Compositions and methods for targeting inflammatory or activated cells and treating or ameliorating inflammatory conditions and pain - Patents.com

Abstract

Methods are provided for modifying amino acid sequences and increasing expression levels of ApoA-I binding proteins to treat neuropathic pain, CNS inflammation, allodynia, post-nerve injury pain, post-operative pain, chemotherapy-induced peripheral neuropathy, neurodegeneration, including neurodegenerative diseases or conditions such as Alzheimer's disease, hyperalgesia, primary headaches such as migraine and cluster headaches, glaucoma or other ocular inflammatory diseases, pulmonary inflammation, asthma, HIV infection, vascular inflammation, atherosclerosis and cardiovascular disease.

Description

[Technical field]

[0001] Related Applications This PCT international application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. (USSN) 63 / 162,714, filed March 18, 2021. The foregoing application is expressly incorporated by reference herein in its entirety for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated by reference herein for all purposes. Government Rights

[0002] This invention was made with Government support under Grants NS102432, NS104769, HL135737, AI147879 and HL136275 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention. Technical Field

[0003] The present invention generally relates to medicine, inflammation, pain management and cell biology.In particular, in alternative embodiments, a method is provided for modifying the structure and increasing the expression level of ApoA-I binding protein (APOA1BP, AIBP or AI-BP, also known as NAD(P)HX epimerase or NAXE) to treat, ameliorate, prevent, reverse, or reduce the severity and / or duration of neuropathic pain, CNS inflammation, allodynia, post-nerve injury pain, post-operative pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced allodynia), neurodegeneration, including neurodegenerative diseases or conditions such as Alzheimer's disease, hyperalgesia, primary headaches such as migraine and cluster headache, glaucoma, pulmonary inflammation and asthma, HIV infection and its comorbidities, and / or vascular inflammation and cardiovascular disease. In alternative embodiments, methods are provided for treating, ameliorating, preventing, reversing, reducing the severity and / or duration of neuropathic pain, allodynia, hyperalgesia, neurodegenerative diseases or conditions such as Alzheimer's disease, primary headaches such as migraine, glaucoma or other ocular inflammatory diseases, pulmonary inflammation and asthma, acute respiratory distress syndrome (ARDS), sepsis, influenza, viral infections including coronavirus (e.g., COVID-19) or HIV infection, or co-morbidities thereof, and / or vascular inflammation, atherosclerosis and cardiovascular disease, comprising structural modification and administration of formulations and pharmaceutical compositions comprising a recombinantly modified APOA1BP polypeptide or protein, or a peptidomimetic or synthetic APOA1BP, or a biological equivalent thereof, that is a human or mammalian APOA1BP. [Background technology]

[0004] background Apolipoprotein AI-binding protein or ApoA-I-binding protein (AIBP), also called NAXE, NAD(P)HX epimerase, is a protein discovered in a screen for proteins that physically associate with Apo AI.

[0005] Regulation of cholesterol metabolism in the context of neurodegeneration, especially Alzheimer's disease (AD), has received ample attention due in part to the strong association of APOE gene polymorphisms with AD risk. However, the role of cholesterol regulation as a factor in the development of chronic pain conditions remains unknown. Chemotherapy-induced peripheral neuropathy (CIPN) is one of the debilitating adverse effects of antineoplastic drug use during cancer treatment, affecting more than 50% of patients undergoing chemotherapy (Seretny et al., 2014). Neuroinflammation mediated by glial cell activation and infiltrating immune cells in the spinal cord and dorsal root ganglia is a key component of CIPN and other neuropathies (Lees et al., 2017; Makker et al., 2017). Glial cells express Toll-like receptor 4 (TLR4), which mediates the secretion of inflammatory cytokines, chemokines and bioactive lipids (Bruno et al., 2018; Gregus et al., 2018; Papageorgiou et al., 2016). Furthermore, CIPN-associated activation of TLR4 signaling has been reported in dorsal root ganglion nociceptors (Chen et al., 2017; Li et al., 2021). Systemic deficiency of TLR4 or its signaling adaptor molecules MyD88 and TRIF, alone or in combination, reduced the CIPN-associated activation of TLR4 in dorsal root ganglion nociceptors (Chen et al., 2017; Li et al., 2021). It attenuates and prevents hyperalgesia and allodynia in treated mice ( Hu et al., 2018 ; Pevida et al., 2013 ; Yan et al., 2019 ), however, the cell type in which TLR4 activation induces allodynia is unknown. Summary of the Invention

[0006] overview In an alternative embodiment, an isolated or recombinant polypeptide, or a chimeric polypeptide is provided, the polypeptide consisting of (or including) an ApoA-I binding protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence, The amino acid sequence on the N-terminal side of the AIBP amino acid sequence is composed of at least 8 amino acids, or the amino acid sequence on the N-terminal side of 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; an amino acid sequence N-terminal to the AIBP amino acid sequence is capable of inducing unfolding of, exposing or otherwise making accessible a cryptic domain in the AIBP amino acid sequence for binding of the polypeptide to TLR4 under relevant physiological conditions; Optionally, "relevant physiological conditions" refers to the relevant physiological conditions experienced by a polypeptide compound in vivo when provided to a subject in need thereof by administration; However, the amino acid sequence on the N-terminal side of the AIBP amino acid sequence is not composed of a His tag and a protein cleavage site that will result in the loss of the His tag when acted on under the above-mentioned conditions.

[0007] In an alternative embodiment of the isolated or recombinant polypeptide, or chimeric polypeptide provided herein, -The amino acid sequence on the N-terminus of the AIBP amino acid sequence is composed of about 8 to about 40 consecutive amino acid residues (or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive amino acid residues), of which about 3 to about 12, or about 8 to about 20, or about 10 to about 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 includes a secretion signal amino acid sequence, optionally the secretion signal amino acid sequence is (or includes) 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, optionally the fibronectin secretion signal domain is MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 24): - the AIBP sequence is hAIBP (SEQ ID NO: 6, or encoded by SEQ ID NO: 5) or d24hAIBP (SEQ ID NO: 21, encoded by SEQ ID NO: 20); - the amino acid sequence on the N-terminus of the AIBP amino acid sequence is composed of about 6, or between about 5 and 40, consecutive histidine amino acid residues (e.g., HHHHHH (SEQ ID NO: 1)) on the N-terminus of the TLR4-binding domain of the AIBP amino acid sequence; - the polypeptide has (or comprises) a thrombin cleavage domain interposed between the N-terminus of the TLR4 binding domain of the ApoA-I binding protein sequence, the thrombin cleavage domain having one or more amino acid deletions and / or mutations within said domain that render it functionally inoperable; The amino acid sequence on the N-terminal side of the -AIBP amino acid sequence is MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) or MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), each having an amino acid mutation in its thrombin cleavage domain that renders it functionally inoperable; The amino acid sequence of the N-terminal side of the AIBP amino acid sequence is TETGKSKR (SEQ ID NO: 26). MDYKDHDGDYKDHDIDYKDDDDKLAAANS (SEQ ID NO: 33), or Selected from the group consisting of MSPIDPMGHHHHHHGRRRASVAAGILVPAASPGLDGICSR (SEQ ID NO: 7); - the AIBP amino acid sequence is (or is derived from) a mammalian AIBP amino acid sequence, optionally the mammalian AIBP amino acid sequence is (or is derived from) a human AIBP amino acid sequence; and / or - the human AIBP amino acid sequence is (or includes) the full-length amino acid sequence of 288 amino acid residues having NCBI Reference Sequence: NP_658985.2, or optionally the human AIBP amino acid sequence is (or includes) the human AIBP amino acid sequence having NCBI Reference Sequence: NP_658985.2, in which amino acids 1 to 24 are deleted from said AIBP amino acid sequence.

[0008] In alternative embodiments, there is provided a pharmaceutical composition or formulation comprised of (or including) a polypeptide compound provided herein and at least one excipient suitable for (formulated for) parenteral administration, hi alternative embodiments, parenteral administration is by intrathecal injection or implant, or by intravenous or intraocular injection.

[0009] In an alternative embodiment, a nucleic acid is provided that consists of (or comprises) a nucleic acid sequence that encodes a polypeptide provided herein.

[0010] In an alternative embodiment, an expression vector is provided that is composed of (or comprises or has) a nucleic acid sequence encoding a polypeptide as provided herein. The expression vector may be a recombinant virus, such as a recombinant adenovirus or a recombinant lentivirus.

[0011] In alternative embodiments, methods and uses are provided for treating, ameliorating, preventing, reversing, or reducing the severity or duration of, or reducing the severity of, the following conditions: -Neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain includes Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - Neurological or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. -Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the pain following nerve or tissue injury or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection; - postoperative or neuropathic pain, Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, as appropriate, a chronic or progressive neurodegenerative disease or condition, as appropriate, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS); - Primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - Glaucoma or other ocular inflammatory disease, - Pulmonary inflammation and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - viral infection, where appropriate the virus is influenza or a coronavirus (where appropriate, the coronavirus is COVID-19) or human immunodeficiency virus (HIV) or a virus that causes HIV infection, (where appropriate, influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), Cytomegalovirus (CMV), Rubivirus or rubella virus, Enterovirus, viral meningitis, rhinovirus, varicella zoster virus or chickenpox virus, Orthopoxvirus or smallpox or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or a comorbidity thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, (by adding an ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) or by increasing the level of an ApoA-I binding protein in the subject); wherein the method includes: (a) providing a formulation or pharmaceutical composition comprising: (i) has a heterologous (or non-native, or non-AIBP, or non-wild-type (wt), or any sequence not present in wild-type (wt) AIBP) amino-terminal amino acid sequence of at least about 10 amino acids, or between about 5 and 20 amino acids, or between about 10 and 100 amino acids, or between about 20 and 80 amino acids, or between about 30 and 50 amino acids; 2. A recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition having 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 at the amino terminus of AIBP that are not present in AIBP or are non-native (to AIBP) amino acid residues or peptides (also referred to as AIBP variants as provided herein), Optionally, the amino acid sequence N-terminal to the AIBP amino acid sequence does not consist of a His tag and a proteolytic cleavage site that, when acted upon under physiological conditions (e.g., in a cellular environment or equivalent, or inside a cell), results in the loss of the His tag; Optionally, the heterologous (or non-wild-type, or non-naturally occurring) amino-terminal amino acid sequence (or amino acid residue) comprises a peptide tag, optionally wherein the peptide tag comprises a multi-histidine (multi-his) tag, optionally wherein the multi-his tag comprises 6 histidines (HHHHHH (SEQ ID NO: 1)), or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more histidine residues; Optionally, the heterologous (or non-wild-type) amino-terminal amino acid sequence comprises an enzymatic cleavage site, optionally, the enzymatic cleavage site comprises a thrombin cleavage site; Optionally, the heterologous (or non-wild-type) amino-terminal amino acid sequence comprises a secretory signal, optionally comprising a fibronectin secretory signal (e.g., SEQ ID NO: 24), an immunoglobulin heavy chain secretory signal or an immunoglobulin kappa light chain secretory peptide, or an interleukin-2 signal peptide; Optionally, the heterologous (or non-wild type) amino-terminal amino acid sequence comprises the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO:2); wherein all of these AIBP variants provided herein (or all AIBP amino acids, including amino-terminal sequences not present in wt AIBP or including heterologous amino-terminal peptides or amino acid residues) are capable of unfolding, exposing, or making accessible, under physiological conditions, a potential domain within the AIBP molecule including amino acids 25-51 that mediates binding of AIBP to a Toll-like receptor 4 (TLR4) polypeptide (in other words, the AIBP variants provided herein have a TLR4-binding domain exposed to the extracellular environment such that the AIBP variants provided herein can bind to a TLR4 polypeptide under physiological conditions), and a recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition; (ii) a recombinant nucleic acid encoding the APOA1BP polypeptide of (i), Optionally, the nucleic acid expressing or encoding an APOA1BP polypeptide or a polypeptide having APOA1BP polypeptide activity is included in an expression vehicle, vector, recombinant virus, or equivalent, Optionally, the vector or virus is or comprises an adenoviral or adeno-associated viral (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector; Optionally, the AAV vector comprises or is: Adeno-associated virus (AAV), or adenovirus vectors, AAV serotypes or variants AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ / 8™ (Cell Biolabs, Inc., San Diego, Calif.); Rhesus monkey-derived AAV, or the rhesus monkey-derived AAV AAVrh.10hCLN2, AAV capsid mutants or AAV hybrid serotypes, an organ-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41 mutant; wherein, optionally, the AAV has been engineered to increase the efficiency of targeting a particular cell type that is non-permissive to wild-type (wt) AAV and / or to improve the efficiency of infecting only a cell type of interest; Optionally, the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications, including: 1) transencapsidation, 2) adsorption of bispecific antibodies to the capsid surface, 3) engineering of a mosaic capsid, and / or 4) engineering of a chimeric capsid; and (iii) a formulation or pharmaceutical composition comprising a recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein of (i), or a recombinant nucleic acid of (ii), where optionally the recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein is or comprises all or a portion of a human or mammalian APOA1BP, or AIBP1 or AIBP2 sequence; (iv) a formulation or pharmaceutical composition of (iii) formulated for in vivo administration; or a formulation or pharmaceutical composition of (iii) formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, Optionally, the formulation or pharmaceutical composition, or the recombinant, peptidomimetic or synthetic APOA1BP, or a biological equivalent of APOA1BP, or a nucleic acid encoding APOA1BP, or a vector containing a nucleic acid encoding APOA1BP, is carried in a nanoparticle, particle, micelle, or liposome or lipoplex, polymersome, polyplex, or dendrimer, and optionally further comprises or expresses a cell or CNS penetrating moiety or peptide, or a CNS targeting moiety or peptide, formulated (iii); (v) providing a formulation or pharmaceutical composition comprising any of the formulations or pharmaceutical compositions of (iii)-(iv) formulated as a nanoparticle, liposome, tablet, pill, capsule, gel, geltab, liquid, powder, emulsion, lotion, aerosol, spray, lozenge, aqueous or sterile or injectable solution, or implant (e.g., intrathecal implant); (b) administering a recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein of (a)(i), or a recombinant nucleic acid of (a)(ii), or a formulation or pharmaceutical composition of (a)(iii) or (a)(iv) to a subject or individual in need thereof, where the subject or individual is a mammal, human or animal, as appropriate; thereby treating, ameliorating, preventing, reversing or reducing the severity or duration of, or reducing the severity of, the following conditions: -Neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain includes Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - Neurological or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. -Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the pain following nerve or tissue injury or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection; - postoperative or neuropathic pain, Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, as appropriate, a chronic or progressive neurodegenerative disease or condition, as appropriate, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS); - Primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - Glaucoma or other ocular inflammatory disease, - Pulmonary inflammation and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - viral infection, optionally the virus being influenza or a coronavirus (optionally the coronavirus is COVID-19) or human immunodeficiency virus (HIV) or a virus causing HIV infection, (optionally influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), Cytomegalovirus (CMV), Rubivirus or rubella virus, Enterovirus, viral meningitis, rhinovirus, varicella zoster virus or chickenpox virus, Orthopoxvirus or smallpox or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, and / or - Vascular inflammation, atherosclerosis and cardiovascular diseases.

