIBP:
  • FIG. 21 A illustrates images of IHC of spinal cord frozen sections from vehicle and tamoxifen induced AIBP-imKO mice, showing colocalization of AIBP staining with IBA1 (microglia), NeuN (neurons) and GFAP (astrocytes);
  • FIG. 21B graphically illustrates data of a sandwich ELISA using BE-1 as a capture antibody in a microtiter plate, dose response curves to wtAIBP and mutAIBP were detected using a rabbit polyclonal anti-AIBP antibody, as discussed in further detail in Example 1, below.
  • FIG. 22A-C graphically illustrate reduced AIBP expression in bronchial epithelium:
  • FIG. 22A graphically illustrates AIBP+ bronchial epithelium in non-asthma and asthma samples
  • FIG. 22B graphically illustrates APOAIBP/HPRTI mRNA in non-asthma and asthma samples
  • FIG. 22C graphically illustrates AIBP expression in bronchial epithelium, as discussed in further detail in Example 3, below.
  • FIG. 23 A-F graphically illustrate that Compound 7 reduces airway hyper responsiveness and eosinophilic pulmonary inflammation in an HDM model of asthma in female and male mice, as discussed in further detail in Example 3, below.
  • FIG. 24A-M illustrate that AIPB reduces retinal neurodegeneration in D2 glaucomatous mice, as discussed in further detail in Example 4, below.
  • FIG. 25A-D illustrates the AIPB reduces retinal neurodegeneration and improves visual function in a microbead-induced hypertension mouse model, as discussed in further detail in Example 4, below.
  • FIG. 26A-B illustrate that AIPB reduces retinal neurodegeneration in a mouse nerve crash model, as discussed in further detail in Example 4, below.
  • compositions and methods using pharmaceutical compounds and formulations comprising nucleic acids, polypeptides, and gene and polypeptide delivery vehicles for regulating or manipulating, including modification of amino acid sequence, adding, maintaining, enhancing or upregulating, the expression of recombinant ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP), and kits comprising all or some of the components for practicing these compositions and methods.
  • APIBP ApoA-I Binding Protein
  • compositions and methods for altering AIBP sequence and structure and delivering therapeutic levels of recombinant AIBP to the body, including the brain and CNS including use of delivery vehicles targeting and/or capable of penetrating the blood brain barrier, and nucleic acid (gene) delivery vehicles such as vectors and viruses such as an adeno-associated virus (AAV) delivery vehicle having contained within an AIBP expressing nucleic acid; and for direct delivery of either AIBP polypeptide or AIBP-expressing nucleic acid directly via intrathecal (i.t.) administration.
  • nucleic acid (gene) delivery vehicles such as vectors and viruses such as an adeno-associated virus (AAV) delivery vehicle having contained within an AIBP expressing nucleic acid
  • AAV adeno-associated virus
  • Example 1 describes studies using a mouse model of chemotherapy-induced peripheral neuropathy, where spinal microglia are characterized by the presence of inflammarafts - enlarged, cholesterol-enriched lipid rafts, which organize the inflammatory response. Manipulation of specific mechanisms regulated cholesterol metabolism and normalized inflammarafts and reprogramed microglia, resulting in a long-lasting alleviation of neuropathic pain.
  • AIBP binding to TLR4 is important because this innate immune receptor is highly expressed in inflammatory cells and concentrates in lipid rafts on the cell surface and mediates inflammatory responses. Enlarged/clustered lipid rafts with increased content of TLR4 and the evidence of TLR4 dimerization are called “inflammarafts”.
  • AIBP targets inflammatory cells, disrupts inflammarafts and inhibits inflammation - spinal neuroinflammation and neuropathic pain, and the effect is applicable to many inflammatory disease states mediated by TLR4.
  • FIG. 11 is a graphical representation of this model.
  • an engineered AIBP comprising an amino acid sequence from the commercial pAcHLT-C vector (BD Biosciences).
  • TLR4 receptors localize to and dimerize in membrane lipid rafts.
  • AIBP apoA-I binding protein
  • Abcgl knockdown induces pain in naive mice and prevents AIBP from reversing CIPN allodynia, highlighting the importance of microglial cholesterol homeostasis in the development of neuropathic pain. Furthermore, characterization of CIPN-associated changes in gene expression in microglia suggests impaired cholesterol metabolism.
  • engineered protein sequences comprised of a ApoA-I Binding Protein (AIBP) amino acid sequence and an amino acid sequence N- terminal to the AIBP amino acid sequence, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence comprises a peptide tag, wherein the peptide tag comprises a multi-histidine (multi-his) tag, in particular, the multi-his tag comprises six contiguous histidine residues (HHHHHH (SEQ ID NO:l)).
  • AIBP ApoA-I Binding Protein
  • heterologous amino terminus amino acid sequence comprises the amino acid sequence
  • MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO:2) having mutation of its thrombin cleavage site so as to render it inoperable.
  • AIBP ApoA-I Binding Protein
  • a recombinant or synthetic ApoA-I Binding Protein (APOAIBP, AIBP, or AI-BP) polypeptide compound or composition having a heterologous amino terminus amino acid sequence of at least about ten amino acid, or between about 10 to 100 amino acids, or between about 20 to 80 amino acids, or between about 30 to 50 amino acids, or any heterologous amino acid sequence sufficient to result in the unfolding and exposing of the cryptic (or hidden, unexposed) N-terminal TLR4 binding domain of the AIBP polypeptide.
  • APIBP ApoA-I Binding Protein
  • murine AIBP is used, for example, a murine AIBP having a sequence encoded by SEQ ID NO:3, and/or an amino acid sequence of SEQ ID NO:4, which optionally can be supplemented with (i.e., further comprise) a fibronectin secretion signal (italic) at the N-terminus, and/or with the His tag (underlined) at the C- terminus; the product is abbreviated as FIB-mAIBP-His:
  • SEQ ID NO:4 MLRGPGPGRLLLLA VLCLGTSVRC TETGKSKRQQSVCRARPTWW GTQRRGS ETMAGAAVKYLSQEEAQAVDQELFNEYQFSVDQLMELAGLSCATAIAKAYPPTS MSKSPPTVLVICGPGNNGGDGLVCARHLKLFGYQPTIYYPKRPNKPLFTGLVTQC QKMDIPFLGEMPPEPMMVDELYELVVDAIFGFSFKGDVREPFHSILSVLSGLTVPI ASIDIPSGWDVEKGNPSGIQPDLLISLTAPKKSATHFTGRYHYLGGRFVPPALEKK YOLNLPSYPDTECVYRLOHHHHHHHH
  • a variant of human AIBP (hAIBP) polypeptide as provided herein for example, a human AIBP having heterologous amino acid sequence that results in exposure of a TLR4 (otherwise cryptic) binding site), or a nucleic acid encoding a variant AIBP as provided herein, is administered to a patient or an individual in need thereof, or is used to manufacture a formulation or pharmaceutical, or is used to make a vector or expression vehicle for administration, or is included in a kit as provided herein, and the AIBP variant can comprise or be encoded by:
  • a modified hAIBP that retains the TLR4-binding domain and has N-terminal residues replaced with a native signal peptide
  • the hAIBP comprises amino acids 25-288 of the hAIBP sequence, also known as d24hAIBP (encoding nucleic acid):
  • GCAG (SEQ ID NO:20) wherein the corresponding d24hAIBP polypeptide is: QTIACRSGPTWWGPQRLNSGGRWDSEVMASTVVKYLSQEEAQAVDQELFNEYQ FSVDQLMELAGLSCATAIAKAYPPTSMSRSPPTVLVICGPGNNGGDGLVCARHLK LF GYEPTI YYPKRPNKPLF T AL VTQC QKMDIPFLGEMP AEPMTIDEL YEL VVD AIF GFSFKGDVREPFHSILSVLKGLTVPIASIDIPSGWDVEKGNAGGIQPDLLISLTAPK K S AT QF T GRYHYLGGRF VPP ALEKK Y QLNLPP YPD TEC VYRLQ (SEQ ID NO:21).
  • the hAIBP fragment comprises amino acids 25 to 288 (also known as d24hAIBP) and the N-terminal modification is:
  • a secretion signal is added to ensure robust secretion of AIBP, for example, a fibronectin secretion signal is added to N terminus of AIBP (see italicized sequences in SEQ ID NO:3 and SEQ ID NO:4); or a nucleic acid encoding a secretion signal is added to the AIBP coding sequence.
  • a secretion signal is a fibronectin secretion signal, an immunoglobulin heavy chain secretion signal or an immunoglobulin kappa light chain secretory peptide (see, for example, PLoS One. 2015; 10(2): eOl 16878), or an interleukin-2 signal peptide (see, for example, J. Gene Med. 2005 Mar;7(3):354-65).
  • polypeptide coding sequences are operatively linked to a promoter, for example, a constitutive, inducible, tissue specific (for example, nerve or brain tissue specific) or ubiquitous promoter or other transcriptional activating agent.
  • a promoter for example, a constitutive, inducible, tissue specific (for example, nerve or brain tissue specific) or ubiquitous promoter or other transcriptional activating agent.
  • the product from post-translational modification of the fibronectin-hAIBP construct has an amino acid sequence (Compound 2): 7 TGA5XRQTIACRSGPTWWGPQRLNSGGRWDSEVMASTVVKYLSQEEAQAVD QELFNEYQFSVDQLMELAGLSCATAIAKAYPPTSMSRSPPTVLVICGPGNNGGDG LVCARHLKLFGYEPTIYYPKRPNKPLFTALVTQCQKMDIPFLGEMPAEPMTIDEL YELVVDAIFGFSFKGDVREPFHSILSVLKGLTVPIASIDIPSGWDVEKGNAGGIQPD LLISLTAPKKSATQFTGRYHYLGGRFVPPALEKKYQLNLPPYPDTECVYRLQ (SEQ ID NO:25), wherein the hAIBP fragment is d24hAIBP and the N-terminal modification is TET GKSKR (SEQ ID NO:26),
  • the sequence of the AIBP polypeptide is modified at its C- terminus to incorporate additional peptidic fragments. This is exemplified by addition of a C -terminal His Tag (underlined in the corresponding amino acid sequence):
  • the post-translational modification of the signal peptide provides a compound (Compound 4):
  • polypeptide coding sequences are operatively linked to a promoter, e.g., a constitutive, inducible, tissue specific (e.g., nerve or brain tissue specific) or ubiquitous promoter or other transcriptional activating agent.