[0012] In alternative embodiments, kits are provided that contain recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptides or proteins; recombinant nucleic acids; and / or formulations or pharmaceutical compositions for use in the methods provided herein, and, optionally, instructions for practicing the methods provided herein.

[0013] In an alternative embodiment, there is provided a use of a formulation or pharmaceutical composition provided herein in the manufacture of a medicament.

[0014] In an alternative embodiment, there is provided a use of a formulation or pharmaceutical composition provided herein in the manufacture of a medicament for treating, ameliorating, preventing, reversing or reducing the severity or duration of, or reducing the severity of, the following symptoms: -Neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain includes Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - Neurological or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. -Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the pain following nerve or tissue injury or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection; - postoperative or neuropathic pain, Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, as appropriate, a chronic or progressive neurodegenerative disease or condition, as appropriate, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS); - Primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - Glaucoma or other ocular inflammatory disease, - Pulmonary inflammation and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - viral infection, where appropriate the virus is influenza or a coronavirus (where appropriate, the coronavirus is COVID-19) or human immunodeficiency virus (HIV) or a virus that causes HIV infection, (where appropriate, influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), Cytomegalovirus (CMV), Rubivirus or rubella virus, Enterovirus, viral meningitis, rhinovirus, varicella zoster virus or chickenpox virus, Orthopoxvirus or smallpox or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or a comorbidity thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular diseases.

[0015] In alternative embodiments, there is provided a formulation, pharmaceutical composition or therapeutic combination for use in a method for treating, ameliorating, preventing, reversing or reducing the severity or duration of, or reducing the severity of, the following conditions: -Neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain includes Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - Neurological or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. -Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the pain following nerve or tissue injury or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection; - postoperative or neuropathic pain, Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, as appropriate, a chronic or progressive neurodegenerative disease or condition, as appropriate, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS); - Primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - Glaucoma or other ocular inflammatory disease, - Pulmonary inflammation and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - viral infection, where appropriate the virus is influenza or a coronavirus (where appropriate, the coronavirus is COVID-19) or human immunodeficiency virus (HIV) or a virus that causes HIV infection, (where appropriate, influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), Cytomegalovirus (CMV), Rubivirus or rubella virus, Enterovirus, viral meningitis, rhinovirus, varicella zoster virus or chickenpox virus, Orthopoxvirus or smallpox or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or a comorbidity thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, The formulation or therapeutic combination includes a formulation or therapeutic combination provided herein, The formulation or therapeutic combination is administered to an individual or patient in need thereof.

[0016] In an alternative embodiment, a method for exposing a latent (or hidden, unexposed, inaccessible) N-terminal TLR4-binding domain of an ApoA-I binding protein (APOA1BP, 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-terminal amino acid sequence of at least about 10 amino acids, or between about 5-50 amino acids, or between about 10-100 amino acids, or between about 20-80 amino acids, or between about 30-50 amino acids, or adding 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 (non-AIBP) amino acid residues or peptides to the AIBP amino terminus, Optionally, the heterologous amino terminal amino acid sequence comprises a peptide tag, optionally wherein the peptide tag comprises a multi-histidine (multi-his) tag, optionally wherein the multi-his tag comprises at least 6 histidines (HHHHHH (SEQ ID NO: 1)), or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more histidine residues; Optionally, the heterologous amino terminal amino acid sequence comprises an enzymatic cleavage site, and optionally, the enzymatic cleavage site comprises a thrombin cleavage site; Optionally, the heterologous amino-terminal amino acid sequence comprises a secretory signal, optionally comprising a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin kappa light chain secretory peptide, or an interleukin-2 signal peptide; Optionally, the heterologous amino-terminal amino acid sequence can be the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) A method is provided that includes:

[0017] In an alternative embodiment, a polypeptide compound is provided, wherein the polypeptide compound is comprised of an ApoA-I binding protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence, The amino acid sequence N-terminal to the AIBP amino acid sequence is composed of at least 8 amino acids, or between 4 and 12 amino acids, or between 5 and 10 amino acids, and the amino acid sequence N-terminal to the AIBP amino acid sequence is capable of inducing unfolding of, exposing or otherwise making accessible a cryptic domain in the AIBP amino acid sequence for binding of the polypeptide to TLR4 under relevant physiological conditions, provided that the amino acid sequence N-terminal to the AIBP amino acid sequence does not consist of a His tag and a proteolytic cleavage site that results in the loss of the His tag when acted upon under said physiological conditions.

[0018] In an alternative embodiment, a subject in need thereof is administered one or more of the following to treat, ameliorate, prevent, reverse, or reduce the severity or duration of a TLR4 mediated disease or condition: A pharma- ceutically acceptable composition comprising a polypeptide compound or a nucleic acid compound provided herein, wherein the nucleic acid sequence of the nucleic acid compound encodes the amino acid sequence of the polypeptide, comprising: A method is provided by providing 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, pulmonary inflammatory diseases or conditions, ocular inflammatory diseases and conditions, sepsis, vascular inflammatory diseases and conditions, post-traumatic stress disorder, post-traumatic war neurosis, post-traumatic stress syndrome (PTSS), and diseases and conditions resulting or caused by viral infections, or sequelae thereof.

[0019] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0020] All publications, patents, and patent applications cited in this specification are herein incorporated by reference for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing one or more color drawings will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0022] The drawings described herein are illustrative of the embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims. [Brief description of the drawings]

[0023] [Figure 1] Figure 1A-F show data demonstrating that chemotherapy-induced peripheral neuropathy alters TLR4 dimerization and lipid rafts in spinal microglia and is reversed by AIBP.

[0024] Figure 1A graphically depicts data showing the withdrawal thresholds of wild-type (WT) mice in response to intraperitoneal (ip) cisplatin (two injections of 2.3 mg / kg / day) followed by a single intrathecal (it) dose of saline (5 μl) or AIBP (0.5 μg / 5 μl); naive mice were not injected.

[0025] Figure 1B-C shows TLR4 dimerization (Figure 1B) and lipid raft content measured by CTxB staining (Figure 1C). + / TMEM119 + Data are presented graphically showing analysis of spinal cord microglial cells 24 hours after it administration of saline or AIBP, ie, on day 8 of the time course shown in FIG. 1A.

[0026] Figure 1C shows images of BV-2 microglial cells incubated with AIBP (0.2 μg / mL) or vehicle in complete medium for 30 min followed by incubation with LPS (100 ng / mL) for 5 min (left panel) and graphically depicts data showing Manders' coefficient (colocalization analysis) with or without LPS and / or AIBP (right panel).

[0027] 1E-F graphically depict data showing AIBP levels over time in the CSF (FIG. 1E) and lumbar spinal cord (FIG. 1F). These are discussed in detail in Example 1 below.

[0028] [Diagram 2] 2A-C show data showing gene expression in spinal microglia of CIPN mice.

[0029] Figure 2A-B shows the microglia (CD11b + TEMEM119 + ) from the three groups shown in FIG. 1A.

[0030] Figure 1A shows an image of a heatmap plot of DEGs across all samples.

[0031] FIG. 1B graphically depicts data showing that groups of significant DEGs were clustered based on the expression profile patterns of different treatment conditions.

[0032] FIG. 1C graphically depicts data showing pathway and GO enrichment analysis of upregulated (group 1 in the right panel) and downregulated (group 2 in the left panel) genes induced by cisplatin treatment, with upregulated pathways shown in “group 1” (red) in the right panel and downregulated pathways shown in “group 2” (blue) in the left panel. These are discussed in detail in Example 1 below.

[0033] [Diagram 3] Figure 3A-H shows data showing disease-associated microglia (DAM) and lipid-related gene expression and lipid droplets in spinal microglia of CIPN mice.

[0034] Figures 3A-C show the same groups as in Figure 2: Figure 3A shows an image of a volcano plot of up- and down-regulated genes in spinal microglia of cisplatin-treated mice versus naïve mice; Figure 3B shows an image of a heatmap representing disease-associated microglia (DAM) signature genes; Figure 3B shows an image of a heatmap of log2-normalized gene counts scaled per row showing the lipid-related gene set.

[0035] Figures 3D-H graphically depict data showing lipid droplet accumulation in spinal microglia as measured by PLIN2 immunostaining in spinal cord sections co-stained with IBA1 and DAPI; Figure 3E graphically depicts IBA1+ / PLIN2+ cells out of total IBA1+ cells per field with or without cisplatin and / or AIBP; Figure 3F graphically depicts average LD number / cell with or without AIBP; Figure 3G graphically depicts average LD size with or without cisplatin and / or AIBP; and Figure 3H graphically depicts normalized Plin2 gene counts with or without AIBP. These are discussed in detail in Example 1 below.

[0036] [Figure 4-1] 4A-H show data showing gene expression in spinal microglia of CIPN mice and the effect of AIBP.

[0037] FIG. 4A shows pathway and gene ontology (GO) enrichment analysis of CIPN up-regulated genes that were down-regulated by AIBP (see group 3 in FIG. 2B) and CIPN down-regulated genes that were up-regulated by AIBP (group 4).

[0038] Figure 4B shows differentially expressed genes (DEGs) in spinal microglia induced by itAIBP, a volcano plot of up- and down-regulated genes in cisplatin / AIBP-treated mice versus (vs.) cisplatin / saline-treated mice.

[0039] FIG. 4C shows a heatmap of group 3 inflammatory genes upregulated in CIPN and downregulated by AIBP.

[0040] [Figure 4-2] FIG. 4D graphically depicts data showing cytokine protein expression in spinal cord tissues from WT naive, cisplatin / saline, and cisplatin / AIBP groups.

[0041] FIG. 4E shows a heatmap of inflammatory genes that are not induced by cisplatin but are downregulated by AIBP.

[0042] FIG. 4F graphically depicts the pathway and GO enrichment analysis of all genes downregulated by AIBP.

[0043] FIG. 4G shows a heat map of non-inflammatory genes downregulated by AIBP that are included in the most enriched pathway, the peptidase inhibitor activity pathway.

[0044] FIG. 4H shows a heatmap of genes whose downregulation in CIPN is reversed by AIBP. These are discussed in detail in Example 1 below.

[0045] [Figure 5-1] Figure 5A-J shows data demonstrating that expression of ABCA1 and ABCG1 in microglia controls nociception and is required for the reversal of AIBP-mediated allodynia in a mouse model of CIPN.

[0046] Figure 5A-B illustrates data from BV-2 cells incubated with AIBP (0.2 µg / mL) or vehicle for 30 min in complete medium, followed by incubation with LPS (100 ng / mL) for 5 min, demonstrating colocalization of cholesterol accessible to ABCA1 (Figure 5A) and APOA1 (Figure 5B) in lipid rafts.

[0047] FIG. 5C illustrates a schematic of an exemplary experimental design and timeline of tamoxifen, cisplatin, AIBP or saline injections in mice.

[0048] FIG. 5D graphically depicts data showing baseline (day 0) withdrawal thresholds prior to the initiation of cisplatin intervention.

[0049] FIG. 5E shows CD11b expression in naive WT and ABC-imKO mice at baseline (day 0). + TMEM119 + Data showing TLR4 surface expression and dimerization and lipid rafts (CTxB) in spinal microglia are shown graphically (n=5 for TLR4 surface expression and lipid raft content analysis in both groups).

[0050] [Figure 5-2] FIG. 5F graphically depicts data showing withdrawal thresholds following it saline or AIBP (0.5 μg / 5 μl) followed by it LPS (0.1 μg / 5 μl) in TAM-induced ABC-imKO mice.

[0051] Figures 5G-H graphically show data demonstrating withdrawal thresholds following ip cisplatin and it saline or AIBP (0.5 µg / 5 µl) injections in TAM-induced ABC-imKO mice (Figure 5G) and non-induced (vehicle) ABC-imKO mice (Figure 5H).

[0052] Figures 5I-J show CD11b immunization on day 8 in the groups shown in panels 5G and 5H. + TEMEM119 + Data showing TLR4 dimerization (FIG. 5I) and lipid rafts (FIG. 5J) in spinal microglia are presented graphically. These are discussed in detail in Example 1 below.

[0053] [Figure 6-1] 6A-G show data showing expression in spinal microglia of ABC-imKO mice.

[0054] The top image in Figure 6A shows a schematic of overlapping genes and pathways induced in naïve ABC-imKO microglia and shared with WT microglia from cisplatin-treated mice with purple (dark, upper) lines connecting overlapping genes and blue (light, lower) lines connecting overlapping enriched pathways, and the bottom image in Figure 6A shows a Venn diagram of genes upregulated in spinal microglia from WT cisplatin- and ABC-imKO naïve mice.

[0055] FIG. 6B shows enrichment pathway analysis of up- and down-regulated genes induced by ABCA1 and ABCG1 knockdown in microglia.

[0056] Figure 6C shows DEGs in naive spinal microglia of TAM-induced ABC-imKO mice.

[0057] FIG. 6D shows a schematic of overlapping genes and pathways induced by cisplatin treatment in ABC-imKO microglia and shared with WT microglia from cisplatin-treated mice.

[0058] FIG. 6E shows DEGs in spinal microglia in cisplatin-treated, TAM-induced ABC-imKO mice compared to cisplatin-treated, WT mice.

[0059] [Figure 6-2] Figures 6F-G show heatmaps of DEG upregulated (Figure 6F") or downregulated (Figure 6G) genes in ABC-imKO microglia in either naïve or cisplatin conditions. These are discussed in detail in Example 1 below.

[0060] [Figure 7] 7A-F show data demonstrating that microglial reprogramming by AIBP is dependent on ABCA1 / ABCG1 expression.

[0061] FIG. 7A shows a schematic Venn diagram comparing the effect of AIBP treatment on gene expression in cisplatin-induced CIPN in WT and ABC-imKO mice.

[0062] FIG. 7B shows a schematic volcano plot representation of up- and down-regulated genes by AIBP treatment in CIPN, comparing AIBP effects in ABC-imKO and WT mice.

[0063] Figure 7C shows a schematic 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).

[0064] FIG. 7D shows a schematic heatmap of cholesterol synthesis and LXR-related genes comparing cisplatin and AIBP effects in wild type and ABC-imKO.

[0065] FIG. 7E shows a schematic heatmap of non-inflammatory genes regulated by AIBP in an ABC-dependent manner.

[0066] FIG. 7F shows a schematic of enrichment pathway analysis of genes upregulated by AIBP in ABC-imKO microglia. These are discussed in detail in Example 1 below.

[0067] [Figure 8] 8A-G show that endogenous AIBP and TLR4 in microglia are important for nociception.

[0068] FIG. 8A shows a schematic of an exemplary experimental design and timeline: tamoxifen; cisplatin; AIBP; and / or saline are injected.

[0069] FIG. 8B graphically depicts baseline (day 0 in FIG. 8A) withdrawal thresholds prior to the initiation of cisplatin intervention.

[0070] FIG. 8C shows WT and Cx3cr1-Cre mice before (naive, day −7 of panel A timeline) and after (TAM, day 0) a tamoxifen injection regimen. ERT2 The graph shows that the withdrawal threshold of (floxed gene free) mice was tested.

[0071] Figures 8D-F graphically show the withdrawal thresholds following ip cisplatin and it saline or AIBP injections in (Figure 8D) TAM-induced AIBP-imKO mice; non-induced (vehicle) AIBP-imKO mice (Figure 8E); and in-house bred whole-body AIBP knockout mice (Figure 8F).

[0072] FIG. 8G graphically depicts the withdrawal thresholds in WT and tamoxifen-induced TLR4-imKO mice following cisplatin injection. These are discussed in detail in Example 1 below.

[0073] [Figure 9-1] 9A-H show data demonstrating the identification of the domain in the AIBP molecule responsible for TLR4 binding.

[0074] Figure 9A shows a schematic representation of human AIBP with a signal peptide, amino acids (aa) 1-24, a previously uncharacterized N-terminal domain (aa 25-51), and the YjeF_N domain (aa 52-288).

[0075] Figure 9B shows an image of a PAGE separation of flag-tagged deletion mutants of human AIBP coexpressed with Flag-tagged TLR4 ectodomain (eTLR4) in HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-TLR4 antibody and immunoblotted (IB) with anti-Flag antibody.

[0076] Figure 9C shows an image of a PAGE separation of his-tagged human (hu), mouse (mo), and zebrafish (zf) AIBPs, all lacking the signal peptide, expressed in the baculovirus / insect cell system and bound to eTLR4-His in vitro, followed by IP with anti-TLR4 antibody and IB with anti-His antibody.

[0077] Figure 9D-H shows data from three independent experiments showing binding of His-tagged wild-type (wt, 25-288 aa) and deletion mutant (mut, 52-288 aa) human AIBP to eTLR4, APOA1 and microglia, as well as immunoprecipitation (IP), blot and quantification of eTLR4 with wtAIBP or mutAIBP in vitro with anti-AIBP antibody.

[0078] FIG. 9D shows a PAGE separation (left image) in which plates were coated with eTLR4 and incubated with wt or mut AIBP and ELISA was performed, and the right image graphically depicts the amount of TLR4 / AIBP in wt and mu AIBP.

[0079] FIG. 9E graphically depicts the binding of wt or mutAIBP (or no AIBP) and AIBP at immobilized eTLR4 in ELISA performed with plates coated with BSA, wtAIBP, or mutAIBP and incubated with APOA1.

[0080] [Figure 9-2] Figure 9F graphically depicts APOA1 bound to AIBP. Figure 9G graphically depicts the number of cells with APOA1 bound to AIBP using flow cytometry (upper graph), and AIBP binding (fold change) relative to wt and mutAIBP in unstimulated LPS-stimulated cells (lower graph).

[0081] FIG. 9H shows APOA1 bound to AIBP using confocal imaging, demonstrating the binding of wtAIBP and mutAIBP to BV-2 microglial cells that were unstimulated or treated with LPS for 15 min. These are discussed in detail in Example 1 below.

[0082] [Figure 10-1] 10A-G show data demonstrating that intrathecal delivery of AIBP lacking the TLR4 binding domain fails to attenuate CIPN allodynia.

[0083] 10A-B graphically show TLR4 dimerization (FIG. 10A) and lipid rafts (FIG. 10B) in BV-2 cells pretreated with wtAIBP or mutAIBP and stimulated with LPS.

[0084] FIG. 10C graphically depicts the withdrawal threshold in WT mice administered itAIBP (0.5 μg / 5 μL) or saline (5 μL) followed by itLPS.

[0085] FIG. 10D graphically depicts the withdrawal threshold in WT mice in response to ip cisplatin followed by itwtAIBP, mutAIBP, or saline.

[0086] FIG. 10E-F shows CD11b cells derived from the lumbar spinal cord of mice on day 21 of the experimental group shown in panel FIG. 10D. + / TMEM119 + Graphical representation of TLR4 dimerization in microglia (FIG. 10E) and lipid rafts (FIG. 10F).

[0087] [Figure 10-2] FIG. 10G is a schematic showing the effect of cisplatin-induced tissue damage (damage-associated molecular patterns (DAMPs)) and chemotherapy-induced peripheral neuropathy (CIPN) with AIBP treatment on microglial gene expression and lipid droplet accumulation, with black dots in the plasma membrane and ER representing cholesterol. Discussed in detail in Example 1 below.

[0088] [Figure 11] Figure 11 shows a schematic model of unfolding or exposing the cryptic N-terminal domain of the AIBP molecule; the figure summarizes the experimental results shown in Figures 12-14, demonstrating that in native AIBP the N-terminal domain (green) is hidden or cryptic or not sufficiently exposed to mediate AIBP binding with TLR4 (upper panel), whereas extending the N-terminus with additional amino acids (orange) changes the conformation of AIBP, making the N-terminal domain of AIBP (green) available for TLR4 binding (lower panel).

[0089] [Figure 12]Figure 12 shows an exemplary amino acid sequence of an engineered AIBP provided herein (SEQ ID NO: 35). The amino acid sequence of this extended AIBP molecule is shown in the lower panel of Figure 11, where blue letters are amino acids from the native AIBP sequence; green boxes are the TLR4 binding sequence (amino acids 25-51 of the human AIBP sequence); black letters and (red) boxes are added amino acids.

[0090] [Figure 13] Figure 13 shows a schematic of TLR4 binding of various exemplary engineered forms of AIBP: All proteins were expressed and purified from the baculovirus / insect cell system: His-d24AIBP: corresponds to the amino acid sequence shown in Figure 12, where the amino acid sequence shows the sequence of the "cleavable His tag" in the orange box, All other figures show various modifications and corresponding changes of the amino acid sequence introduced into the AIBP molecule, where the "N-terminal domain" box in green represents the amino acid 25-51 sequence of native AIBP, and the right column shows the results of co-immunoprecipitation experiments of AIBP variants with recombinant ectodomain of TLR4, For His-24 AIBP: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) For "truncated His-d24 AIBP", GSPGLDGICSR (SEQ ID NO: 9), For "5xD mut His-d24 AIPB", MSPI DPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), for "truncated 5xD His-d24 AIPB" GSDGDDGDDDR (SEQ ID NO: 10), for "2xD mut His-d24 AIBP" MSPI DPMGHHHHHHGRRRASVAAGILVPRGSDGDDGICSR (SEQ ID NO: 11), and for "truncated His-d24 AIBP" GSPGLDGICSR (SEQ ID NO: 9).

[0091] [Figure 14]FIG. 14 shows schematic TLR4 binding of various engineered forms of AIBP: all proteins were co-expressed with full-length TLR4 in mammalian systems: SS, secretion signal, corresponds to amino acids 1-24 in the human AIBP sequence; the right column shows the results of co-immunoprecipitation from cell lysates of AIBP variants with TLR4, MDYKDHKGKYKDHDIDYKDDDDKLAAANS (SEQ ID NO: 14) for "Flag-full length" and MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 24) for fibronectin signal peptide.

[0092] [Figure 15] TLR4 affinity: Schematic representation of various AIBP constructs to optimize the structure for the baculovirus / insect cell expression system: GSDGDDGDDDR (SEQ ID NO: 11), MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) For "PKA site + thrombin cleavage site": MGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 17) For "thrombin cleavage site": MAGILVPRGSPGLDGICSR (SEQ ID NO: 18) for "3xFLAG": GSDGDDGDDDR (SEQ ID NO: 11) for "5XD".

[0093] [Figure 16] Figure 16 shows a schematic of TLR4 binding of various engineered forms of AIBP. All proteins were expressed and purified from E. coli. The right column shows the results of co-immunoprecipitation experiments of recombinant ectodomain of TLR4 with AIBP variants.

[0094] [Figure 17] Figures 17A-D provide validation of the specificity of the TLR4 antibodies used for flow cytometry and microscopy, and also show TLR4 dimerization and lipid rafts measured in dorsal root ganglion macrophages.

[0095] FIG. 17A graphically depicts flow cytometry of single cell suspensions from the spinal cord of WT (left image) and Tlr4− / − mice (right image) showing TLR4-APC and TLR4 / MD2-PE antibody staining of CD11b+ (PercP-Cy5.5) / TMEM199+ (Pe-Cy7) microglia.

[0096] FIG. 17B shows confocal images of peritoneal derived macrophages from WT and Tlr4− / − mice co-stained with F4 / 80-FITC and TLR4-647 antibodies; scale bar, 5 μm.

[0097] 17C-D graphically depict flow cytometry analysis of CD11b+ DRG macrophage cells showing TLR4 dimerization (FIG. 17C) and lipid raft content measured by CTxB staining (FIG. 17D) 24 h after it saline or AIBP. These are discussed in Example 1 below.

[0098] [Figure 18-1] Figure 18A-E (or Figure S2, or Supplementary Figure 2) shows the FACS sorting strategy of spinal microglia, quality control and phenotype control for RNA-seq.

[0099] FIG. 18A shows the sorting strategy for lumbar CD11b+TMEM119+ 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 CD11b and TMEM119.

[0100] FIG. 18B shows flow cytometry analysis of sorted microglia measuring the purity of sorted cells, including TMEM119 and CD11b, SSC-A and GLAST1, and SSC-1 and CD24, and the absence of GLAST1+ astrocytes or CD24+ neurons.

[0101] FIG. 18C shows microglia lineage analysis using a heatmap of microglia-specific genes.

[0102] 18D-E show heat maps of CIPN-suppressed genes upregulated by AIBP (group 4) (FIG. 18D) and CIPN-induced genes downregulated by AIBP (group 3) (FIG. 18E) in wild-type mice. These are discussed in Example 1 below. [Figure 18-2] Same as above.

[0103] [Figure 19-1] Figure 19A-D provides immunohistochemical validation of conditional knockout of ABCA1 and ABCG1 in spinal microglia of tamoxifen-induced ABC-imKO mice.

[0104] FIG. 19A shows DAPI, IBA1, ABCA1, MERGE, and COLOC MASK, with and without tamixifen.

[0105] FIG. 19B shows DAPI, IBA1, ABCG1, merge, and COLOC masks with and without tamoxifen.

[0106] [Figure 19-2] FIG. 19C shows DAPI, NeuN, ABCA1, merge, and COLOC masks with and without tamoxifen.

[0107] FIG. 19D shows DAPI, GFAP, ABCA1, merge, and COLOC masks with and without tamoxifen. These are discussed in further detail in Example 1 below.

[0108] [Figure 20-1]Figure 20A-E shows tactile allodynia data from tamoxifen-treated WT mice in itLPS and CIPN experiments and provides additional RNA-seq data for ABC-imKO-dependent genes and the cisplatin effect of ABC-imKO on WT mice.

[0109] Figures 20A-B graphically depict data from in-house bred WT littermate mice, as controls for ABC-imKO mice, that were administered a tamoxifen regimen (TAM, 200 μL / day, 10 mg / mL, for 5 consecutive days) followed by (Figure 20A) an it injection of AIBP (0.5 μg / 5 μL) or saline (5 μL) and 2 hours later an it LPS (0.1 μg / 5 μL); (Figure 20B) an ip injection of cisplatin (2.3 mg / Kg) on ​​days 1 and 3, followed by an it injection of AIBP (0.5 μg / 5 μL) or saline (5 μL) on day 7.

[0110] FIG. 20C graphically depicts data from day 7 when ABC-imKO mice were injected with TAM, then cisplatin as above, followed by it saline (5 μL), AIBP (0.5 μg / 5 μL) or hp-β-CD (0.25 mg / 5 μL).