  • a promoter e.g., a constitutive, inducible, tissue specific (e.g., nerve or brain tissue specific) or ubiquitous promoter or other transcriptional activating agent.
  • full length human AIBP is modified at its N-terminus, wherein such modification facilitates TLR4 binding, for example Compound 5-encoding nucleic acid (cDNA):
  • hATBP sequence which retains the cryptic TLR4 binding domain is modified at its N-terminus.
  • Example sequences comprise DNA and peptide sequence for amino acids 25-288 of hAIBP (d24hAIBP):
  • CTGCAG (SEQ ID NO:34), wherein, the hATBP fragment is d24hAIBP and the N-terminal modification is: MSPIDPMGHHHHHHGRRRAS VAAGILVPRGSPGLDGIC SR (SEQ ID NO:2), (Compound 8- encoding nucleic acid sequence):
  • GRF VPP ALEKK Y OLNLPP YPDTEC VYRLO (SEQ ID NO: 36), wherein, the hATBP fragment is d24hAIBP and the N-terminal modification is:
  • APIBP ApoA-I Binding Protein
  • the amino acid N-terminal sequence comprises between 3 and 12 basic amino acids selected from histidine (H), lysine (K) or arginine (R).
  • Examples sequences are represented by: (Compound 8 encoding nucleic acid sequence): ATG TCC CCT ATA GAT CCGATG GGA CAT CAT CAT CAT CAC GGA AGGAGA AGG GCCAGTGTTGCG GCG GGA A TT TTG GTC CCT GCT GCA AGC CCA GGA CTC GA T GGC A TA TGC TCG AGG
  • the thrombin cleavage site LVPRGS incorporates described amino acid mutation and prevents cleavage and unexpected loss of TLR4 binding activity as described in Example 2. It should be acknowledged that these sequences are illustrative and are not limiting to the invention.
  • any of the amino acids in the N-terminal modification of hAIBP may be unnatural and inserted by methods known to those skilled in the art.
  • kits for practicing the methods as provided herein.
  • products of manufacture such as implants or pumps, kits and pharmaceuticals for practicing the methods as provided herein.
  • products of manufacture, kits and/or pharmaceuticals comprising all the components needed to practice a method as provided herein.
  • kits also comprise instructions for practicing a method as provided herein,
  • APIBP ApoA-I Binding Protein
  • compositions and formulations used to practice methods and uses as provided herein comprise recombinant APOAIBP nucleic acids and polypeptides or result in an increase in expression or activity of recombinant APOAIBP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate, for example, a neuropathic pain, a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post- traumatic stress syndrome (PTSS), optionally glaucoma or other inflammatory diseases of the eye, optionally lung inflammation and asthma, optionally HIV infection or its comorbidities, and/or optionally vascular inflammation, atherosclerosis and cardiovascular disease.
  • a neuropathic pain a neurodegenerative disease or condition,
  • compositions and formulations used to practice methods and uses as provided herein comprise recombinant APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to prevent or decrease the intensity of and/or frequency of for example, the neuropathic pain or neurodegenerative disease or condition.
  • the pharmaceutical compositions used to practice methods and uses as provided herein can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, /or example , the latest edition of Remington's Pharmaceutical Sciences. Maack Publishing Co., Easton PA (“Remington’s”).
  • these compositions used to practice methods and uses as provided herein are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like.
  • the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo , in vitro or ex vivo conditions, a desired in vivo , in vitro or ex vivo method of administration and the like. Details on techniques for in vivo , in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature.
  • Formulations and/or carriers used to practice methods or uses as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo , in vitro or ex vivo applications.
  • formulations and pharmaceutical compositions used to practice methods and uses as provided herein can comprise a solution of compositions (which include peptidomimetics, racemic mixtures or racemates, isomers, stereoisomers, derivatives and/or analogs of compounds) disposed in or dissolved in a pharmaceutically acceptable carrier, for example, acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride.
  • acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid.
  • solutions and formulations used to practice methods and uses as provided herein are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.
  • solutions and formulations used to practice methods and uses as provided herein can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo , in vitro or ex vivo administration selected and the desired results.
  • compositions and formulations used to practice methods and uses as provided herein can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells (for example, an injured or diseased neuronal cell or CNS tissue), or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo , in vitro or ex vivo application.
  • nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice methods and uses as provided herein, for example, to deliver compositions comprising recombinant APOA1BP nucleic acids and polypeptides in vivo , for example, to the CNS and brain.
  • these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, for example, for targeting a desired cell type or organ, for example, a nerve cell or the CNS, and the like.
  • multilayered liposomes comprising compounds used to practice methods and uses as provided herein, for example, as described in Park, et ah, U.S. Pat. Pub. No. 20070082042.
  • the multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice methods and uses as provided herein.
  • Liposomes can be made using any method, for example, as described in Park, et al., U.S. Pat. Pub. No.
  • 20070042031 including method of producing a liposome by encapsulating an active agent (for example, recombinant APOA1BP nucleic acids and polypeptides), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
  • an active agent for example, recombinant APOA1BP nucleic acids and polypeptides
  • liposome compositions used to practice methods and uses as provided herein comprise a substituted ammonium and/or polyanions, for example, for targeting delivery of a compound (for example, a recombinant APOA1BP nucleic acid and polypeptide) to a desired cell type (for example, an endothelial cell, a nerve cell, or any tissue or area, for example, a CNS, in need thereof), as described for example, in U.S. Pat. Pub. No. 20070110798.
  • a compound for example, a recombinant APOA1BP nucleic acid and polypeptide
  • a desired cell type for example, an endothelial cell, a nerve cell, or any tissue or area, for example, a CNS, in need thereof
  • nanoparticles comprising compounds (for example, recombinant APOA1BP nucleic acids and polypeptides used to practice methods provided herein) in the form of active agent-containing nanoparticles (for example, a secondary nanoparticle), as described, for example, in U.S. Pat. Pub. No. 20070077286.
  • active agent-containing nanoparticles for example, a secondary nanoparticle
  • nanoparticles comprising a fat-soluble active agent or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
  • solid lipid suspensions can be used to formulate and to deliver compositions used to practice methods and uses as provided herein to mammalian cells in vivo , for example, to the CNS, as described, for example, in U.S. Pat. Pub. No. 20050136121.
  • recombinant AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like are modified to facilitate intrathecal injection, for example, delivery into the cerebrospinal fluid or brain.
  • AIBP peptides or polypeptides, or recombinant AIBP-comprising nanoparticles, liposomes and the like are engineered to comprise a moiety that allows the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, to bind to a receptor or cell membrane structure that facilitates delivery into the CNS or brain, for example, where the moiety can comprise a mannose-6-phosphate receptor, a melanotransferrin receptor, a LRP receptor or any other receptor that is ubiquitously expressed on the surface of any CNS or brain cell.
  • conjugation of mannose- 6-phosphate moieties allows the AIBP peptides or polypeptides, or recombinant AIBP- comprising nanoparticles, liposomes and the like, to be taken up by a CNS cell that expresses a mannose-6-phosphate receptor.
  • any protocol or modification of the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, that facilitates entry or delivery into the CNS or brain in vivo can be used, for example, as described in USPN 9,089,566.
  • recombinant AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like are directly or indirectly linked or conjugated to any blood brain barrier (BBB)-targeting agent, for example, a transferrin, an insulin, a leptin, an insulin-like growth factor, a cationic peptide, a lectin, a Receptor- Associated Protein (RAP) (a 39 kD chaperone localized to the endoplasmic reticulum and Golgi, a lipoprotein receptor-related protein (LRP) receptor family ligand), an apolipoprotein B- 100 derived peptide, an antibody (for example, a peptidomimetic monoclonal antibody) to a transferrin receptor, an antibody (for example, a peptidomimetic monoclonal antibody) to a transferrin receptor, an antibody (for example, a peptidomimetic monoclonal antibody) to a
  • any protocol or modification of the AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like, that facilitates crossing of the BBB can be used, for example, as described in US Pat App Pub nos. 20050142141; 20050042227.
  • any protocol can be used, for example: direct intra-cranial injection, transient permeabilization of the BBB, and/or modification of AIBP peptides or polypeptides, or AIBP-comprising nanoparticles, liposomes and the like to alter tissue distribution
  • any delivery vehicle can be used to practice the methods or uses as provided herein, for example, to deliver compositions (for example, recombinant APOA1BP nucleic acids and polypeptides) to a CNS or a brain in vivo.
  • delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used for example as described, for example, in U.S. Pat. Pub. No. 20060083737.
  • a delivery vehicle is a transduced cell engineered to express or overexpress and then secrete an endogenous or exogenous AIBP.
  • a dried polypeptide-surfactant complex is used to formulate a composition used to practice methods as provided herein, for example as described, for example, in U.S. Pat. Pub. No. 20040151766.
  • a composition used to practice methods and uses as provided herein can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, for example, as described in U.S. Patent Nos. 7,306,783; 6,589,503.
  • the composition to be delivered is conjugated to a cell membrane-permeant peptide.
  • the composition to be delivered and/or the delivery vehicle are conjugated to a transport-mediating peptide, for example, as described in U.S. Patent No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to poly-phosphoinositides.
  • cells that will be subsequently delivered to a CNS or a brain are transfected or transduced with an AIBP-expressing nucleic acid, for example, a vector, for example, by electro-permeabilization, which can be used as a primary or adjunctive means to deliver the composition to a cell, for example, using any electroporation system as described for example in U.S. Patent Nos. 7,109,034;
  • the nucleic acids, vectors or recombinant viruses are designed for in vivo or CNS delivery and expression.
  • an expression vehicle for example, vector, recombinant virus, and the like
  • the provided are methods for being able to turn on and turn off AIBP-expressing nucleic acid or gene expression easily and efficiently for tailored treatments and insurance of optimal safety.