[0111] [Figure 20-2] FIG. 20D shows a heatmap of differentially regulated genes across all conditions (naive, induced by cisplatin / saline or cisplatin / AIBP) regulated in the ABC-imKO scheme.

[0112] FIG. 20D shows all significant genes obtained from a likelihood ratio test using a reduced model without the interaction term (condition:genotype).

[0113] [Figure 20-3]Figure 20E shows a heatmap of pathway enrichment of cisplatin-upregulated genes in WT and ABC-imKO microglia using a cutoff P<0.05, enrichment>1.5, and a minimum overlap of 3 genes within a pathway, which are discussed in more detail in Example 1 below.

[0114] [Figure 21] Figures 21A-B provide immunohistochemical validation of AIBP knockout in spinal microglia of tamoxifen-induced AIBP-imKO mice and demonstrate that the BE-1 monoclonal antibody has similar affinity for wtAIBP and mutAIBP.

[0115] FIG. 21A shows images of IHC of spinal cord frozen sections from vehicle and tamoxifen-induced AIBP-imKO mice showing co-localization of AIBP staining with IBA1 (microglia), NeuN (neurons) and GFAP (astrocytes).

[0116] FIG. 21B graphically depicts data from a sandwich ELISA using BE-1 as the capture antibody in a microtiter plate and a dose-response curve for wtAIBP and mutAIBP detected using a rabbit polyclonal anti-AIBP antibody. These are discussed in further detail in Example 1 below.

[0117] [Figure 22] 22A-C graphically show the reduction of AIBP expression in bronchial epithelium.

[0118] FIG. 22A graphically depicts AIBP+ bronchial epithelium in non-asthmatic and asthmatic samples.

[0119] FIG. 22B graphically depicts APOA1BP / HPRT1 mRNA in non-asthmatic and asthmatic samples.

[0120] FIG. 22C graphically depicts AIBP expression in bronchial epithelium. These are discussed in further detail in Example 3 below.

[0121] [Figure 23-1] 23A-F graphically demonstrate that compound 7 reduces airway hyperresponsiveness and eosinophilic lung inflammation in the HDM model of asthma in female and male mice, as discussed in further detail in Example 3 below. [Figure 23-2] Same as above.

[0122] [Figure 24] 24A-M show that AIPB reduces retinal neurodegeneration in D2 glaucomatous mice, as discussed in further detail in Example 4 below.

[0123] [Diagram 25] 25A-D show that AIPB attenuates retinal neurodegeneration and improves visual function in a microbead-induced hypertension mouse model, as discussed in further detail in Example 4 below.

[0124] [Figure 26] 26A-B show that AIPB attenuates retinal neurodegeneration in a mouse nerve crush model, as discussed in further detail in Example 4 below.

[0125] Like reference numbers in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0126] Reference will now be made in detail to various exemplary embodiments provided herein, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to provide the reader with a better understanding of certain details of aspects and embodiments of the invention and should not be construed as a limitation on the scope of the invention.

[0127] Detailed Description In alternative embodiments, compositions and methods (using pharmaceutical compounds and formulations including nucleic acids, polypeptides, and gene and polypeptide delivery vehicles) for regulating or manipulating recombinant ApoA-I binding proteins (APOA1BP, AIBP, or AI-BP), including modifying amino acid sequences, adding, maintaining, enhancing, or upregulating expression of the proteins, and kits containing all or part of the components for carrying out these compositions and methods are provided. In alternative embodiments, 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 the use of delivery vehicles capable of targeting and / or penetrating the blood-brain barrier, and nucleic acid (gene) delivery vehicles such as vectors and viruses, such as adeno-associated virus (AAV) delivery vehicles, contained within AIBP-expressing nucleic acids; and compositions and methods for directly delivering either AIBP polypeptides or AIBP-expressing nucleic acids via intrathecal (it) administration are provided.

[0128] Example 1 describes a study using a mouse model of chemotherapy-induced peripheral neuropathy, in which spinal microglia are characterized by the presence of inflammarafts (enlarged cholesterol-rich lipid rafts) that orchestrate the inflammatory response. Manipulation of specific mechanisms regulated cholesterol metabolism, normalized inflammarafts, and reprogrammed microglia, resulting in long-term relief of neuropathic pain.

[0129] We also show that a deletion mutant of AIBP lacking the TLR4-binding domain does not reverse neuropathic pain in a mouse model of chemotherapy-induced peripheral neuropathy. The binding of AIBP to TLR4 is important because this innate immune receptor is highly expressed in inflammatory cells, concentrated in cell surface lipid rafts, and mediates inflammatory responses. Enlarged / clustered lipid rafts with increased TLR4 content and evidence of TLR4 dimerization are called "inflammatories". By binding to TLR4, AIBP targets inflammatory cells, disrupts inflammatories, and inhibits inflammation-spinal nerve inflammation and neuropathic pain. The effect is applicable to many inflammatory disease states mediated by TLR4.

[0130] We also found that in native AIBP, the N-terminal TLR4-binding domain is cryptic, and native AIBP does not bind to TLR4. The TLR4-binding domain in AIBP is exposed when the N-terminus is extended with additional amino acids, as is the case for the recombinantly engineered form of AIBP provided herein, for example, as shown in Figure 13. Figure 11 is a graphical representation of this model.

[0131] In an alternative embodiment, an engineered AIBP is provided that contains an amino acid sequence derived from the commercially available pAcHLT-C vector (BD Biosciences).

[0132] TLR4 receptors localize and dimerize in membrane lipid rafts. Enlarged, cholesterol-rich lipid rafts with activating receptors and adaptor molecules (herein referred to as inframa-rafts (Miller et al., 2020)) serve as organizing platforms for initiating inflammatory signaling and cellular responses. Modulation of cholesterol content within the plasma membrane can affect inframa-raft and TLR4 dimerization, signaling, and inflammatory responses in various cell types (Karasinska et al., 2013; Tall and Yvan-Charvet, 2015; Yvan-Charvet et al., 2008). In addition to TLR4, inframa-rafts regulate the activation of numerous other receptors and components of signaling pathways, as reviewed in (Miller et al., 2020). Thus, we hypothesized that CIPN is associated with altered cholesterol dynamics in spinal microglia, leading to the formation of inframa-rafts and persistent neuroinflammation in the spinal cord.

[0133] To test this hypothesis, we measured spinal microglial lipid rafts and TLR4 dimerization in CIPN mice. To manipulate cholesterol dynamics, we used intrathecal injections of apoAI-binding protein (AIBP), an effective multiplier of cholesterol removal from several cell types (Choi et al., 2018; Fang et al., 2013; Woller et al., 2018), and mice with inducible microglia-specific knockdown of cholesterol transporters Abca1 and Abcg1. We demonstrate that AIBP induces a redistribution of cholesterol in the microglial membrane and enhances the colocalization of accessible cholesterol with the cholesterol transporter ABCA1. This redistribution depletes cholesterol from the plasma membrane and sets the conditions for reverting inflammatory rafts to physiological lipid rafts. Microglia-specific Abca1 / Abcg1 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 gene expression changes in microglia suggests impaired cholesterol metabolism. Recombinant AIBP sequence

[0134] In an alternative embodiment, an engineered protein sequence is disclosed that is comprised of an 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 consecutive histidine residues (HHHHHH (SEQ ID NO:1)).

[0135] In another embodiment, the heterologous amino terminal amino acid sequence comprises the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), in which a mutation in the thrombin cleavage site renders the thrombin cleavage site non-functional.

[0136] In some embodiments, a peptide is provided having an amino acid sequence produced from a commercially available pAcHLT-C vector (BD Biosciences), the amino acid sequence being composed of an ApoA-I binding protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence, the amino acid sequence N-terminal to the AIBP amino acid sequence comprising a peptide tag, the peptide tag comprising a multi-histidine (multi-his) tag.

[0137] In alternative embodiments, methods are provided for administering in vivo recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compounds or compositions having a heterologous amino-terminal amino acid sequence of at least about 10 amino acids, or between about 10-100 amino acids, or between about 20-80 amino acids, or between about 30-50 amino acids, or any heterologous amino acid sequence sufficient to result in the unfolding and exposure of a latent (or hidden, unexposed) N-terminal TLR4-binding domain of an AIBP polypeptide.

[0138] In an alternative embodiment, a mouse AIBP is used, e.g., a mouse AIBP having the sequence encoded by SEQ ID NO: 3 and / or the amino acid sequence of SEQ ID NO: 4, which can optionally be supplemented (i.e., further comprises) a fibronectin secretion signal (italics) at the N-terminus and / or a His tag (underlined) at the C-terminus; this product is abbreviated as FIB-mAIBP-His. SEQ ID NO:3: [ka] SEQ ID NO:4: [ka]

[0139] In alternative embodiments, a variant of a human AIBP (hAIBP) polypeptide provided herein (e.g., a human AIBP having a heterologous amino acid sequence that exposes a TLR4 (otherwise cryptic) binding site), or a nucleic acid encoding a variant AIBP provided herein, is administered to a patient or individual in need thereof, or used to manufacture a formulation or medicament, or used to generate a vector or expression vehicle for administration, or included in a kit provided herein, wherein the AIBP variant may comprise or be encoded by: Human AIBP-encoding nucleic acid (cDNA) (SEQ ID NO:5) [ka] [ka] Human AIBP polypeptide (SEQ ID NO:6) [ka]

[0140] In some embodiments, a modified hAIBP is used that retains the TLR4 binding domain and replaces the N-terminal residues with the native signal peptide, for example, hAIBP contains amino acids 25-288 of the hAIBP sequence known as d24hAIBP (encoding nucleic acid): [ka] wherein the corresponding d24hAIBP polypeptide is: [ka] It is.

[0141] In some embodiments, human AIBP is provided in which a portion of the N-terminus of AIBP (amino acids 1-24, d24hAIBP) is replaced with (or further comprises) a fibronectin secretion signal (italics); the product is abbreviated as FIB-d24hAIBP and designated compound 1: Human FIB-d24hAIBP (compound 1)-encoding nucleic acid (cDNA): [ka] [ka]

[0142] In this embodiment, the hAIBP fragment comprises amino acids 25 to 288 (also known as d24hAIBP) and the N-terminal modifications are as follows: MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (sequence number 24).

[0143] In one embodiment, a secretion signal is added to ensure strong secretion of AIBP. For example, a fibronectin secretion signal is added to the N-terminus of AIBP (see the italicized sequences of SEQ ID NO: 3 and SEQ ID NO: 4). Alternatively, a nucleic acid encoding a secretion signal is added to the AIBP coding sequence. In an alternative embodiment, the secretion signal is a fibronectin secretion signal, an immunoglobulin heavy chain secretion signal or an immunoglobulin kappa light chain secretion peptide (see, for example, PLoS One. 2015; 10 (2): e0116878), or an interleukin-2 signal peptide (see, for example, J. Gene Med. 2005 Mar; 7 (3): 354-65).

[0144] In alternative embodiments, the polypeptide coding sequence is operably linked to a promoter, e.g., a constitutive, inducible, tissue-specific (e.g., neural or brain tissue-specific) or ubiquitous promoter or other transcriptional activator.

[0145] In other embodiments, the product from the post-translational modification of the fibronectin-hAIBP construct has the following amino acid sequence (Compound 2): [ka] The hAIBP fragment is d24hAIBP and the N-terminal modification is TETGKSKR (SEQ ID NO:26).

[0146] In another embodiment, the sequence of the AIBP polypeptide is modified at its C-terminus to incorporate additional peptide fragments, as exemplified by the addition of a C-terminal His tag (the corresponding amino acid sequence is underlined). (Nucleic acid sequence encoding compound 3): [ka] [ka]

[0147] Amino acid sequence (compound 3): [ka] Here, the hAIBP fragment contains a His tag (FIB-d24 AIBP-His) and an N-terminal modification: LRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 29) It is d24hAIBP containing.

[0148] In another embodiment, post-translational modification of the signal peptide results in the compound (Compound 4): [ka] [ka] Here, the hAIBP fragment is d24hAIBP-His and the N-terminal modification is TETGKSKR (SEQ ID NO: 26).

[0149] In other embodiments, the polypeptide coding sequence is operably linked to a promoter, e.g., a constitutive, inducible, tissue-specific (e.g., neural or brain tissue-specific) or ubiquitous promoter or other transcriptional activator.

[0150] In another embodiment, the full-length human AIBP is modified at its N-terminus. Such modifications enhance TLR4 binding, for example, the nucleic acid (cDNA) encoding compound 5: [ka] It encodes the following amino acid (compound 5): [ka] Here, the hAIBP fragment is full length and the N-terminal modification is MDYKDHDGDYKDHDIDYKDDDDKLAAANS (SEQ ID NO: 33).

[0151] In other embodiments, the hAIBP sequence retaining the potential TLR4 binding domain is modified at its N-terminus. Exemplary sequences include the DNA and peptide sequences of amino acids 25-288 of hAIBP (d24hAIBP): (Nucleic acid sequence encoding compound 6): [ka] Amino acid sequence (compound 7): [ka] [ka] wherein the hAIBP fragment is d24hAIBP and the N-terminal modification is: MDYKDHDGDYKDHDIDYKDDDDKLAAANS (SEQ ID NO: 33) (Nucleic acid sequence encoding compound 7): [ka] wherein the hAIBP fragment is d24hAIBP and the N-terminal modification is: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), (Nucleic acid sequence encoding compound 8): [ka] [ka] (Compound 6): [ka] wherein the hAIBP fragment is d24hAIBP and the N-terminal modification is: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), (Nucleic acid sequence encoding compound 7): [ka] [ka] Amino acid sequence (compound 7): [ka] wherein the hAIBP fragment is d24hAIBP and the N-terminal modification is: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (sequence number 19).

[0152] In other embodiments, compositions are provided for recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compounds or compositions having a heterologous amino-terminal amino acid sequence of at least about 8 amino acids, or between about 8-100 amino acids, or between about 8-40 amino acids, or between about 30-50 amino acids, or any heterologous amino acid sequence sufficient to result in unfolding and exposure of a latent (or hidden, unexposed) N-terminal TLR4 binding domain of an AIBP polypeptide.

[0153] In an alternative embodiment, the N-terminal sequence of amino acids comprises between 3 and 12 basic amino acids selected from histidine (H), lysine (K) or arginine (R).

[0154] In other embodiments, the compounds described herein can be further modified to improve biological activity properties, for example, by removing putative peptide cleavage sites. Exemplary sequences are represented by the following: (Nucleic acid sequence encoding compound 8): [ka] Amino acid sequence (compound 8): [ka] Here, the hAIBP fragment is d24hAIBP and the N-terminal modification is: MSPIDPMGHHHHHHGRRRASVAAGILVPAASPGLDGICSR (SEQ ID NO: 7).

[0155] In this sequence, the thrombin cleavage site LVPRGS (SEQ ID NO: 13) incorporates the amino acid mutations described, preventing cleavage and unexpected loss of TLR4 binding activity, as described in Example 2.

[0156] It should be appreciated that these sequences are exemplary and are not limiting of the invention.

[0157] In other embodiments, any of the amino acids in the N-terminal modification of hAIBP may be non-naturally occurring and may be inserted by methods known to those of skill in the art. Manufacturing products, kits

[0158] Also provided are articles of manufacture, such as implants or pumps, kits and pharmaceuticals for carrying out the methods provided herein. In alternative embodiments, articles of manufacture, kits and / or pharmaceuticals are provided that contain all components necessary to carry out the methods provided herein. In alternative embodiments, the kits also include instructions for carrying out the methods provided herein. Formulations and pharmaceutical compositions

[0159] In alternative embodiments, pharmaceutical formulations or compositions are provided that include nucleic acids and polypeptides for carrying out the methods and uses provided herein for modulating neuropathic pain, the methods comprising upregulating expression of recombinant ApoA-I binding protein (APOA1BP, AIBP, or AI-BP).In alternative embodiments, pharmaceutical formulations or compositions are provided for use in in vivo, in vitro or ex vivo methods for treating, preventing, reversing and / or ameliorating neuropathic pain. In an alternative embodiment, pharmaceutical compositions and formulations used to practice the methods and uses provided herein, comprising recombinant APOA1BP nucleic acids and polypeptides or resulting in increased expression or activity of recombinant APOA1BP 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, neuropathic pain, a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), optionally glaucoma or other ocular inflammatory disease, optionally pulmonary inflammation and asthma, optionally HIV infection or its comorbidities, and / or optionally vascular inflammation, atherosclerosis and cardiovascular disease. In an alternative embodiment, pharmaceutical compositions and formulations used to practice the methods and uses provided herein that comprise recombinant APOA1BP nucleic acids and polypeptides or that result in increased expression or activity of recombinant APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof, for example, in an amount sufficient to prevent or reduce the intensity and / or frequency of neuropathic pain or a neurodegenerative disease or condition.

[0160] In alternative embodiments, the pharmaceutical compositions used to practice the methods and uses provided herein can be administered parenterally, topically, orally, or by local administration, such as by aerosol or transdermal. The pharmaceutical compositions can be formulated in any manner and can be administered in a variety of unit dosage forms depending on the condition or disease and the extent of the illness, the general medical condition of each patient, the preferred method of administration obtained, etc. Details regarding techniques for formulation and administration are fully described in the scientific and patent literature, see, for example, Remington's Pharmaceutical Sciences , Maack Publishing Co., Easton PA ("Remington's"), most recent edition.

[0161] For example, in alternative embodiments, the compositions used to practice the methods and uses provided herein are formulated in buffers, saline solutions, powders, emulsions, vesicles, liposomes, nanoparticles, nanolipid particles, etc. In alternative embodiments, the compositions can be formulated in any manner and applied in various concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, the desired in vivo, in vitro or ex vivo method of administration, etc. Details regarding techniques for in vivo, in vitro or ex vivo formulations and administration are well described in the scientific and patent literature. The formulations and / or carriers used to practice the methods or uses provided herein can be in the form of tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc. suitable for in vivo, in vitro or ex vivo application.

[0162] In alternative embodiments, the formulations and pharmaceutical compositions used to practice the methods and uses provided herein can include solutions of the compositions (including peptidomimetics, racemic mixtures or racemates, isomers, stereoisomers, derivatives and / or analogs of the compounds) placed or dissolved in a pharma- ceutically acceptable carrier; for example, acceptable vehicles and solvents that can be used include water and Ringer's solution, isotonic sodium chloride. Additionally, sterile, fixed oils can be used as a solvent or suspending medium. For this purpose, any fixed oil can be used, including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, the solutions and formulations used to practice the methods and uses provided herein can be prepared as sterile and generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well-known sterilization techniques.

[0163] Solutions and formulations used to practice the methods and uses provided herein may contain auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of active agents in these formulations may vary widely and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.

[0164] The compositions and preparations used for carrying out the methods and uses provided herein can be delivered by using liposomes.By using liposomes, the delivery of active agents can be concentrated to target cells in in vivo, in vitro or ex vivo applications, especially by using liposomes that carry target cell-specific ligands (e.g., damaged or diseased neuronal cells or CNS tissues) on the surface of the liposomes, or that are otherwise preferentially directed to specific tissues or organ types. Nanoparticles, Nanolipid Particles and Liposomes

[0165] Also provided are nanoparticles, nanolipid particles, vesicles and liposomal membranes containing the compounds used to carry out the methods and uses provided herein, for example, to deliver compositions containing recombinant APOA1BP nucleic acids and polypeptides in vivo, for example, to the CNS and brain. In alternative embodiments, these compositions are designed to target specific molecules, including biological molecules such as polypeptides, including cell surface polypeptides, for example, to target desired cell types or organs, such as neural cells or the CNS.

[0166] Multilamellar liposomes containing the compounds used to practice the methods and uses provided herein are provided and are described, for example, in Park et al., U.S. Patent Application Publication No. 20070082042. Multilamellar liposomes can be prepared to a particle size of about 200-5000 nm using a mixture of oil phase components including squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithin to entrap the compositions used to practice the methods and uses provided herein.

[0167] Liposomes can be made using any method, including a method of generating liposomes by encapsulating an active agent (e.g., recombinant APOA1BP nucleic acid and polypeptide), as described, for example, in Park et al., U.S. Patent Application Publication No. 20070042031, which includes providing an aqueous solution to a first reservoir; providing an organic lipid solution to a second reservoir; and then mixing the aqueous solution and the organic lipid solution in a first mixing region to generate a liposome solution, whereby liposomes encapsulating the active agent are generated substantially instantaneously upon mixing of the organic lipid solution with the aqueous solution; and then immediately mixing the liposome solution with a buffer to generate a dilute liposome solution.

[0168] In one embodiment, the liposome compositions used to practice the methods and uses provided herein contain substituted ammonium and / or polyanions, e.g., to target delivery of compounds (e.g., recombinant APOA1BP nucleic acids and polypeptides) to a desired cell type (e.g., endothelial cells, neuronal cells, or any tissue or area in need thereof, e.g., the CNS), e.g., as described in U.S. Patent Application Publication No. 20070110798.

[0169] For example, nanoparticles are provided that include compounds (e.g., recombinant APOA1BP nucleic acids and polypeptides used to practice the methods provided herein) in the form of nanoparticles (e.g., secondary nanoparticles) that include an active agent, as described in U.S. Patent Application Publication No. 20070077286. In one embodiment, nanoparticles are provided that include a lipid-soluble active agent or a lipid-solubilized water-soluble active agent that interacts with a divalent or trivalent metal salt.

[0170] In one embodiment, the compositions used to practice the methods and uses provided herein can be formulated and delivered to mammalian cells, e.g., the CNS, in vivo using solid lipid suspensions, e.g., as described in U.S. Patent Application Publication No. 20050136121. Modification of delivery vehicle and modification of AIBP

[0171] In alternative embodiments, recombinant AIBP peptides or polypeptides, or nanoparticles, liposomes, etc., containing AIBP (e.g., containing or having therein recombinant APOA1BP nucleic acid or polypeptide used to carry out the methods provided herein) are modified to facilitate intrathecal injection, e.g., delivery to the cerebrospinal fluid or brain. For example, in alternative embodiments, AIBP peptides or polypeptides, or nanoparticles, liposomes, etc., containing recombinant AIBP are engineered to contain a moiety that allows the AIBP peptides or polypeptides, or nanoparticles, liposomes, etc., containing AIBP to bind to receptors or cell membrane structures and facilitate delivery to the CNS or brain, e.g., the moiety may include the mannose-6-phosphate receptor, the melanotransferrin receptor, the LRP receptor, or any other receptor ubiquitously expressed on the surface of CNS or brain cells. For example, conjugation of a mannose-6-phosphate moiety allows the AIBP peptides or polypeptides, or recombinant AIBP-containing nanoparticles, liposomes, etc., to be taken up by CNS cells expressing the mannose-6-phosphate receptor. In alternative embodiments, any protocol or modification, such as an AIBP peptide or polypeptide, or nanoparticles, liposomes, etc., containing AIBP, that allows entry or delivery to the CNS or brain in vivo, can be used, e.g., as described in U.S. Pat. No. 9,089,566.

[0172] In alternative embodiments, the recombinant AIBP peptide or polypeptide, or nanoparticles, liposomes, etc. comprising AIBP (e.g., comprising or having a recombinant APOA1BP nucleic acid or polypeptide used to practice the methods provided herein) are directly or indirectly linked or conjugated to a blood-brain barrier (BBB) ​​targeting agent, such as transferrin, insulin, leptin, insulin-like growth factor, cationic peptides, lectins, receptor-associated protein (RAP) (a 39 kD chaperone localized in the endoplasmic reticulum and Golgi, a lipoprotein receptor-related protein (LRP) receptor family ligand), apolipoprotein B-100 derived peptides, antibodies against the transferrin receptor (e.g., peptidomimetic monoclonal antibodies), antibodies against the insulin receptor (e.g., peptidomimetic monoclonal antibodies), antibodies against the insulin-like growth factor receptor (e.g., peptidomimetic monoclonal antibodies), antibodies against the leptin receptor (e.g., peptidomimetic monoclonal antibodies), and the like. In alternative embodiments, any protocol or modification of AIBP peptide or polypeptide, or AIBP-containing nanoparticles, liposomes, etc., that facilitates crossing the BBB can be used, for example, as described in U.S. Patent Application Publication Nos. 20050142141; 20050042227. For example, any protocol, for example, direct intracranial injection, transient permeabilization of the BBB, and / or modification of AIBP peptide or polypeptide, or AIBP-containing nanoparticles, liposomes, etc., to alter tissue distribution can be used to enhance CNS or brain delivery of the compositions used to carry out the methods provided herein. Delivery Cells and Delivery Vehicles

[0173] In alternative embodiments, any delivery vehicle can be used to carry out the methods or uses provided herein, for example, to deliver compositions (e.g., recombinant APOA1BP nucleic acids and polypeptides) to the CNS or brain in vivo. For example, a delivery vehicle comprising a polycation, such as a polyethyleneimine derivative, a cationic polymer and / or a cationic peptide, as described in US Patent Publication No. 20060083737, can be used. In one embodiment, the delivery vehicle is a transduced cell engineered to express or overexpress and then secrete endogenous or exogenous AIBP.

[0174] In one embodiment, the dried polypeptide-surfactant complex is used to formulate compositions used to practice the methods provided herein, e.g., as described in U.S. Patent Application Publication No. 20040151766.

[0175] In one embodiment, the composition used to carry out the methods and uses provided herein can be applied to cells using a vehicle with cell membrane permeable peptide conjugate, for example, as described in U.S. Patent No. 7,306,783;6,589,503.In one aspect, the composition delivered is conjugated to cell membrane permeable peptide.In one embodiment, the composition delivered and / or delivery vehicle is conjugated to a transport mediating peptide that is highly basic and binds to polyphosphoinositides, for example, as described in U.S. Patent No. 5,846,743.

[0176] In one embodiment, the cells to be delivered to the CNS or brain are transfected or transduced with AIBP-expressing nucleic acid, e.g., vector, e.g., by electropermeabilization.This electropermeabilization can be used as a primary or secondary means for delivering the composition to cells, e.g., by using any electroporation system, e.g., as described in U.S. Patent Nos. 7,109,034; 6,261,815; 5,874,268. In vivo delivery of AIBP-encoding nucleic acids

[0177] In alternative embodiments, compositions and methods are provided for delivering nucleic acids encoding AIBP peptides or polypeptides, or nucleic acids encoding peptides or polypeptides having AIBP activity, or vectors or recombinant viruses containing these nucleic acids.In alternative embodiments, nucleic acids, vectors or recombinant viruses are designed for in vivo or CNS delivery and expression.

[0178] In alternative embodiments, compositions and methods are provided for the delivery and controlled expression of a recombinant AIBP-encoding nucleic acid or gene, or an expression vehicle (e.g., a vector, a recombinant virus, etc.) that contains (has contained therein) a recombinant AIBP-encoding nucleic acid or gene, so that the AIBP protein can be released into the bloodstream or systemic circulation and produce a beneficial effect in the body, e.g., in the CNS, brain or other targets.

[0179] In an alternative embodiment, methods are provided whereby AIBP-expressing nucleic acid or gene expression can be easily and efficiently turned on and off for tailored treatment and to ensure optimal safety.

[0180] In alternative embodiments, recombinant AIBP protein or proteins expressed by one or more AIBP-expressing nucleic acids or genes have a beneficial or favorable effect (e.g., therapeutic or prophylactic) on a tissue or organ, e.g., the brain, CNS, or other target, even if secreted into the blood or systemic circulation distant (e.g., anatomically distant) from their site(s) of action.