  • recombinant AIBP protein or proteins expressed by the AIBP-expressing nucleic acid(s) or gene(s) have a beneficial or favorable effects (for example, therapeutic or prophylactic) on a tissue or an organ, for example, the brain,
  • CNS CNS, or other targets, even though secreted into the blood or general circulation at a distance (for example, anatomically remote) from their site or sites of action.
  • a recombinant virus and the like for in vivo expression of a recombinant AIBP-encoding nucleic acid or gene to practice the methods as provide herein.
  • the expression vehicles, vectors, recombinant viruses and the like expressing the an AIBP nucleic acid or gene can be delivered by intramuscular (IM) injection, by intravenous (IV) injection, by subcutaneous injection, by inhalation, by a biolistic particle delivery system (for example, a so-called “gene gun”), and the like, for example, as an outpatient, for example, during an office visit.
  • IM intramuscular
  • IV intravenous
  • a biolistic particle delivery system for example, a so-called “gene gun”
  • this “peripheral” mode of delivery for example, expression vehicles, vectors, recombinant viruses and the like injected IM or IV, can circumvent problems encountered when genes or nucleic acids are expressed directly in an organ (for example, the brain or CNS) itself. Sustained secretion of an AIBP in the bloodstream or general circulation also circumvents the difficulties and expense of administering proteins by infusion.
  • a recombinant virus for example, a long-term virus or viral vector
  • a vector, or an expression vector, and the like can be injected, for example, in a systemic vein (for example, IV), or by intramuscular (IM) injection, by inhalation, or by a biolistic particle delivery system (for example, a so-called “gene gun”), for example, as an outpatient, for example, in a physician's office.
  • a systemic vein for example, IV
  • IM intramuscular
  • a biolistic particle delivery system for example, a so-called “gene gun”
  • the individual, patient or subject is administered (for example, inhales, is injected or swallows), a chemical or pharmaceutical that induces expression of the AIBP-expressing nucleic acids or genes; for example, an oral antibiotic (for example, doxycycline or rapamycin) is administered once daily (or more or less often), which will activate the expression of the gene.
  • a chemical or pharmaceutical that induces expression of the AIBP-expressing nucleic acids or genes; for example, an oral antibiotic (for example, doxycycline or rapamycin) is administered once daily (or more or less often), which will activate the expression of the gene.
  • an AIBP protein is synthesized and released into the subject's circulation (for example, into the blood), and subsequently has favorable physiological effects, for example, therapeutic or prophylactic, that benefit the individual or patient (for example, benefit heart, kidney or lung function).
  • the physician or subject desires discontinuation of the AIBP treatment, the subject simply stops taking the activating chemical or pharmaceutical, for example, antibiotic.
  • Alternative embodiments comprise use of "expression cassettes" comprising or having contained therein a nucleotide sequence used to practice methods provided herein, for example, an AIBP-expressing nucleic acid, which can be capable of affecting expression of the nucleic acid, for example, as a structural gene or a transcript (for example, encoding an AIBP protein) in a host compatible with such sequences.
  • Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, for example, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like.
  • a "vector" can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell.
  • a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • vectors can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (for example, a cell membrane, a viral lipid envelope, etc.).
  • vectors can include, but are not limited to replicons (for example, RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example, plasmids, viruses, and the like, see, for example, U.S. Patent No. 5,217,879), and can include both the expression and non-expression plasmids.
  • a vector can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
  • promoters include all sequences capable of driving transcription of a coding sequence in a cell, for example, a mammalian cell such as a muscle, nerve or brain cell. Promoters used in the constructs provided herein include ex acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a nucleic acid, for example, an AIBP-encoding nucleic acid.
  • a promoter can be a cis- acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3’ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
  • “constitutive” promoters can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
  • “inducible” or “regulatable” promoters can direct expression of a nucleic acid, for example, an AIBP-encoding nucleic acid, under the influence of environmental conditions, administered chemical agents, or developmental conditions.
  • methods of the invention comprise use of nucleic acid (for example, gene or polypeptide encoding a recombinant AIBP-encoding nucleic acid) delivery systems to deliver a payload of the nucleic acid or gene, or AIBP-expressing nucleic acid, transcript or message, to a cell or cells in vitro , ex vivo , or in vivo , for example, as gene therapy delivery vehicles.
  • nucleic acid for example, gene or polypeptide encoding a recombinant AIBP-encoding nucleic acid
  • methods of the invention comprise use of nucleic acid (for example, gene or polypeptide encoding a recombinant AIBP-encoding nucleic acid) delivery systems to deliver a payload of the nucleic acid or gene, or AIBP-expressing nucleic acid, transcript or message, to a cell or cells in vitro , ex vivo , or in vivo , for example, as gene therapy delivery vehicles.
  • nucleic acid for example, gene or
  • expression vehicle, vector, recombinant virus, or equivalents used to practice methods provided herein are or comprise: an adeno- associated virus (AAV), a lentiviral vector or an adenovirus vector; an AAV serotype AAV5, AAV6, AAV8 or AAV9; a rhesus-derived AAV, or the rhesus-derived AAV AAVrh.l0hCLN2; an organ-tropic AAV, or a neurotropic AAV; and/or an AAV capsid mutant or AAV hybrid serotype.
  • AAV adeno- associated virus
  • the AAV is engineered to increase efficiency in targeting a specific cell type that is non-permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest.
  • the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications comprising: 1) a transcapsidation, 2) adsorption of a bi- specific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid.
  • AAV adeno-associated virus
  • serotypes AAV-8, AAV-9, AAV-DJ or AAV-DJ/8TM are used to deliver an AIBP-encoding nucleic acid payload for expression in the CNS.
  • serotypes, or variants thereof are used for targeting a specific tissue:
  • the rhesus-derived AAV AAVrh.l0hCLN2 or equivalents thereof can be used, wherein the rhesus-derived AAV may not be inhibited by any pre-existing immunity in a human; see for example, Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct;23(5):324-35, Epub 2012 Nov 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct 17; teaching that direct administration of AAVrh.l0hCLN2 to the CNS of rats and non-human primates at doses scalable to humans has an acceptable safety profile and mediates significant payload expression in the CNS.
  • AAVs adeno-associated viruses
  • NAbs neutralizing antibodies
  • methods provided herein can comprise screening of patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy; see, for example, Sun, et al., J. Immunol. Methods. 2013 Jan 31 ;387(l-2): 114-20, Epub 2012 Oct 11
  • compositions and formulations used to practice methods and uses as provided herein can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject already suffering from a disease, condition, infection or defect in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disease, condition, infection or disease and its complications (a “therapeutically effective amount”), including for example, a neuropathic pain.
  • recombinant APOAIBP nucleic acid- or polypeptide- comprising pharmaceutical compositions and formulations as provided herein are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate a neuropathic pain, an inflammation-induced neuropathic pain, an inflammation-induced neuropathic pain, a nerve or CNS inflammation, a allodynia, a post nerve injury pain or neuropathic pain, a post-surgical pain or neuropathic pain, a chemotherapeutic-induced peripheral neuropathy (CIPN) (for example, cisplatin-induced allodynia), a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer’s disease or a Chronic Traumatic Encephalopathy (CTE) or a related tauopathy, a traumatic brain injury (TBI), a posttraumatic stress disorder, a traumatic war neurosis, or a post-traumatic stress syndrome (PTSS), a
  • CIPN
  • the amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • viral vectors such as adenovirus or AAV vectors are administered to an individual in need therein, and in alternative embodiment the dosage administered to a human comprises: a dose of about 2 c 10 12 vector genomes per kg body weight (vg/kg), or between about 10 10 and 10 14 vector genomes per kg body weight (vg/kg), or about 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or more vg/kg, which can be administered as a single dosage or in multiple dosages, as needed. In alternative embodiments, these dosages are administered orally, IM, IV, or intrathecally.
  • the vectors are delivered as formulations or pharmaceutical preparations, for example, where the vectors are contained in a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
  • these dosages are administered once a day, once a week, or any variation thereof as needed to maintain in vivo expression levels of recombinant AIBP, which can be monitored by measuring actually expression of AIBP or by monitoring of therapeutic effect, for example, diminishing of pain.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, for example, Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur.
  • the active agents rate of absorption, bioavailability, metabolism, clearance, and the like
  • formulations can be given depending on the dosage and frequency as required and tolerated by the patient.
  • the formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate a conditions, diseases or symptoms as described herein.
  • alternative exemplary pharmaceutical formulations for oral administration of compositions used to practice methods as provided herein are in a daily amount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram of body weight per day.
  • dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation.
  • Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra.
  • the methods as provided herein can further comprise co-administration with other drugs or pharmaceuticals, for example, compositions for treating any neurological or neuromuscular disease, condition, infection or injury, including related inflammatory and autoimmune diseases and conditions, and the like.
  • the methods and/or compositions and formulations as provided herein can be co-formulated with and/or co administered with, fluids, antibiotics, cytokines, immunoregulatory agents, anti inflammatory agents, pain alleviating compounds, complement activating agents, such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (for example, a ficolin), carbohydrate-binding domains, and the like and combinations thereof.
  • bioisosteres of compounds used to practice the methods provided herein for example, polypeptides having a recombinant APOA1BP activity.
  • Bioisosteres used to practice methods as provided herein include bioisosteres of, for example, recombinant APOAIBP nucleic acids and polypeptides, which in alternative embodiments can comprise one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to compounds used to practice methods or uses as provided herein.
  • the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structures.
  • one or more hydrogen atom(s) is replaced with one or more fluorine atom(s), for example, at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place.
  • fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.
  • compositions and formulations used to practice methods as provided herein are delivered directly into a CNS or a brain, for example, either by injection intravenously or intrathecally, or by various devices known in the art.
  • U.S. Pat. App. Pub. No. 20080140056 describes a rostrally advancing catheter in the intrathecal space for direct brain delivery of pharmaceuticals and formulations.
  • Implantable infusion devices can also be used; for example, a catheter to deliver fluid from the infusion device to the brain can be tunneled subcutaneously from the abdomen to the patient's skull, where the catheter can gain access to the individual's brain via a drilled hole.