[0181] In alternative embodiments, expression vehicles, vectors, recombinant viruses, etc. are provided for in vivo expression of recombinant AIBP-encoding nucleic acids or genes for carrying out the methods provided herein. In alternative embodiments, expression vehicles, vectors, recombinant viruses, etc. expressing AIBP nucleic acids or genes can be delivered, for example, during a clinic visit, e.g., as an outpatient, by intramuscular (IM) injection, intravenous (IV) injection, subcutaneous injection, inhalation, biolistic particle delivery system (e.g., so-called "gene gun"), etc.

[0182] In an alternative embodiment, this "peripheral" delivery mode, such as expression vehicles, vectors, recombinant viruses, etc., injected IM or IV, can avoid problems encountered when genes or nucleic acids are directly expressed in the organ itself (e.g., brain or CNS). Continuous secretion of AIBP in the bloodstream or systemic circulation also avoids the difficulty and expense of administering proteins by injection.

[0183] In alternative embodiments, recombinant virus (e.g., long-term virus or viral vector), or vector, or expression vector, etc., can be injected, for example, as an outpatient, for example, in a doctor's office, for example, by systemic vein (e.g., IV), or by intramuscular (IM) injection, by inhalation, or by biolistic particle delivery system (e.g., so-called "gene gun"). In alternative embodiments, several days or weeks later (e.g., 4 weeks later), the individual, patient, or subject is administered (e.g., inhaled, injected, or swallowed) a chemical or pharmaceutical agent that induces expression of AIBP-expressing nucleic acid or gene. For example, an oral antibiotic (e.g., doxycycline or rapamycin) that activates expression of the gene is administered once a day (or more or less frequently). In an alternative embodiment, after "activation" or induction of expression of a nucleic acid or gene (e.g., by an inducible promoter), an AIBP protein is synthesized and released into the circulation (e.g., into the blood) of a subject, which then has a favorable physiological effect, e.g., a therapeutic or preventive effect, that benefits the individual or patient (e.g., beneficial to cardiac, renal or pulmonary function). If the physician or subject desires to discontinue AIBP treatment, the subject simply stops taking the activating chemical or pharmaceutical drug, e.g., an antibiotic. Alternative embodiments include the use of "expression cassettes" that contain or have nucleotide sequences used to carry out the methods provided herein, e.g., an AIBP-expressing nucleic acid that can affect expression of a nucleic acid, e.g., as a structural gene or transcript (e.g., encoding an AIBP protein) in a host compatible with such sequences. An expression cassette can include at least one promoter operably linked to a polypeptide coding sequence or an inhibitory sequence; further, in one aspect, it includes other sequences, e.g., a transcription termination signal. Additional factors necessary or useful for effecting expression, e.g., enhancers, can also be used.

[0184] In alternative embodiments, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. In alternative embodiments, a "vector" may include a nucleic acid capable of infecting, transfecting, transiently or permanently transducing a cell. In alternative embodiments, a vector may be a naked nucleic acid, or a nucleic acid complexed with a protein or lipid. In alternative embodiments, a vector may include viral or bacterial nucleic acid and / or protein, and / or membrane (e.g., cell membrane, viral lipid envelope, etc.). In alternative embodiments, a vector may include, but is not limited to, a replicon (e.g., RNA replicon, bacteriophage) to which a piece of DNA may be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Pat. No. 5,217,879), and may include both expression and non-expression plasmids. In alternative embodiments, vectors may be stably replicated by cells during mitosis as autonomous structures, or may be integrated into the host genome.

[0185] In alternative embodiments, a "promoter" includes any sequence capable of driving transcription of a coding sequence in a cell, e.g., a mammalian cell, such as a muscle, nerve or brain cell. The promoters used in the constructs provided herein include cis-acting transcriptional control elements and regulatory sequences involved in regulating or modulating the timing and / or rate of transcription of a nucleic acid, e.g., an AIBP-encoding nucleic acid. For example, a promoter can be a cis-acting transcriptional control element, which includes enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences involved in transcriptional regulation. These cis-acting sequences usually interact with proteins or other biomolecules to effect transcription (switch on / off, regulate, modulate, etc.).

[0186] In alternative embodiments, a "constitutive" promoter can be a promoter that drives expression continuously under most environmental conditions and states of development or cell differentiation. In alternative embodiments, an "inducible" or "regulatable" promoter can direct expression of a nucleic acid, such as an AIBP-encoding nucleic acid, under the influence of an environmental condition, an administered chemical, or a developmental condition. Gene Therapy and Gene Delivery Vehicles

[0187] In alternative embodiments, the methods of the invention include the use of a nucleic acid (e.g., a gene or polypeptide encoding a recombinant AIBP-encoding nucleic acid) delivery system to deliver a nucleic acid or gene, or a payload of an AIBP-expressing nucleic acid, transcript or message, to one or more cells in vitro, ex vivo, or in vivo, e.g., as a gene therapy delivery vehicle.

[0188] In alternative embodiments, the expression vehicle, vector, recombinant virus, or equivalent used to carry out the methods provided herein is or comprises: adeno-associated virus (AAV), lentiviral vector, or adenoviral vector; AAV serotype AAV5, AAV6, AAV8, or AAV9; Rhesus monkey-derived AAV, or Rhesus monkey-derived AAV AAVrh.10hCLN2; organotropic AAV, or neurotropic AAV; and / or AAV capsid mutant or AAV hybrid serotype. In alternative embodiments, AAV is engineered to increase the efficiency of targeting specific cell types that are non-permissive to wild-type (wt) AAV, and / or to improve the efficiency of infecting only the cell type of interest. In alternative embodiments, hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications, including: 1) trans-encapsidation, 2) adsorption of bispecific antibodies to the capsid surface, 3) engineering of mosaic capsid, and / or 4) engineering of chimeric capsid.Methods for engineering adeno-associated virus (AAV) capsid to increase the efficiency of targeting specific cell types that are non-permissive to wild-type (wt) viruses and to improve the efficiency of infecting only the cell types of interest are well known in the art.See, for example, Wu et al., Mol.Ther.2006 Sep;14(3):316-27.Epub 2006 Jul 7;Choi, et al., Curr.Gene Ther.2005 Jun;5(3):299-310.

[0189] For example, in alternative embodiments, serotypes AAV-8, AAV-9, AAV-DJ or AAV-DJ / 8™ (Cell Biolabs, Inc., San Diego, Calif.), which have increased in vivo uptake into brain tissue, are used to deliver AIBP-encoding nucleic acid payloads for expression in the CNS. In alternative embodiments, the following serotypes or variants thereof are used to target specific tissues: [Table 1]

[0190] In an alternative embodiment, the rhesus monkey-derived AAV AAVrh.10hCLN2 or its equivalent can be used, which may not be inhibited by any pre-existing immunity in humans; see, e.g., 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; which teach that administration of AAVrh.10hCLN2 directly to the CNS of rats and non-human primates at doses scalable to humans results in an acceptable safety profile and mediates significant payload expression in the CNS.

[0191] Because adeno-associated virus (AAV) is a common infectious agent in primates, and therefore healthy primates harbor a large pool of AAV-specific neutralizing antibodies (NAbs) that inhibit AAV-mediated gene transfer therapeutic strategies, the methods provided herein may include screening patient candidates for AAV-specific NAbs prior to treatment, particularly using the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy; see, e.g., Sun, et al., J. Immunol. Methods. 2013 Jan 31;387(1-2):114-20, Epub 2012 Oct 11. dosage

[0192] The pharmaceutical compositions and formulations used to carry out the methods and uses provided herein can be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the compositions are administered to a subject already suffering from a disease, condition, infection or deficiency in an amount sufficient to cure, alleviate or partially arrest the clinical signs of the disease, condition, infection or disease and its complications, including, for example, neuropathic pain ("therapeutically effective amount"). For example, in alternative embodiments, the pharmaceutical compositions and formulations comprising the recombinant APOA1BP nucleic acid or polypeptide provided herein are used to treat neuropathic pain, inflammation-induced neuropathic pain, inflammation-induced neuropathic pain, inflammation of the nerve or CNS, allodynia, post-nerve injury pain or neuropathic pain, post-operative pain or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced allodynia), neurodegenerative diseases or conditions, optionally chronic or progressive neurodegenerative diseases or conditions, optionally Alzheimer's disease, and the like. or administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and / or ameliorate chronic traumatic encephalopathy (CTE) or related tauopathies, traumatic brain injury (TBI), post traumatic stress disorder, post traumatic war neurosis, or post traumatic stress syndrome (PTSS), migraine headaches, hyperalgesia, optionally glaucoma or other ocular inflammatory diseases, optionally pulmonary inflammation and asthma, optionally HIV infection or its comorbidities, and / or optionally vascular inflammation, atherosclerosis and cardiovascular disease.

[0193] The amount of pharmaceutical composition sufficient to accomplish this is defined as a "therapeutically effective amount." Effective dosing schedules and amounts for this use, i.e., "dosing regimens," depend on 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 condition, age, etc. The mode of administration is also taken into consideration when calculating a patient's dosing regimen.

[0194] In an alternative embodiment, a viral vector, such as an adenovirus or AAV vector, is administered to an individual in need thereof, and in an alternative embodiment, the dosage administered to a human is about 2×10 per kg of body weight.12 Vector genomes (vg / kg), or approximately 10 per kg of body weight 10 ~10 14 between vector genomes (vg / kg) or ca. 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15vg / kg or more, which can be administered as a single dose or multiple doses as needed. In alternative embodiments, these dosages are administered orally, IM, IV, or intrathecally. In alternative embodiments, the vector is delivered as a formulation or pharmaceutical, for example, the vector is contained in a nanoparticle, particle, micelle, or liposome or lipoplex, polymersome, polyplex, or dendrimer. In alternative embodiments, these dosages are administered once a day, once a week, or any variation thereof as needed to maintain the in vivo expression of recombinant AIBP, which can be monitored by actually measuring the expression of AIBP or by monitoring the therapeutic effect, e.g., pain relief. Dosing regimens also take into account pharmacokinetic parameters well known in the art, i.e., rate of absorption, bioavailability, metabolism, clearance, etc. of the active agent (see, e.g., 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. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). The current state of the art allows clinicians to determine the dosage regimen for each individual patient, active agent, and disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidelines to determine the dosage regimen, i.e., dosage schedule and dosage level, administered in carrying out the methods provided herein is accurate and appropriate.

[0195] The formulation may be administered once or multiple times, depending on the dosage and frequency required and tolerated by the patient. The formulation should provide a sufficient amount of active agent to effectively treat, prevent or ameliorate the conditions, diseases or symptoms described herein. For example, the daily dose of another exemplary pharmaceutical formulation for oral administration of the composition used to practice the methods provided herein is between about 0.1-0.5 to about 20, 50, 100 or 1000 ug per kilogram of body weight per day or more. In alternative embodiments, a dosage of about 1 mg to about 4 mg per kg of body weight per patient per day is used. In contrast to oral administration, lower dosages can be used in the bloodstream, in a body cavity, or in the lumen of an organ. Substantially higher dosages can be used for topical or oral administration, or administration by powder, spray, or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's, supra.

[0196] The methods provided herein can further include co-administration with other drugs or pharmaceuticals, e.g., compositions for treating any neurological or neuromuscular disease, condition, infection or injury, including associated inflammatory and autoimmune diseases and conditions, etc. For example, the methods and / or compositions and formulations provided herein can be co-formulated and / or co-administered with fluids, antibiotics, cytokines, immunomodulatory agents, anti-inflammatory agents, pain relieving compounds, complement activators, peptides or proteins containing collagen-like or fibrinogen-like domains (e.g., ficolins), carbohydrate binding domains, and the like, and combinations thereof. Bioisosteres of compounds

[0197] In alternative embodiments, the compounds used to carry out the methods provided herein, such as biological equivalents of polypeptides having recombinant APOA1BP activity, are also provided. The biological equivalents used to carry out the methods provided herein include, for example, biological equivalents of recombinant APOA1BP nucleic acids and polypeptides, which in alternative embodiments may include the replacement of one or more substituents and / or groups with substituents that produce substantially similar biological properties and / or groups with substantially similar physical or chemical properties as the compounds used to carry out the methods or uses provided herein. In one embodiment, the purpose of exchanging one biological equivalent for another is to improve the desired biological or physical properties of the compound without significant changes to the chemical structure.

[0198] For example, in one embodiment, one or more hydrogen atoms are replaced with one or more fluorine atoms, for example at the site of metabolic oxidation; this can prevent metabolism (catabolism) from occurring.Since fluorine atoms are similar in size to hydrogen atoms, the overall topology of the molecule is not significantly affected, and the desired biological activity is not affected.However, molecules that have been blocked from the pathway for metabolism may have a longer half-life or be less toxic. Devices for delivering therapeutics directly to the CNS or brain

[0199] In alternative embodiments, pharmaceutical compositions and formulations, including nanoparticles and liposomes, used to practice the methods provided herein are delivered directly to the CNS or brain, for example, by intravenous or intrathecal injection, or by various devices known in the art. For example, US Patent Application Publication No. 20080140056 describes a catheter that advances rostrally in the intrathecal cavity to deliver pharmaceuticals and formulations directly to the brain. Implantable infusion devices can also be used. For example, a catheter for delivering fluid from an infusion device to the brain can be threaded subcutaneously from the abdomen to the patient's skull, and the catheter can access the individual's brain through a drill hole. Alternatively, the catheter can be implanted to deliver agents intrathecally in the patient's spinal canal. Flexible guide catheters can also be used, with a distal end for introduction under the patient's skull and a proximal end that remains outside the patient's body. See, for example, US Patent Application Publication No. 20060129126.

[0200] In alternative embodiments, the pharmaceutical compositions and formulations used to practice the methods provided herein are delivered via direct delivery of pharmaceutical compositions and formulations including nanoparticles and liposomes, or direct implantation of cells expressing AIBP into the brain, e.g., using a cell implantation cannula, syringe, etc., as described, for example, in U.S. Patent Application Publication No. 20080132878; or an elongated medical insertion device, e.g., as described, for example, in U.S. Patent No. 7,343,205; or a surgical cannula, e.g., as described, for example, in U.S. Patent No. 4,899,729. Implantation cannulas, syringes, etc. can also be used for direct injection of liquids, e.g., fluid suspensions.

[0201] In alternative embodiments, the pharmaceutical compositions and formulations used to practice the methods provided herein are delivered with a tracer detectable, for example, by magnetic resonance imaging (MRI) and / or X-ray computed tomography (CT). The tracer can be co-injected with the therapeutic agent and used to monitor the distribution of the therapeutic agent as it travels through the target tissue, for example, as described in U.S. Patent No. 7,371,225. Kit and Instructions

[0202] For example, kits are provided that include compositions (including devices described herein) and / or instructions for practicing the methods provided herein for treating, ameliorating or preventing neuropathic pain. Thus, kits, cells, vectors, etc. may also be provided. In alternative embodiments, kits are provided that include compositions used to practice the methods provided herein, or compositions, pharmaceutical compositions or formulations provided herein, and optionally instructions for their use.

[0203] The present invention will be further described with reference to the examples described herein, however it will be understood that the invention is not limited to such examples. EXAMPLES

[0204] Working Example Example 1: Efficacy demonstrated in an exemplary method for treating pain This example describes and demonstrates the efficacy of exemplary embodiments and methods provided herein for treating or ameliorating neuropathic pain, including, for example, allodynia and TLR4-mediated inflammation-induced neuropathic pain.

[0205] Neuroinflammation is a major component in the transition to and maintenance of neuropathic pain states. Spinal neuroinflammation is accompanied by activation of TLR4, which localizes to enlarged cholesterol-enriched lipid rafts, referred to here as inframalads. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia led to the formation of inframalads and induced tactile allodynia in naive mice. ApoA-I binding protein (AIBP) promoted cholesterol depletion from inframalads and reversed neuropathic pain in a chemotherapy-induced peripheral neuropathy (CIPN) model in wild-type mice, but AIBP (compound 7) failed to reverse allodynia in mice with ABCA1 / ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind to microglia or reverse CIPN allodynia. The sustained therapeutic effect of a single dose of AIBP (compound 7) in CIPN was associated with anti-inflammatory and reprogramming of cholesterol metabolism and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling TLR4 inflammation and gene expression programs in microglia and blocking the persistence of neuroinflammation. result Chemotherapy-induced peripheral neuropathy alters lipid rafts and TLR4 dimerization in spinal microglia

[0206] In a chemotherapy-induced peripheral neuropathy model (Woller et al., 2018), intraperitoneal injection of cisplatin induced severe tactile allodynia in male mice (Figure 1A). This condition was associated with increased formation of lipid rafts in spinal microglia, suggesting altered membrane cholesterol dynamics, and increased dimerization of TLR4 (Figure 1B and 1C). Intrathecal AIBP (compound 7) reversed CIPN-associated allodynia in spinal microglia and normalized lipid raft and TLR4 dimer levels (Figure 1A-C). These data suggest that TLR4 receptor dimerization, the first step in the activation of the TLR4 inflammatory cascade, occurs in lipid rafts in microglia, as demonstrated in other cell types (Cheng et al., 2012; Zhu et al., 2010). This idea was supported by in vitro experiments, in which TLR4 localization in lipid rafts was significantly increased in BV-2 microglial cells treated with LPS, and AIBP (compound 7) prevented LPS-induced TLR4-CTxB colocalization (Figure 1D). The specificity of the TLR4 antibody used in flow cytometry and microscopy experiments was verified by the TLR4 antibody. - / - We validated this using cells derived from mice (Fig. S1A and B). Because dorsal root ganglion (DRG) macrophages are also involved in nociceptive responses and express TLR4, we assessed their TLR4 dimerization and lipid raft content. However, DRG CD11b + No significant changes were observed in myeloid cells at this time point (fig. S1C and D). AIBP in CSF and spinal cord (Compound 7) Short-term exposure to

[0207] A single intrathecal administration of AIBP (compound 7) had a sustained therapeutic effect that reversed allodynia in CIPN mice, lasting for at least 2 months (Woller et al., 2018). This could be explained either by a prolonged exposure of AIBP (compound 7) in the spinal cord upon it delivery or by a disease-modifying effect reflected in changes in gene expression profiles. To test the former, we measured the pharmacokinetics of AIBP in CSF and lumbar spinal cord homogenates after it delivery of recombinant AIBP. In these experiments, to avoid cross-reaction of endogenous mouse AIBP in spinal cord tissue with the antibody we use, we used Apoa1bp - / - Mice were used. This study demonstrated short-term exposure of AIBP (compound 7) in CSF and spinal cord tissue, reaching peak levels by 30 min and already undetectable after 4 h (Figures 1E and 1F). These results are consistent with recent reports of rapid clearance of macromolecules from the CSF (Ahn et al., 2019) and suggest that reprogramming of spinal microglia and / or other cell types occurs as a result of relaxation of TLR4 dynamics in the membrane and possibly other effects of AIBP. Chemotherapy-induced peripheral neuropathy alters gene expression profiles in spinal microglia

[0208] To characterize spinal microglia in CIPN, we performed RNA-seq and differential gene expression analysis of spinal microglia isolated from naive, cisplatin / saline and cisplatin / AIBP (compound 7)-treated wild-type (WT) mice (see Methods and Figure S2 for quality control and dataset characterization). We used likelihood ratio tests (LRT) to identify genes regulated by any condition across all samples and identified 3254 differentially expressed genes (DEGs) showing a main effect of CIPN and AIBP (compound 7) in the transcriptome of spinal microglia (Figure 2A). The majority of these changes were caused by the CIPN condition, with little effect of AIBP (compound 7) (Figures 2A and 2B, groups 1 and 2). However, there was a smaller group of CIPN-regulated genes, whose changes were completely reversed by AIBP (compound 7) treatment (Figures 2A and B, groups 3 and 4). Among the pathways and gene ontology (GO) biological processes upregulated in CIPN were replication, translation and mitochondrial function. Several of the enriched pathways were related to microglial phenotypic changes associated with CNS diseases such as Parkinson's and Alzheimer's (Figure 2C). The cholesterol transporters Abca1 and Abcg1 were downregulated in microglia from cisplatin-treated mice, indicating impaired membrane cholesterol transport (Figures 3A and 3C). Among the downregulated genes were lysosomal genes important in autophagy and lipophagy, suggesting lipid storage dysregulation. Arachidonic acid metabolism genes were also upregulated (Figure 3C), suggesting the release of bioactive lipid mediators and inflammation.

[0209] Using pairwise comparisons of CIPN and naïve groups after LRT analysis, we also observed downregulation of Cx3cr1, P2ry12 and Tmem119 homeostasis markers (Figure 3A and B), a phenotype associated with transition to neurodegenerative disease-associated microglia (DAM) (Masuda et al., 2019; Nugent et al., 2020; Prinz et al., 2019). A subset of DAM signature genes in microglia revealed a DAM signature with decreased homeostatic genes and increased inflammatory and cholesterol metabolism genes. Modulation of the phagocytic TAM receptors Tyrobp, Axl and Mertk partially mimicked DAM characteristic of neurodegenerative diseases in CIPN microglia, except that we observed downregulation of Trem2 with no significant effect on its partner receptor gene, Tyrobp (Figure 3B). This suggests a decreased phagocytic phenotype associated with altered lipid homeostasis and increased lipid accumulation in macroglia and macrophages (Jaitin et al., 2019; Marschallinger et al., 2020).

[0210] Interestingly, some of the DAM signatures associated with lipid storage genes enriched in CIPN were downregulated by AIBP (compound 7), including the gene encoding the lipid droplet protein PLIN2 (Figures 3B and 3C). PLIN2 immunohistochemistry validated the RNA-seq results, showing an increase in the number and size of lipid droplets in spinal microglia of cisplatin-treated mice, and the reversal of this effect by AIBP (compound 7) (Figures 3D-H). AIBP (Compound 7) Selective reversal of CIPN-induced changes in inflammatory gene expression by

[0211] When analyzing the gene group upregulated by CIPN and reversed by AIBP (group 3 in Figure 2B and Figure S2F), pathways of inflammatory response, leukocyte chemotaxis and neutrophil degranulation pathways were found to be enriched (Figure 4A). Some of the genes in these enriched pathways include Il1b, Ccl2, Glipr1 and Glipr2, Gpnmb, Cxcl2, Cxcl3, S100a8, Il22ra2, Il1r2, Fpr1, Apoe, Ccl9 and the TLR4 interactor gene Tril (Figure 4B and C). Examining cytokine protein expression in spinal cord tissue, we confirmed the regulation of CCL2 (MCP-1) and CXCL2 (MIP2) expression by AIBP (compound 7) (Figure 4D). AIBP (compound 7) also downregulated inflammatory and non-inflammatory genes that were not induced by cisplatin. These include Ccl24, Il3ra, Xcr1 and the TLR4 pathway-related gene Ptpn22 (Figure 4E). Pathway and GO analysis of all genes downregulated by AIBP (compound 7) show enrichment in the TLR4 signaling pathway, along with cytokine-cytokine receptor interactions, protein kinases A and C and MAPK regulatory pathways, receptor-mediated endocytosis and other membrane signaling pathways (Figure 4F). Regulation of calcium and membrane potential was also downregulated, and enrichment in peptidase inhibitor pathways indicates the effect of AIBP (compound 7) on recently reported pain-related peptidase inhibition-related genes such as Pi16 and the α-synuclein gene Snca, which interact with lipid membranes and regulate vesicle trafficking and neurotransmitter release (Figure 4G).

[0212] Differential expression analysis of microglia from AIBP (compound 7)-treated mice in the CIPN model also revealed 40 genes that were downregulated in CIPN and reversed by AIBP (compound 7) (Figure S2E). Enriched pathways included regulation of kinase and phosphatase activity (Figure 4A), as well as regulation of actin cytoskeleton, membrane reorganization and nuclear signaling genes Bin1, Pak1, Vav2 and Ccdc88a and membrane lipid signaling cascade-associated protein Dgka (Figure 4H). Overall, these results suggest that AIBP (compound 7)-induced reversal of microglial reprogramming induced in CIPN mice involves regulation of lipid metabolism and transport from the membrane to lipid droplets and / or extracellular receptors. AIBP (Compound 7) fails to reverse allodynia in mice with ABCA1 / ABCG1-deficient microglia

[0213] To assess the role of microglial cholesterol dynamics in nociception, we first measured the colocalization of membrane cholesterol available for efflux or trafficking to the endoplasmic reticulum with the ABCA1 cholesterol transporter, as detected by binding of ALOD4 (He et al., 2017). Treatment of BV-2 cells with LPS reduced the colocalization of ALOD4 with ABCA1 and APOA1 in lipid raft domains, an effect that was reversed by AIBP (compound 7) (Figures 5A and B).

[0214] Next, we generated tamoxifen-induced microglia-specific ABCA1 and ABCG1 double knockout mice (ABC-imKO, Fig. 5C), and demonstrated that ABCA1 and ABCG1 knockdown significantly increased the spinal cord IBA1 + It was observed in microglia, but GFAP + Astrocytes or NeuN +We confirmed that this was not observed in neurons (Figure S3). Knockdown of ABCA1 and ABCG1 cholesterol transporters in microglia resulted in basal allodynia even in the absence of stimulus loading (day 0) (Figure 5D). These results add to the evidence that impaired microglial cholesterol transport leads to a promoted state. Indeed, we observed that spinal microglia from naive ABC-imKO mice had increased TLR4 surface expression, increased TLR4 dimerization, and higher lipid raft content compared to those from WT mice (Figure 5E).

[0215] Notably, unlike WT mice, itAIBP (compound 7) failed to prevent mechanical allodynia induced by itLPS in ABC-imKO mice (Figures 5F and S5A). Next, we induced CIPN in ABC-imKO mice with cisplatin and observed a further rapid onset of allodynia. Furthermore, although it delivery of AIBP in ABC-imKO mice on day 7 of the CIPN model did not reverse mechanical allodynia (Figure 5G), itAIBP was effective in reversing CIPN allodynia in transgenic (littermate) mice treated with vehicle instead of tamoxifen (Figure 5H) and in wild-type mice treated with tamoxifen (Figure S5B). It injection of 2-hydroxypropyl-β-cyclodextrin (hp-β-CD), which depletes cholesterol from the plasma membrane but does not require expression of ABCA1 or ABCG1, attenuated allodynia in ABC-imKO mice (Figure S4C). In naive ABC-imKO mice, TLR4 dimerization and lipid raft abundance were significantly higher than in naive WT mice, and they were not significantly altered by either cisplatin or AIBP in ABC-imKO microglia (Figure 5I and J). These results support the idea that AIBP requires cholesterol transporters to alter inflammatories and TLR4 dimerization dynamics in microglia and reverse allodynia. ABCA1 / ABCG1 deficiency reprograms microglia towards a CIPN-like phenotype

[0216] To understand the effect of cholesterol transport on CIPN- and AIBP-induced transcriptional changes, we analyzed differential gene expression in ABC-imKO microglia. We identified 121 genes that were significantly altered across the two genotypes and three experimental conditions (Figure S4D). In spinal microglia of naïve ABC-imKO mice not loaded with cisplatin, the majority of upregulated genes and enriched pathways overlapped with upregulated genes induced by cisplatin in WT mice (Figure 6A and B). Among the enriched pathways in naïve ABC-imKO mice, we identified the response to interferon, inflammatory response, complement activation, and arachidonic acid metabolism pathways (Figure 6B). Upregulated interferon genes included Ifi207 and Ifi27l2a, as well as inflammatory genes Xcr1, Cb4, C3, and Klrb1b. The lipid metabolism-related genes Apoe and Ch25h were significantly upregulated in naive ABC-imKO mice (Figure 6C and F), similar to the changes induced by cisplatin in WT mice. This microglial reprogramming may at least partially explain the pain behavior observed in naive ABC-imKO mice.