  • a catheter may be implanted such that it delivers the agent intrathecally within the patient's spinal canal.
  • Flexible guide catheters having a distal end for introduction beneath the skull of a patient and a proximal end remaining external of the patient also can be used, for example, see U.S. Pat. App. Pub. No. 20060129126.
  • compositions and formulations used to practice methods as provided herein are delivered via direct delivery of pharmaceutical compositions and formulations, including nanoparticles and liposomes, or direct implantation of cells that express AIBP into a brain, for example, using any cell implantation cannula, syringe and the like, as described for example, in U.S. Pat. App. Pub. No. 20080132878; or elongate medical insertion devices as described for example, in U.S. Pat. No. 7,343,205; or a surgical cannula as described for example, in U.S. Pat. No. 4,899,729.
  • Implantation cannulas, syringes and the like also can be used for direct injection of liquids, for example, as fluid suspensions.
  • pharmaceutical compositions and formulations used to practice methods as provided herein are delivered with tracers that are detectable, for example, by magnetic resonance imaging (MRI) and/or by X-ray computed tomography (CT); the tracers can be co-infused with the therapeutic agent and used to monitor the distribution of the therapeutic agent as it moves through the target tissue, as described for example, in U.S. Pat. No. 7,371,225.
  • MRI magnetic resonance imaging
  • CT X-ray computed tomography
  • kits comprising compositions (including the devices as described herein) and/or instructions for practicing methods as provided herein to for example, treat, ameliorate or prevent a neuropathic pain.
  • kits, cells, vectors and the like can also be provided.
  • kits comprising: a composition used to practice a method as provided herein, or a composition, a pharmaceutical composition or a formulation as provided herein, and optionally comprising instructions for use thereof.
  • Example 1 Efficacy demonstrated in exemplary methods for treating pain
  • This example describes and demonstrates exemplary embodiments, and the efficacy of methods as provided herein to for example, treat or ameliorate a neuropathic pain, including for example, allodynia and TLR4-mediated inflammation-induced neuropathic pain.
  • Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states.
  • Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts.
  • AIBP apoA-I binding protein
  • Chemotherapy-induced peripheral neuropathy alters lipid rafts and TLR4 dimerization in spinal microglia
  • TLR4 receptor dimerization which is the first step in the activation of a TLR4 inflammatory cascade, occurs in microglial lipid rafts, as is demonstrated in other cell types (Cheng et al., 2012; Zhu et al., 2010).
  • This notion was supported in in vitro experiments in which localization of TLR4 in lipid rafts was significantly increased in BV-2 microglia cells treated with LPS, and AIBP (compound 7) prevented LPS-induced TLR4-CTxB colocalization (Fig. ID).
  • the specificity of the TLR4 antibodies used in flow cytometry and microscopy experiments was verified with cells from TIr4 ⁇ mice (Fig. SI A and B).
  • AIBP compound 7
  • a single intrathecal dose of AIBP (compound 7) had a long-lasting therapeutic effect of reversing allodynia in CIPN mice sustained for at least 2 months (Woller et al., 2018). This can be explained either by AIBP (compound 7) long exposure in the spinal cord upon i.t. delivery or by a disease-modifying effect reflected in changes in gene expression profile.
  • AIBP compound 7
  • Apoalbp ⁇ mice in these experiments to avoid cross-reactivity of the antibodies we use with endogenous mouse AIBP in the spinal cord tissue.
  • DAM neurodegenerative disease-associated microglia
  • Fig. 4A enriched pathways of inflammatory response, leukocyte chemotaxis and neutrophil degranulation pathways.
  • Some of the genes in these enriched pathways include Illb, Ccl2, Gliprl and Glipr2, Gpnmb, Cxcl2, Cxcl3, S100a8, Il22ra2, Illr2, Fprl, Apoe, Ccl9 and the TLR4 interactor gene Tril (Fig. 4B and C).
  • AIBP compound 7
  • Fig. 4D cytokine protein expression in spinal cord tissue
  • Fig. 4D cytokine protein expression in spinal cord tissue
  • Fig. 4E cytokine protein expression in spinal cord tissue
  • Fig. 4E cytokine protein expression in spinal cord tissue
  • Fig. 4E cytokine protein expression in spinal cord tissue
  • Fig. 4E cytokine protein expression in spinal cord tissue
  • Fig. 4E The pathway and GO analysis of all genes downregulated by AIBP (compound 7) show enrichment in the TLR4 signaling pathway, together with cytokine-cytokine receptor interaction, protein kinase A and C and MAPK regulation pathways, receptor mediated endocytosis and other membrane signaling pathways
  • AIBP (compound 7) cannot reverse allodynia in mice with ABCAl/ABCGl deficient microglia
  • i.t. AIBP compound 7
  • Fig. 5F and S5A mechanical allodynia induced by i.t. LPS in ABC-imKO mice
  • i.t. delivery of AIBP in ABC-imKO mice at day 7 of the CIPN model did not reverse mechanical allodynia (Fig. 5G)
  • i.t. AIBP was effective in reversing CIPN allodynia in transgenic (littermates) mice treated with vehicle instead of tamoxifen (Fig.
  • Upregulated interferon genes included Ifi207 and Ifi27l2a , and inflammatory genes Xcrl, Cb4, C3, and Klrblb. Lipid metabolism related genes Apoe and Ch25h were significantly upregulated in naive ABC-imKO, similar to the changes induced by cisplatin in WT mice (Fig. 6C and F). This microglia reprogramming might explain, at least in part, the pain behavior observed in naive ABC-imKO mice
  • Fig. 6E and G is an LXR agonist and a key regulator of macrophage foam cell transcriptome in atherosclerosis (Span et all, 2012).
  • Impaired phagocytosis and upregulation of Tnfrsf26, Trpv4, Il3ra, 1115a , and Shtnl may indicate a differential role of membrane dynamics for nociceptive processes in ABC-imKO mice.
  • Microglial reprogramming by AIBP (compound 7) is dependent on ABCA1 and ABCG1 expression
  • zebrafish AIBP did not bind human eTLR4 (Fig. 9C).
  • wtAIBP baculovirus/insect cell system
  • mutAIBP aa 52-288
  • mutAIBP did not bind eTLR4 in a pull-down assay (Fig. 9D) nor in ELISA with eTLR4-coated plates and detection of bound AIBP with the BE-1 anti- AIBP monoclonal antibody (mAh) developed in our lab (Choi et ah, 2020) (Fig. 9E).
  • the BE-1 mAh had equal affinity to wtAIBP and mutAIBP (Fig. S5B).
  • Binding of mutAIBP to APOAl remained unchanged when compared to wtAIBP (Fig. 9F).
  • wtAIBP but not mutAIBP bound to BV-2 microglia stimulated with LPS Fig. 9G and H).
  • AIBP lacking its TLR4 binding domain cannot alleviate CIPN allodynia
  • mutAIBP lacking the TLR4 binding site was unable to inhibit LPS-induced TLR4 dimerization in BV-2 microglia (Fig. 10A) but retained the overall ability to reduce lipid rafts (Fig. 10B).
  • this TLR4 targeting mediates the therapeutic effect of AIBP.
  • Mice that received i.t. saline or mutAIBP prior to i.t. LPS developed allodynia rapidly and to the same extent, whereas i.t. wtAIBP prevented mechanical allodynia induced by LPS (Fig. IOC).
  • wtAIBP reversed established allodynia, with the sustained therapeutic effect lasting for at least 14 days (Fig. 10D).
  • i.t. mutAIBP induced only a modest and transient reversal in mechanical thresholds that did not reach naive or baseline levels and lasted only for 2-3 days (Fig. 10D).
  • the mice were terminated, and lumbar spinal cords analyzed.
  • cisplatin-induced polyneuropathy continued to be associated with increased TLR4 dimerization and lipid rafts in spinal microglia, which were significantly reduced by i.t. wtAIBP but not mutAIBP (Fig. 10E and F), similar to the effect observed on day 8 (Fig. IB and C).
  • AIBP has a singular ability to disrupt inflammarafts in activated cells but has little effect on physiological lipid rafts in quiescent cells. We proposed that this is due to AIBP binding to TLR4, which is highly expressed on the surface of inflammatory cells, directing cholesterol depletion to these cells (Miller et al., 2020; Woller et al., 2018). In this work, we identified the N-terminal domain of AIBP as the binding site for TLR4 and demonstrated the critical role of this domain in enabling AIBP binding to activated microglia and its therapeutic effect in CIPN.
  • AIBP a selective therapy directed to inflammarafts as opposed to non-selective cholesterol removal effected by cyclodextrins, APOAl and APOA1 mimetic peptides, or LXR agonists.
  • the mutated human AIBP lacking the N-terminal domain still binds to APOAl, and the wild type zebrafish Aibp in which this N-terminal domain is naturally absent, still augments cholesterol efflux from endothelial cells and regulates angiogenesis and orchestrates emergence of hematopoietic stem and progenitor cells from hemogenic endothelium (Fang et al., 2013; Gu et al., 2019), suggesting a different, TLR4-independent mechanism of AIBP interaction with endothelial cells.
  • Intrathecal delivery of AIBP has a lasting therapeutic effect in a mouse model of CIPN, observed for as long as 10 weeks in our earlier work (Woller et al., 2018) and for 2 weeks in this study. This is in contrast to a short exposure of i.t. AIBP, peaking at 30 min and largely gone by 4 hours from both CSF and lumbar spinal cord tissue. The dissociation between exposure and therapeutic effect suggests a disease-modifying action of AIBP.
  • the reduced CTxB binding and reduced percentage of TLR4 dimers in spinal microglia were observed for as long as 24 hours and even 2 weeks after a single i.t.
  • AIBP injection indicating sustained disruption of inflammarafts by AIBP, in contrast to their persistent presence in microglia of i.t. saline injected CIPN mice.
  • AIBP disease-modifying effect likely involves reprogramming of gene expression profile in spinal microglia.
  • AIBP reversed only 3% of all genes whose expression in spinal microglia was affected by CIPN, AIBP significantly reduced the inflammatory gene expression and the levels of inflammatory cytokines in spinal tissue induced by the cisplatin regimen.
  • cytokines and chemokines that have been described to have a role in CIPN, such as Illb, Cxcl2 and Ccl2 (Brandolini et ah, 2019; Oliveira et ah, 2014; Pevida et ah, 2013; Yan et ah, 2019).
  • CIPN neurodegenerative microglia
  • a similar microglia lipid droplets phenotype and the transcriptome was recently described as associated with aging and neurodegeneration (Marschallinger et al., 2020; Nugent et ah, 2020).
  • AIBP compound 7
  • APOAl APOAl
  • LXR LXR agonist
  • mxABCAl single nucleotide variant has been found with quality of life scores in painful bone metastasis patients (Furfari et al., 2017).
  • AIBP failed to downregulate inflammatory genes and even upregulated some of them and upregulated non-inflammatory, pain-related Arc, and Pil6 genes that regulate synaptic plasticity (Hossaini et al., 2010; Singhmar et al., 2020).
  • Differential reprograming by AIBP of WT and ABCAl/ABCGl -deficient microglia could be dependent on the desmosterol converting enzyme Dhcr24 , which regulates desmosterol and cholesterol content and when decreased is associated with foam cell formation and homeostatic anti-inflammatory response (Spann et al., 2012).
  • AIBP (compound 7) was also unable to reverse CIPN or LPS induced allodynia in ABC- imKO mice.
  • mice Wild type, Abca A Abcg A , llr4 n n , Slcla3-Cre ERT and Cx3crl-Cre ERT2 mice, all on the C57BL/6 background, were purchased from the Jackson Lab (Bar Harbor, ME) or bred and weaned in-house. Hr4 ⁇ mice were a gift from Dr. Akira (Osaka University). The ApoalbpAA mouse was previously generated in our laboratory using ES cells derived from C57BL/6 mice.
  • mice were cross-bred in our laboratories:, ApoalbpflA Cx3crl-Cre ERT2 (AIBP-imKO), Tlr A Cx3crl-Cre ERT2 (TLR4- imKO), Abca A Abcg A Cx3crl-Cre ERT2 (ABC-imKO), and Abca A Abcg A Slcla3- Cre ERT ( ABC-iaKO). All microglia conditional knockout mice used in experiments had only one allele of Cx3crl-Cre ERT2 to avoid generating a Cx3crl knockout. Mice were housed up to 4 per standard cage at room temperature and maintained on a 12:12 hour light: dark cycle. All behavioral testing was performed during the light cycle. Both food and water were available ad libitum. All experiments were conducted with male mice and according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California
  • BV-2 immortalized microglia cell line (Blasi et al., 1990) was cultured in Dulbecco’s MEM with 5% fetal bovine serum (FBS). Thioglycollate-elicited peritoneal macrophages were harvested from C57BL/6 or Hr 4 mice and maintained in DMEM (Cellgro) supplemented with 10% heat-inactivated FBS (Cellgro) and 50pg/mL gentamicin (Omega Scientific). HEK293 cells (RR ⁇ D:CVCL_0045) were cultured in DMEM supplemented with 10% FBS and 50pg/mL gentamicin. All cells were cultured in 5% CO2 atmosphere at 37°C.
  • Chemotherapy-induced peripheral neuropathy model To develop chemotherapy- induced peripheral neuropathy (CIPN), intraperitoneal (i.p.) injections of cisplatin (2.3 mg/kg/injection; Spectrum Chemical MFG) were performed on day 1 and day 3. During the period of cisplatin administration, weight loss, behavioral changes and mechanical allodynia were monitored and measured. The criteria for euthanasia were the weight loss in excess of 20% body weight and erratic behavior; however, no animals required euthanasia.
  • CIPN chemotherapy- induced peripheral neuropathy
  • CIPN chemotherapy- induced peripheral neuropathy
  • cisplatin 2.3 mg/kg/injection; Spectrum Chemical MFG
  • Intrathecal delivery of AIBP (compound 7) or saline Mice were anesthetized using 5% isoflurane in oxygen for induction and 2% isoflurane in oxygen for maintenance of anesthesia. Intrathecal injections were performed according to (Hylden and Wilcox, 1980). Briefly, the lower back was shaven and disinfected, and the animals were placed in a prone posture holding the pelvis between the thumb and forefinger. The L5 and L6 vertebrae were identified by palpation and a 30G needle was inserted percutaneously on the midline between the L5 and L6 vertebrae. Successful entry was assessed by the observation of a tail flick. Injections of 5pL were administered over an interval of ⁇ 30 seconds.
  • Drugs for intrathecal delivery were formulated in physiological sterile 0.9% NaCl. Based on previous study (Woller et al., 2018), AIBP (compound 7) dosing for spinal delivery in these studies was 0.5pg/5pL. Following recovery from anesthesia, mice were evaluated for normal motor coordination and muscle tone.
  • TLR4 dimerization and lipid rafts assays Ex-vivo and in vitro TLR4 dimerization and lipid rafts assays.
  • the TLR4 dimerization assay uses two TLR4 antibodies for flow cytometry: MTS510 recognizes TLR4/MD2 as a monomer (in TLR4 units) but not a dimer; SA15-21 binds to any cell surface TLR4 irrespective of its dimerization status (Akashi et al., 2003; Zanoni et al., 2016). The percentage of TLR4 dimers was then calculated from MTS510 and SA15-21 measured in the same cell suspension. Lipid raft content was measured using CTxB, which binds to ganglioside GM1.
  • BV-2 cells were preincubated with 0.2 pg/ml AIBP (compound 7) in serum-containing medium for 30 min, followed by a 15 min incubation with LPS 100 ng/mL. At the end of incubation, cells were immediately put on ice, washed once with PBS and fixed for 10 min with 4 % formaldehyde.
  • AIBP compound 7
  • spinal cords were harvested by hydro extrusion (Kennedy et al., 2013), fixed with 4% formaldehyde and put on ice while processing.
  • Single-cell suspensions from lumbar tissue were obtained using a Neural Tissue Dissociation kit (Miltenyi Biotec) according to the manufacturer’s protocol.
  • Myelin Removal Beads II (Miltenyi Biotec) were added to samples and incubated for 15 min at 4°C, followed by separation with LS column and a MACS Separator (Miltenyi Biotec).
  • BV-2 cells were plated on coverslips in 12-well plates and preincubated with 0.2 pg/ml AIBP in 5% serum-containing medium for 30 min, followed by a 5- or 15-min incubation with 100 ng/mL LPS. At the end of incubation, cells were immediately put on ice, washed once with PBS and fixed for 10 min with 4 % formaldehyde.
  • slides were incubated with either 1:100 Alexa488 conjugated anti-NeuN antibody (Cell Signaling, RRID:AB_2799470) or 1:100 Alexa488 conjugated anti-GFAP antibody (Cell Signaling, RRID:AB_2263284).
  • Slides were washed 3 times with PBS and mounted with Prolonged Gold with DAPI (Cell Signaling).
  • Image acquisitions of at least one slide of each animal were performed using a 63X objective and a Leica SP8 confocal microscope with Lightening deconvolution. Colocalization analyses were performed in ImageJ/FIJI (NIH, RRID:SCR_003070/
  • the pALOD4 plasmid (Gay A., 2015) was obtained from Addgene (Cat no #111026, RRID: Addgene l 11026) and used to transform E. coli competent cells BL21(DE3), and positive colonies were selected in Amp + LB plates. After induction of the expression with ImM isopropyl b-d-l-thiogalactopyranoside (IPTG) and lysis, His-tagged ALOD4 was purified using an Ni-NTA agarose column with imidazole elution. Protein was dialyzed against PBS and concentration measured. Aliquots were stored at -80°C.
  • AIBP baculovirus/insect cell system
  • compound 7 Cloning and expression of wtAIBP and mutAIBP in baculovirus/insect cell system AIBP (compound 7) was produced in a baculovirus/insect cell system to ensure posttranslational modification and endotoxin-free preparation as described in (Choi et ah, 2018; Woller et ah, 2018).
  • Human wild type (wt) AIBP and mutant (mut) AIBP, mouse wild type AIBP, and zebrafish wild type AIBP (Fang et ak, 2013) were cloned into a pAcHLT-C vector behind the polyhedrin promoter.
  • the vector contains an N-terminal His-tag to enable purification and detection.
  • Insect Sf9 cells were transfected with BestBac baculovirus DNA (Expression Systems) and the AIBP vector. After 4-5 days, the supernatant was collected to afford a baculovirus stock. Fresh Sf9 cells were infected with the AIBP producing baculovirus, cell pellets were collected after 3 days, lysed, sonicated, cleared by centrifugation, and the supernatants loaded onto a Ni-NTA agarose column eluted with imidazole. Protein was dialyzed against saline, and concentration measured. Aliquots were stored at - 80°C.
  • AIBP compound 7
  • Rathamout AIBP mice were used for the pharmacokinetic study. Intrathecal injections of AIBP (2.5pg/5 pL) were performed as previously described (Hylden and Wilcox, 1980), and the CSF was collected after 15min, 30min, lh, 4h or 8h, as described (Liu and Duff, 2008). Briefly, capillary tubes (0.8x100mm) were pulled using a micropipette puller. Mice were anesthetized using 3% isoflurane with mixture of 50% oxygen and 50% room air. The skin of the neck was shaved, and the mouse was placed on the stereotaxic instrument.
  • N-PERTM Neuronal Protein Extraction Reagent (Thermo Fisher) at lg/lOmL on ice. After 10 min incubation on ice, samples were centrifuged (10,000 xg for lOmin at 4°C) to pellet the cell debris, and supernatants were diluted 1:1 with 1% BSA-TBS. Plates were coated with BE-1 anti-AIBP monoclonal antibody (5pg/mL), incubated for 3h with spinal cord extracts or CSF samples and detected with a rabbit polyclonal anti-AIBP antibody, followed by a goat-anti-rabbit-ALP antibody(Sigma Aldrich, RRID: AB 258103). Plates were read as above.
  • RNA-seq library prep sequencing and quality control We followed the low input bulk seq SmartSeq2 protocol from (Rosales et al., 2018). Cells sorted into a lysis buffer containing Triton X-100, RNase Inhibitor, and Oligo(dT)30-VN were hybridized with oligo(dT)+ to the poly(A) tails of the mRNA.
  • Determination of DEGs was performed by DEseq2 binomial modeling using a likelihood ratio test (LRT) including all samples across all factors and using a reduced design without condition factor to determine the main effect of cisplatin and AIBP and all significant genes altered by these conditions.
  • LRT likelihood ratio test
  • Volcano plots include genes significantly different with an absolute fold change >1.5. Pathway enrichment and GO analysis were performed in metascape.org (RRID:SCR_016620) using a minimum of 3 genes and.P ⁇ 0.05 (Zhou et al., 2019).
  • Co-immunoprecipitation assays for TLR4 binding were performed by mixing 1 pg of eTLR4 (Sino Biological) and AIBP in PBS containing 0.5% Triton X-100 and incubating for 1 hour at room temperature. Samples were precleared by adding Protein A/G Sepharose beads at room temperature for 30min, followed by addition of 1 pg of BE-1 monoclonal anti- AIBP antibody and incubation for 2 hours. Protein A/G Sepharose beads were added and incubated for an additional one hour, followed by 5 washes with PBS containing 0.5% Triton X-100 and immunoblot of samples.
  • HEK293 cells (RRTD:CVCL_0045) were transfected with Flag-eTLR4 and a Flag-AIBP (wild type or one of the mutants) construct.
  • a Flag-AIBP construct wild type or one of the mutants construct.
  • cells were harvested and lysed with an ice-cold lysis buffer (50mM Tris-HCl, pH7.5, 1% NP-40, 150mM NaCl, ImM EDTA, ImM EGTA, 5mMNa3V04, ImMNaF, and a protease inhibitor cocktail from Sigma).
  • Cell lysates were preincubated with protein A/G Sepharose beads for 30 min at 4°C and immunoprecipitated with a mouse anti-TLR4 antibody (Abeam) overnight at 4°C.
  • the lysates were incubated with protein A/G beads for 1 hour at 4°C. Unbound proteins were removed by washing with lysis buffer, and the beads were run on a Bolt Bis-Tris gel (Invitrogen); the bound AIBP was detected by immunoblotting with an anti-Flag antibody (Sigma).
  • ELISA binding assays To assess AIBP-TLR4 binding, 96-well plates were coated with 5pg/ml of eTLR4, washed three times with PBS containing 0.05% Tween-20, blocked with PBS containing 1% BSA, and incubated with wtAIBP or mutAIBP, followed by 2pg/ml of a biotinylated BE-1 anti-AIBP monoclonal antibody. To assess AIBP-APOAl binding, plates were coated with BSA, wtAIBP or mutAIBP, washed, blocked, and incubated with 5pg/ml of human APOAl (a gift from Dmitri Sviridov,
  • BV-2 microglia cells stimulated or not with lOOng/mL LPS for 15 min were blocked with Tris-buffered saline (TBS) containing 1% BSA for 60 min on ice and incubated with either 2pg/mL BSA or 2pg/mL AIBP for 2h on ice.
  • TBS Tris-buffered saline
  • LSBio FITC-conjugated anti- His antibody
  • Cytokine measurement in spinal tissue by ELISA Levels of IL-6 (DY406), IL-Ib (DY401), MCP-1 (DY479) and MIP2 (DY452) in spinal cord lysates were measured using a mouse DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.
  • Figure 1 demonstrates reversal of pain behavior by wild type (wt) AIBP protein in a mouse model of chemotherapy -induced peripheral neuropathy (CIPN) and a reduction of activated TLR4 dimers associated with pro-inflammatory lipid rafts (Inflammarafts):
  • saline or AIBP i.e. at day 8 of the time course shown in A.
  • D BV-2 microglia cells were incubated for 30 min with AIBP (compound 7) (02pg/mL) or vehicle in complete media, followed by a 5 min incubation with LPS (lOOng/mL). Scale bar, 5 mih. Bar graph shows Manders’ tMl coefficient.
  • Figure 2 compares the change in gene signature of naive mice to those treated with chemotherapy agent cisplatin to mice treated with cisplatin and a wild type (wt) AIBP protein:
  • A Heatmap of DEGs across all samples (all technical replicates are presented in columns). Significant (adjusted ⁇ 0.01) up or down regulated genes showing main effect tested by LRT (likelihood ratio test). Log2 relative expression, B, Groups of significant DEGs clustered based on expression profile patterns in different treatment conditions. C, Pathway and GO enrichment analysis of upregulated (group 1 in panel 2B) and downregulated (group2) genes induced by cisplatin treatment, using adjusted P ⁇ 0.05 and absolute fold change >1.5 and a minimum overlap of 3 genes in the pathway. Upregulated pathways are shown in red and downregulated in blue.
  • FIG. 3 compares differences in disease associated microglia (DAM) gene expression signature and lipid droplets in mice receiving chemotherapy to naive mice and CIPN mice treated with a wt AIBP:
  • DAM disease associated microglia
  • FIG. 3 DAM and lipid related gene expression and lipid droplets in spinal microglia of CIPN mice.
  • A-C Same groups as in Fig. 2.
  • A Volcano plot of upregulated and downregulated genes in spinal microglia of cisplatin-treated vs. naive mice. Cutoff of adjusted P ⁇ 0.05 and absolute fold change >1.5 represented in light green dots.
  • B Heatmap depicting disease associated microglia (DAM) signature genes.
  • C Heatmap of log2 normalized gene counts scaled by row showing lipid related gene sets.
  • D-H Lipid droplet accumulation in spinal microglia measured by PLIN2 immunostaining in spinal cord sections co-stained with IBA1 and DAPI. Experimental conditions as in Fig.
  • Figure 4 summarizes changes in gene expression in CIPN mice that have been treated with a wt AIBP protein:
  • A pathway and GO enrichment analysis of CIPN-upregulated genes that were downregulated by AIBP (compound 7) (group 3 in Fig. 2B)) and CIPN-downregulated genes that were upregulated by AIBP (compound 7) (group 4), using adjusted P ⁇ 0.05 and absolute fold change >1.5 and a minimum overlap of 3 genes in the pathway. Upregulated pathways are shown in red and downregulated in blue.
  • B DEGs in spinal microglia induced by i.t. AIBP.
  • Adjusted P ⁇ 0.05 and Benjamini-Hochberg FDR ⁇ 5% represented in a volcano plot of up and down regulated genes in cisplatin/AIBP vs. cisplatin/saline treated mice. Cutoff adjusted P ⁇ 0.05 and absolute fold change >1.5 shown in light green dots.
  • C Heatmap of inflammatory genes in group 3 upregulated in CIPN and downregulated by AIBP.
  • E Heatmap of inflammatory genes not induced by cisplatin but downregulated by AIBP (compound 7).
  • F Pathway and GO enrichment analysis of all genes downregulated by AIBP (compound 7) using adjusted P ⁇ 0.05 and absolute fold change >1.5 and a minimum overlap of 3 genes in a pathway.
  • G Heatmap of non-inflammatory genes downregulated by AIBP (compound 7) included in the most enriched pathway: peptidase inhibitor activity pathway.
  • H Heatmap of genes whose downregulation in CIPN was reversed by AIBP (compound 7). Mean ⁇ S.E.M.; * P ⁇ 0.05 comparing to naive group and cisplatin/i.t. saline group.
  • Figure 5 demonstrates that the cholesterol transporters ABCAl and ABCGl are necessary for AIBP-mediated reversal of pain in a model of mouse CIPN:
  • FIG. 5 ABCAl and ABCGl expression in microglia controls nociception and is required for AIBP (compound 7)-mediated reversal of allodynia in a mouse model of CIPN.
  • A-B, BV-2 cells were incubated for 30 min with AIBP (compound 7) (0 2pg/mL) or vehicle in complete media, followed by a 5 min incubation with LPS (lOOng/mL).
  • Scale bar, 7pm Bar graphs show Manders’ tMl coefficient.
  • TAM Tamoxifen
  • cisplatin 2.3mg/Kg
  • AIBP compound 7
  • saline 5m1
  • D Baseline (day 0) withdrawal thresholds before the start of cisplatin intervention.
  • G-H Withdrawal thresholds following i.p.
  • FIG. 6 Gene expression in spinal microglia of ABC-imKO mice.
  • RNA-seq data sets from ABC- imKO and WT (not littermates) mice were acquired in the same experiment.
  • A Top: Overlapping genes and pathways induced in naive ABC-imKO microglia and shared with WT microglia in mice treated with cisplatin, showed in purple lines connecting overlapping genes and in blue lines connecting the overlapping enriched pathways.
  • Bottom Venn diagram of upregulated genes in spinal microglia from WT cisplatin and ABC-imKO naive mice.
  • B Enrichment pathway analysis of up and down regulated genes induced by ABCAl and ABCGl knockdown in microglia, using cutoff E ⁇ 0.05, enrichment >1.5 and a minimum overlap of 3 genes in the pathway.
  • C DEGs in naive spinal microglia of TAM-induced ABC-imKO mice. Adjusted ⁇ 0.05 and Benjamini- Hochberg FDR ⁇ 5%.
  • D Overlapping genes and pathways induced by cisplatin treatment in ABC-imKO microglia and shared with WT microglia in mice treated with cisplatin.
  • E DEGs in spinal microglia of cisplatin-treated, TAM-induced ABC-imKO mice compared to cisplatin-treated WT mice. Adjusted P ⁇ 0.05 and Benjamini-Hochberg FDR ⁇ 5%.
  • F-G Heatmap of DEGs upregulated (F) or downregulated (G) in ABC-imKO microglia either in naive or cisplatin condition.
  • Figure 7 Compares gene expression of wild type and ABC knockout mice treated with an AIBP protein (compound 7) as provided herein:
  • FIG. 7 Microglial reprogramming by AIBP is dependent on ABCAl/ABCGl expression.
  • A Venn diagram comparing the effect of AIBP treatment on gene expression in WT and ABC-imKO mice in which CIPN was induced by cisplatin.
  • B Volcano plot representation of up and down regulated genes by AIBP treatment in CIPN comparing AIBP effect on ABC-imKO vs. WT mice. Cutoff of adjusted P ⁇ 0.05 and absolute fold change >1.5 shown in light green dots.
  • C Heatmap of log2 normalized gene counts of inflammatory genes altered by AIBP in an ABC-dependent manner (downregulated by AIBP in WT microglia but upregulated by AIBP in ABC-imKO.
  • D Heatmap of cholesterol synthesis and LXR related genes comparing cisplatin and AIBP effect in wild type and ABC-imKO.
  • E Heatmap of non-inflammatory genes regulated by AIBP in an ABC-dependent manner.
  • F Enrichment pathway analysis of upregulated genes by AIBP in ABC-imKO microglia, using cutoff ⁇ 0.05, enrichment >1.5 and a minimum overlap of 3 genes in the pathway.
  • Figure 8 demonstrates that knockout of either AIBP or TLR4 contributes to pain behavior (nociception):
  • FIG. 8 Endogenous AIBP and TLR4 in microglia are important in nociception.
  • A Experimental design and timeline. Tamoxifen (TAM, 10 mg/mL, 200pL/day); cisplatin (2.3 mg/kg/day); AIBP (compound 7) (0.5pg/5pl); saline (5m1).
  • D-F Withdrawal thresholds following i.p. cisplatin and i.t.
  • Figure 9 Identifies sequence motifs that contribute to AIBP binding to TLR4 : Figure 9 Identification of the domain in the AIBP molecule responsible for TLR4 binding.
  • A Human AIBP: signal peptide (aa 1-24), previously uncharacterized N- terminal domain (aa 25-51), and YjeF N domain (aa 52-288).
  • B Flag-tagged deletion mutants of human AIBP were co-expressed in HEK293 cells with the Flag-tagged TLR4 ectodomain (eTLR4). Cell lysates were immunoprecipitated (IP) with an anti-TLR4 antibody and immunoblotted (IB) with an anti-Flag antibody.
  • IP immunoprecipitated
  • IB immunoblotted
  • C His-tagged human (hu), mouse (mo) and zebrafish (zf) AIBP, all lacking the signal peptide, expressed in a baculovirus/insect cell system, were combined in a test-tube with eTLR4-His, followed by IP with an anti-TLR4 antibody and IB with an anti -His antibody.
  • D-H Binding of His- tagged wild type (wt, 25-288 aa) and the deletion mutant (mut, 52-288 aa) human AIBP to eTLR4, APOAl and microglia.
  • IP of eTLR4 and wtAIBP or mutAIBP in a test tube with an anti-AIBP antibody blot and quantification from 3 independent experiments (D).
  • ELISA with plates coated with eTLR4 and incubated with wtAIBP or mutAIBP (n 3)
  • FIG. 10 Intrathecal delivery of AIBP lacking the TLR4 binding domain cannot alleviate CIPN allodynia.
  • G Diagram illustrating the effect of CIPN and AIBP (compound 7) treatment on microglia gene expression and lipid droplet accumulation. Black dots in the plasma membrane and the ER depict cholesterol.
  • Figure 11 postulates a model for exposure of the TLR4 binding site of AIBP in a modified AIBP sequence: Figure 11.
  • Model of unfolding or exposing a cryptic N-terminal domain in the AIBP molecule The diagram summarizes and illustrates results of experiments shown in Figs. 12-14, which demonstrate that in native AIBP the N-terminal domain (green) is hidden or cryptic or not sufficiently exposed to mediate AIBP binding to TLR4 (top panel). Extending the N-terminus with additional amino acids (orange) changes the AIBP conformation and makes the N-terminal domain of AIBP (green) accessible for TLR4 binding (bottom panel).
  • Figure 12 One example of the amino acid sequence of an exemplary engineered AIBP as provided herein.
  • FIG. 13 TLR4 binding of various engineered forms of AIBP. All proteins were expressed and purified from a baculovirus/insect cell system.
  • the top drawing for His- d24AIBP corresponds to the amino acid sequence shown in Fig. 12.
  • the amino acid sequence below the top drawing shows the sequence of the orange box “cleavable His tag.” All other drawings show different modifications and corresponding changes in the amino acid sequence introduced to the AIBP molecule.
  • the green “N-terminal domain” box depicts the amino acid 25-51 sequence of native AIBP.
  • Figure 14 demonstrates TLR4 binding of certain modified AIBP sequences from a mammalian expression system:
  • TLR4 binding of various engineered forms of AIBP continued 1. All proteins were co-expressed with the full-length TLR4 in a mammalian system. SS, secretion signal, corresponding to the amino acids 1-24 in the human AIBP sequence. The column on the right shows the results of co-immunoprecipitation from cell lysates of the AIBP variants with TLR4.
  • FIG. 15 Various AIBP constructs to optimize the structure for TLR4 affinity: baculovirus/insect cell expression system.
  • Figure 16 confirms that N-terminal modification to AIBP is necessary for TLR4 binding in an E. coli expression system:
  • TLR4 binding of various engineered forms of AIBP continued 2. All proteins were expressed and purified from an E.coli. The column on the right shows the results of co-immunoprecipitation experiments of the AIBP variants with a recombinant ectodomain of TLR4, there was no TLR4 binding using the AIBP variants d24 AIBP-his or d51 AIBP-his
  • FIG. 17A-D (or, Fig. SI, or supplementary figure 1) provides validation of the specificity of TLR4 antibodies used for flow cytometry and microscopy, and also shows TLR4 dimerization and lipid rafts measured in dorsal root ganglia macrophages:
  • FIG. 17A graphically illustrates flow cytometry of single cell suspensions from spinal cords of WT (left images) and Tlr4-/- mice (right images) showing TLR4-APC and TLR4/MD2-PE antibodies staining of CDllb+(PercP-Cy5.5)/TMEM199+(Pe-Cy7) microglia;
  • FIG. 17B illustrates confocal images of peritoneal elicited macrophages from WT and Tlr4-/ ⁇ mice co-stained with F4/80-FITC and TLR4-647 antibodies; Scale bar, 5 pm; and
  • FIG. 18A-E shows FACS sorting strategy for spinal microglia, quality controls and phenotypic controls for RNA-seq:
  • FIG. 18A illustrates sorting strategy for lumbar CD1 lb+TMEMl 19+ spinal microglia, including: SSC-A and FSC-A, SSC-W and SSC-H, UVE/DEAD (APC-Cy7-A) and SSC-A, GLAST1 and CD24, and, CDllb and TMEM119;
  • FIG. 18B illustrates flow cytometry analysis of sorted microglia measuring purity of sorted cells and absence of GLAST1+ astrocytes or CD24+ neurons, including TMEM1 19 and CD1 lb, SSC-A and GLAST1, and SSC-1 and CD24;
  • FIG. 18C illustrates microglial linage analysis with a heatmap of microglia specific genes. Log+1 of normalized counts from all samples was calculated for the 40 microglia specific genes listed in Butovsky et. al, 2014, as well as for the 3 genes that are expressed at low levels in microglia but at high levels specifically in neurons ( Nefl ), oligodendrocytes ( Omg ) or astrocytes ( Slc6al );
  • FIG. 18D-E illustrate heatmaps of CIPN-repressed genes that were up-regulated by AIBP (group 4) (FIG. 18D) and CIPN-induced genes that were downregulated by AIBP (group 3) in wildtype mice (FIG. 18E); Log2 normalized gene counts scaled by row and columns represent all technical replicates of the 3 biological samples.
  • FIG. 19A-D (or, Fig. S3, or supplementary figure 3) provide immunohistochemical validation of conditional knockout of ABCAl and ABCGl in spinal microglia of tamoxifen-induced ABC-imKO mice:
  • IHC of spinal cord frozen sections from vehicle and tamoxifen induced ABC- imKO mice showing colocalization of ABCAl and ABCGl staining with IBA1 (microglia), NeuN (neurons) and GFAP (astrocytes).
  • Slides were mounted with Prolog Gold with DAPI.
  • Confocal images were acquired with a 63x objective and analyzed with ImageJ software for colocalization.
  • Colocalization masks and Pearson’s R-values, Manders’ colocalization coefficients above threshold and randomization Costes P values were calculated as described in Methods for at least 1 slide for each animal in the experiment. Representative images and values shown correspond to one animal per condition. Scale bar, 50 pm.
  • FIG. 20A-E shows tactile allodynia data for tamoxifen-treated WT mice in i.t. LPS and CIPN experiments. It also provides additional RNA-seq data for ABC-imKO dependent genes and the cisplatin effect on ABC-imKO vs. WT mice.
  • TAM tamoxifen regimen
  • FIG. 20A i.t. injection of AIBP (0.5pg/5pL) or saline (5pL) and i.t.
  • FIG. 20D Heatmap of differentially regulated genes across all conditions (naive, induced by cisplatin/saline or cisplatin/ AIBP) regulated in an ABC-imKO manner.
  • FIG. 20E Heatmap of pathway enrichment of cisplatin upregulated genes in WT and ABC-imKO microglia using cutoff C ⁇ 005, enrichment >1.5 and a minimum overlap of 3 genes in the pathway. Heatmap depicts common and specific pathways enriched by cisplatin in both genotypes.
  • FIG. 21 (or, Fig. S5, or supplementary figure 5) provides immunohistochemical validation of AIBP knockout in spinal microglia of tamoxifen-induced AIBP-imKO mice. It also demonstrates that the BE-1 monoclonal antibody has similar affinity to wtAIBP and mutAIBP.
  • FIG. 21A IHC of spinal cord frozen sections from vehicle and tamoxifen induced AIBP-imKO mice, showing colocalization of AIBP staining with IBAl (microglia), NeuN (neurons) and GFAP (astrocytes). Slides were mounted with Prolog Gold with DAPI. Confocal images were acquired with a 63x objective and analyzed with ImageJ software for colocalization.
  • FIG. 2 IB Sandwich ELISA using BE-1 as a capture antibody in a microtiter plate. Dose response curves to wtAIBP and mutAIBP were detected using a rabbit polyclonal anti-AIBP antibody. No statistical differences were found for BE-1 affinity to wtAIBP and mutAIBP using two-way ANOVA with Bonferroni post hoc test for multiple comparisons.
  • a pulldown assay was performed using compounds as provided herein.
  • Compounds 3, 7, 8 or 9 and other constructs described in Figures 13 and 14 were purified from either a baculovirus (BD Bioscience) or CHO (ExpiCHO, Expression Systems, ThermoFisher) cell expression system and incubated with TLR4 protein (Sino biological). Pull-down was performed with anti-AIBP antibody described. Bound TLR4 to AIBP was detected by western blot with an anti-his antibody (both modified AIBP and TLR4 have his-tag). Detailed experimental information is provided in example 1 as to pull down method.
  • Example 3 Efficacy demonstrated in exemplary models: asthma Reduced AIBP expression in bronchial epithelial cells of asthmatic patients:
  • Apolipoprotein A-I binding protein (AIBP; gene nam e APOA1BP or NAXE) is a secreted protein (1), which facilitates removal of excess cholesterol from activated cells, including primary alveolar macrophages, endothelial cells, and microglia (2-4).
  • AIBP bronchoalveolar lavage fluid
  • BALF bronchoalveolar lavage fluid
  • AIBP is secreted into BALF (4).
  • AIBP facilitates mitophagy, helps maintain mitochondrial function and reduces oxidative stress in macrophages (6).
  • the hypothesis that AIBP expression serves to protect against inflammation implies that raising AIBP levels in the lung may have a therapeutic effect.
  • Compound 7 was administered 2 hours before the administration of HDM.
  • intranasal HDM intranasal HDM in female mice induce lung eosinophilic inflammation and the airway hyperresponsiveness (AHR) to methacholine challenge (8).
  • Two doses of Compound 7, 2.5 and 25 pg, or vehicle (PBS) were administered to 8-week-old C57BL/6J female and male mice weekly, via intranasal instillation, 2 hours before the intranasal HDM.
  • Intranasal Compound 7 produced no apparent adverse effects.
  • ICS inhaled corticosteroids
  • bronchial epithelial cells were isolated from bronchi of postmortem lungs. In brief, bronchi were dissected, and the interior of each bronchus was scraped with a Cell Lifter (Corning, Inc.) to obtain bronchial epithelial cells. The bronchial epithelial cells were collected and cultured in CnT-17 media (Cellntec, Bern, Switzerland). These primary bronchial epithelial cells were of >95% pure as assessed by E-cadherin expression by flow cytometry. Human and mouse lung immunohistochemistry
  • Paraffin-embedded lung sections were stained using a cocktail of mouse anti-human and anti-mouse AIBP monoclonal antibodies A7 and BE-1 developed in our lab (6, 7) and mixed at 1 :2 ratio. Due to close homology of mouse and human AIBP, both antibodies recognize the mouse and the human protein. Quantification of AIBP-positive staining in epithelial cells was performed for each lung section using an image analysis system (Image-Pro plus, Media Cybernetics), and results were expressed as AIBP-positive area of bronchial epithelium per pm length of the bronchial basal membrane in human specimens.
  • AIBP expression in the mouse lung was measured using a mean grey value tool in Image J (NIH), and the values in the cytosol of bronchial epithelium of bronchiole with a 150-200 pm internal diameter were normalized to that in adjacent alveolae. The operators were blinded to the identity of samples.
  • NASH image J
  • RNA from each cell sample was processed for RT-qPCR as previously described (8).
  • samples were treated with RNA-STAT-60 (TelTest), and reverse-transcribed with Oligo-dT and Superscript II kit (Life Technologies).
  • qPCR was performed with TaqMan PCR Master Mix and TaqMan primers for human APOA1BP (Hs.PT.58.22278956, Integrated DNA Technologies, Coralville, IA).
  • the relative amounts of APOA1BP mRNA were normalized to those of the housekeeping gene hypoxanthine phosphoribosyltransf erase- 1 ( HPRT1 ).
  • Compound 7 was expressed in a baculovirus/insect cell system to ensure posttranslational modification and endotoxin-free preparation and purified by affinity chromatography using a Ni-NTA agarose column, followed by ion exchange chromatography and buffer replacement.
  • the product was greater than 90% pure, with no detectable aggregates (HPLC-SEC) and residual endotoxin less than 0.2 EU/mg.
  • BAL was collected by lavage of 1 ml PBS via tracheal catheter, centrifuged and the pellet was resuspended in 1 ml PBS.
  • differential cell counts were quantified in Wright-Giemsa stained slides(8).
  • Lung eosinophil counts were quantified in the peribronchial space in lung paraffin-embedded sections stained with an anti-mouse major basic protein (MBP) rabbit polyclonal antibody (kindly provided by Mayo Foundation for Medical Education and Research). Results are expressed as the number of peribronchial cells staining positive per bronchiole with a 150-200 pm internal diameter. At least 5 bronchioles were counted in each slide. The operator was blinded to the identity of samples.
  • MBP major basic protein
  • Figure 22 Reduced AIBP expression in bronchial epithelium.
  • RFT1081 reduces airway hyperresponsiveness and eosinophilic pulmonary inflammation in an acute HDM model of asthma in female and male mice.
  • AAV-AIBP protects retinal ganglion cells and their axons and improves visual function in experimental glaucoma:
  • Glaucomatous DBA/2 J ( D2 ) mouse model The advantage of using the genetic D2 model, together with age-matched non-glaucomatous control D2 -Gpnmb + mice, is that it replicates the chronic IOP elevation of human glaucoma, with retinal pathology developing with age, at around 9-10 months (1, 2).
  • D2 has its limitations as the glaucoma-like pathology develops in these mice secondary to anterior segment anomalies with synechiae and pigment dispersion (1, 2).
  • we observed significantly elevated cholesterol content in the retina of Apoalbp / compared with WT mice see Fig. 24A), suggesting that AIBP deficiency induces excessive cholesterol accumulation in the retina.
  • AAV-Null or AAV-AIBP we intravitreally injected AAV-Null or AAV-AIBP at the age of 5 months and analyzed tissue samples (retina, optic nerve head and brain) at the age of 10 months.
  • RBP RNA-binding protein with multiple splicing
  • NF68 neurofilament 68
  • CTB cholera toxin subunit B
  • SC superior colliculus
  • Microbead-induced ocular hypertension model Recently, we successfully developed a mouse model of microbead-induced ocular hypertension, which showed a significant loss of RGCs at 6 weeks post procedure in 4-mo-old C57BL/6J mice (Fig. 25). To further validate the protective effects of AAV-AIBP on RGCs and visual function in vivo , we intravitreally injected AAV-Null or AAV-AIBP 3 weeks before the microbead injection. AAV-AIBP significantly reduced RGC death (see Fig. 25C) and importantly, ameliorated visual dysfunction (see Figure 25D).
  • ONC Oytic nerve crush
  • AAV-delivered AIBP expression reduces cholesterol content, protects RGC and their axons, inhibits microglial activation (not shown), and preserves visual function.
  • FIG 24 AAV-AIBP reduces retinal neurodegeneration in glaucomatous DBA/2J (D2) mice.
  • a and B Apoalbp mice.
  • Filipin staining for cholesterol A
  • Quantification of filipin intensity in the inner retina B
  • C-M Glaucomatous D2 mice.
  • Filipin staining for cholesterol C
  • Quantification of filipin intensity in the inner retina D
  • Confirmation of AIBP expression in the retina by immunoblot with anti-His antibody E).
  • IOP measurements F).
  • RBPMS green
  • RGCs Quantitative analysis of RGC survival in the middle and peripheral retinas (H).
  • FIG. 25 AAV-AIBP reduces retinal neurodegeneration and improves visual function in a microbead-induced hypertension mouse model.
  • A IOP time course in microbead- injected eyes.
  • B Representative images from the peripheral area of the retina by TUJ1 staining at 6 weeks after microbead injection.
  • C Quantitative analysis of RGC survival in the middle area of the retina.
  • D Visual function measurement by PERG analysis.
  • FIG. 26 AAV-AIBP reduces retinal neurodegeneration and a mouse optic nerve crash model.
  • A Representative images for RBPMS-positive RGCs in the middle area of the retina following ONC injury;
  • TLR4 toll-like receptor-4
  • Apolipoprotein A-I attenuates palmitate-mediatedNF- kappaB activation by reducing Toll-like receptor-4 recruitment into lipid rafts.
  • AIBP Apolipoprotein A-I Binding Protein Regulates Oxidized LDL (Low-Density Lipoprotein)-Induced Mitophagy in Macrophages. Arterioscler Thromb Vase Biol 0, ATVBAHA.120.315485. Choi, S.H., etal. (2018). AIBP augments cholesterol efflux from alveolar macrophages to surfactant and reduces acute lung inflammation. JCI Insight 3, el20519.
  • MultiQC summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047-3048.
  • AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate. Science 363, 1085-1088.
  • mice Hylden, J.L., and Wilcox, G.L. (1980). Intrathecal morphine in mice: a new technique. Eur J Pharmacol 67, 313-316.
  • Chemotherapy-induced peripheral neuropathy in a dish dorsal root ganglion cells treated in vitro with paclitaxel show biochemical and physiological responses parallel to that seen in vivo. Pain 162, 84-96.
  • Lipid rafts in glial cells role in neuroinflammation and pain processing. J Lipid Res 61, 655-666.
  • TREM2 Regulates Microglial Cholesterol Metabolism upon Chronic Phagocytic Challenge. Neuron 105, 837-854 e839.
  • Apolipoprotein A-l binding protein promotes macrophage cholesterol efflux by facilitating apolipoprotein A-l binding to ABCA1 and preventing ABCA1 degradation.

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EP4203987A4 (en) * 2020-08-25 2024-10-16 The Regents of The University of California COMPOSITIONS AND METHODS FOR THE TREATMENT OF OCULAR NEUROINFLAMMATION
WO2025076208A1 (en) * 2023-10-03 2025-04-10 Raft Pharmaceuticals, Inc. Compositions and methods for treating pain disorders
WO2026006723A1 (en) * 2024-06-27 2026-01-02 Anand Rene Reagents and methods for treating headache and migraine pain and co-morbidities thereof

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