[0217] Induction of CIPN in ABC-imKO mice also upregulated several gene sets and pathways common to both ABC-imKO and WT microglia. However, unlike WT, pathways such as phagosome, actin dynamics for phagocytic cup formation and cell cycle pathways were not enriched in ABC-imKO microglia from cisplatin-treated mice (Figures 6D and S5E). Cisplatin in ABC-imKO microglia failed to induce the expression of several inflammatory genes and downregulated the expression of Cxcl3, Xrip1 and phagosome-associated Fcrls and Cybb (NOX2) (Figures 6F and G). In the absence of cholesterol transporters, cisplatin did not induce cholesterol synthesis pathway genes and downregulated Ch25h and Dhcr24, suggesting that excess free cholesterol was present to favor the accumulation of desmosterol, an LXR agonist and key regulator of macrophage foam cell transcriptome in atherosclerosis (Figure 6E and G), (Span et al., 2012). Impaired phagocytosis and upregulation of Tnfrsf26, Trpv4, Il3ra, Il15a, and Shtn1 (Figure 6E-G) may indicate a differential role of membrane dynamics on nociceptive processes in ABC-imKO mice. AIBP (Compound 7) Microglial reprogramming by ABCA1 depends on the expression of ABCG1 and ABCA2

[0218] To understand the differential effects of AIBP (compound 7) in WT and ABC-imKO mice, we compared the up- and down-regulated genes induced by AIBP treatment in both genotypes (Figures 7A and B). The effects of AIBP (compound 7) on gene regulation were significantly different, with only a few common genes being down-regulated in both genotypes (Figure 7A). In the absence of cholesterol transport machinery, AIBP was unable to regulate inflammatory genes, instead inducing their expression (Figures 7B and 7C). The induction of inflammatory genes correlates with an increase in other cholesterol biosynthesis genes, including Dhcr24 and Srebf2, which were down-regulated in WT microglia (Figure 7D). This suggests that the reduction in desmosterol and the increase in cholesterol content regulate the expression of inflammatory genes in microglia. Consistent with this, induction of genes controlled by LXR, such as Apoe, Apoc1 and Pparg, was observed in microglia of ABC-imKO mice but not in WT mice (Figure 7D). Other non-inflammatory genes regulated in the opposite direction in ABC-imKO by AIBP (compound 7) compared to WT include the endopeptidase activity genes Pi16 and Capn11 and the AMPA receptor and synaptic regulator Arc (Figure 7E). In ABC-imKO microglia, AIBP (compound 7) upregulated cholesterol metabolism pathways, cytokine release and chemokine signaling regulation, kinase and endopeptidase activity, and lamellipodia and fiber organization pathways (Figure 7F). Most of these pathways were downregulated by AIBP in WT microglia (Figure 4F). Taken together, these data indicate that reprogramming of microglial gene expression induced by AIBP (compound 7) depends on cholesterol homeostasis regulated by cholesterol transporters ABCA1 and ABCG1. Microglial AIBP and TLR4 regulate nociception

[0219] As the above experiments implied the activation of microglial TLR4 in AIBP (compound 7)-regulated cholesterol homeostasis and nociception, we investigated whether microglia-specific knockout of AIBP or TLR4 would affect CIPN allodynia. We generated tamoxifen-induced microglia-specific Apoa1bp knockout and TLR4 knockout mice (AIBP-imKO and TLR4-imKO, Fig. 8A). Knockdown of endogenous AIBP in microglia induced mechanical allodynia even before the mice were challenged with cisplatin (day 0, Fig. 8B). This suggests that AIBP plays a role in maintaining microglial homeostasis function in mechanical nociception. Control Cx3cr1-Cre mice without floxed genes showed no significant effect on CIPN allodynia. ERT2 In mice, injection of tamoxifen did not induce mechanical allodynia (Figure 8C). After cisplatin challenge, microglial AIBP knockdown showed a faster decrease in mechanical threshold than control mice (compare day 2 in Figure 8D with day 6 in Figure 8E), indicating higher sensitization in microglial AIBP knockdown mice. Intrathecal injection of recombinant AIBP on day 7 equally reversed CIPN-related allodynia in both vehicle and tamoxifen-induced AIBP-imKO mice (Figure 8C and Figure 8D). In systemic Apoa1bp knockout mice, we did not observe basal allodynia compared to WT mice, and itAIBP rescued CIPN-induced allodynia (Figure 8F). In contrast to AIBP-imKO or ABC-imKO, TLR4-imKO mice were protected from the rapid onset of cisplatin-induced allodynia and showed delayed-onset, mild allodynia ( Figure 8G ), suggesting a role for TLR4 expression in microglia in mediating pain sensitization. Identification of AIBP domains involved in TLR4 binding

[0220] Because TLR4-imKO mice were protected from early / acute CIPN (Figure 8G), we were unable to use this model to assess the in vivo significance of AIBP-TLR4 binding as previously reported (Woller et al., 2018). Here, we took a different approach and created AIBP mutants that did not bind to TLR4. To elucidate which domains of AIBP are involved in binding to TLR4, we started by mutating amino acids predicted from the crystal structure (Jha et al., 2008) of the YjeF_N domain of AIBP (Figure 9A) that are involved in protein-protein interactions, but these mutants retained TLR4-binding properties (not shown). Next, we developed a series of deletion mutants of AIBP scanning the entire length of the protein and tested them in pull-down assays with the TLR4 ectodomain (eTLR4) (Figure 9B). These experiments suggested that the 25-51 amino acid N-terminal domain located after the aa 1-24 signal peptide is involved in eTLR4 binding (Figure 9A and Figure 9B). The aa 25-51 N-terminal domain was not structured in the published crystal structure of mouse AIBP (Jha et al., 2008). Both human and mouse AIBP contain a homologous aa 25-51 N-terminal domain, but zebrafish AIBP does not. Indeed, unlike human and mouse, zebrafish AIBP did not bind to human eTLR4 (Figure 9C). For further experiments, we expressed and purified wtAIBP (aa 25-288), which lacks the signal peptide, and mutAIBP (aa 52-288), which lacks both the signal peptide and the N-terminal domain, from a baculovirus / insect cell system.

[0221] Unlike wtAIBP, mutAIBP did not bind to eTLR4 in pull-down assays (Figure 9D), in ELISA using eTLR4-coated plates, or in detection of bound AIBP using the BE-1 anti-AIBP monoclonal antibody (mAb) developed in our laboratory (Choi et al., 2020) (Figure 9E). The BE-1 mAb had equal affinity for wtAIBP and mutAIBP (Figure S5B). Binding of mutAIBP to APOA1 was unchanged compared to wtAIBP (Figure 9F). In cell culture experiments, wtAIBP, but not mutAIBP, bound to LPS-stimulated BV-2 microglia (Figures 9G and 9H). The increase in wtAIBP binding in response to LPS can be explained by the recruitment of TLR4 to the cell surface and its localization to the inframaft (Yvan-Charvet et al., 2008; Zhang et al., 2018; Zhu et al., 2010). Collectively, these results suggest a role for the aa 25–51 N-terminal domain of AIBP in TLR4 binding. AIBP lacking the TLR4-binding domain fails to alleviate CIPN allodynia

[0222] Unlike wtAIBP, mutAIBP, which lacks TLR4 binding sites, failed to inhibit LPS-induced TLR4 dimerization in BV-2 microglia (Figure 10A), but retained the overall ability to reduce lipid rafts (Figure 10B). Next, we tested the hypothesis that this targeting of TLR4 mediates the therapeutic effects of AIBP. Mice administered saline or mutAIBP it prior to LPS rapidly and to the same extent developed allodynia, whereas itwtAIBP prevented mechanical allodynia induced by LPS (Figure 10C). In a CIPN mouse model, itwtAIBP reversed established allodynia, with a sustained therapeutic effect that lasted for at least 14 days (Figure 10D). However, itmutAIBP induced only a modest, transient reversal of mechanical thresholds, which did not reach naive or baseline levels and lasted only for 2–3 days (Figure 10D). Mice were sacrificed on day 21 and lumbar spinal cords were analyzed. Notably, even at this late time point, cisplatin-induced polyneuritis continued to be associated with increased TLR4 dimerization and lipid rafts in spinal microglia, an effect observed at day 8 (Figure 1B and C), which was significantly reduced by itwtAIBP, but not mutAIBP (Figure 10E and F). These results support the hypothesis that AIBP targeting of TLR4 inflammasome mediates the majority of AIBP's therapeutic effects in mouse models of CIPN. Consideration

[0223] In this study, we report a novel mechanism of selective cholesterol depletion from inflammatories hosting TLR4 in spinal microglia as a new level of regulation of neuropathic pain in chemotherapy-induced peripheral neuropathy (Figure 10G) and possibly other neuropathies. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia induced spontaneous allodynia in naive mice showing similarity to cisplatin effects, and importantly, lack of expression of ABCA1 and ABCG1 in microglia abolished the ability of AIBP to reverse allodynia induced by LPS or cisplatin or to reduce inflammatories and TLR4 dimerization in spinal microglia. This singular effect on behavior and TLR4 dynamics was accompanied by differential gene expression in ABC-imKO microglia and the inability of AIBP to suppress inflammatory genes.

[0224] AIBP has a singular ability to disrupt inflammatories in activated cells, but has little effect on physiological lipid rafts in resting cells. We proposed that this is due to AIBP binding to TLR4, which is highly expressed on the surface of inflammatory cells, instructing these cells to deplete cholesterol (Miller et al., 2020; Woller et al., 2018). In this study, 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. This leads us to propose that AIBP represents a selective treatment directed at inflammatories, in contrast to the nonselective cholesterol removal provided by cyclodextrins, APOA1 and APOA1 mimetic peptides, or LXR agonists. Although mutated human AIBP lacking the N-terminal domain still binds to APOA1, and wild-type zebrafish Aibp, which does not naturally lack this N-terminal domain, still increases cholesterol efflux from endothelial cells, regulates angiogenesis, and orchestrates the emergence of hematopoietic stem and progenitor cells from hemogenic endothelial cells ( Fang et al., 2013 ; Gu et al., 2019 ), suggesting that there is a distinct, TLR4-independent mechanism for AIBP interaction with endothelial cells.

[0225] Intrathecal delivery of AIBP has a sustained therapeutic effect in mouse models of CIPN, observed over as long a period as 10 weeks in our previous study (Woller et al., 2018) and over a 2-week period in the present study. This is in contrast to the brief exposure of itAIBP. AIBP peaked at 30 min and nearly disappeared from both CSF and lumbar spinal cord tissue by 4 h. The dissociation between exposure and therapeutic effect suggests a disease-modifying effect of AIBP. Decreased CTxB binding and decreased percentage of TLR4 dimers in spinal microglia were observed for 24 h and even 2 weeks after a single itAIBP injection, indicating persistent disruption of inflammatories by AIBP, in contrast to their persistent presence in microglia from CIPN mice it-injected with saline. In addition to the targeted effect on TLR4 inflammatories, the AIBP disease-modifying effect may involve reprogramming of gene expression profiles in spinal microglia. Although AIBP reversed only 3% of all genes whose expression in spinal microglia was affected by CIPN, AIBP significantly reduced inflammatory gene expression and levels of inflammatory cytokines in spinal tissue induced by the cisplatin regimen. These include genes encoding cytokines and chemokines that have been described to have a role in CIPN, such as Il1b, Cxcl2, and Ccl2 (Brandolini et al., 2019; Oliveira et al., 2014; Pevida et al., 2013; Yan et al., 2019).

[0226] In addition to inflammatory genes, the cisplatin regimen induced transcriptional changes resembling gene signatures of disease-associated and neurodegenerative microglia (DAMs). CIPN was associated with altered expression of lipid metabolism genes and lipid droplet accumulation in microglia, which was reduced by AIBP (compound 7) treatment. Similar microglial lipid droplet phenotypes and transcriptomes were recently described to be associated with aging and neurodegeneration (Marschallinger et al., 2020; Nugent et al., 2020). Homeostatic genes that were downregulated during microglial transition to these pathological phenotypes (Masuda et al., 2019; Nugent et al., 2020; Prinz et al., 2019) were also downregulated in microglia from CIPN mice. CIPN-induced downregulation of microglial Abca1 and Abcg1 expression is a key factor for understanding the effects of AIBP. Although AIBP (compound 7) did not reverse the CIPN-associated reduction of Abca1 or Abcg1 mRNA, its ability to stabilize ABCA1 protein and promote cholesterol efflux (Zhang et al., 2016) may be sufficient to normalize microglial cholesterol metabolism. The effect of AIBP (compound 7) on allodynia was reproduced by itAPOA1 or LXR agonists, albeit transiently (Woller et al., 2018). Furthermore, a negative association between ABCA1 single nucleotide variants and quality of life scores in patients with painful bone metastases has been found (Furfari et al., 2017). However, we cannot exclude other mechanisms, unrelated to the reversal of a subset of genes affected by CIPN, by which AIBP (compound 7) reprograms microglia to confer a protective phenotype in pain-promoted conditions.

[0227] One of the key findings of this study was that in the absence of ABCA1 and ABCG1 in microglia, AIBP was unable to downregulate inflammatory genes and even upregulated some of them, as well as non-inflammatory pain-related Arc and Pi16 genes that regulate synaptic plasticity (Hossaini et al., 2010; Singhmar et al., 2020). Differential reprogramming by AIBP in WT and ABCA1 / ABCG1-deficient microglia may be dependent on the desmosterol converting enzyme Dhcr24, which regulates desmosterol and cholesterol content and, when reduced, is associated with foam cell formation and homeostatic anti-inflammatory responses (Spann et al., 2012). Importantly, AIBP (compound 7) also failed to reverse CIPN- or LPS-induced allodynia in ABC-imKO mice. These results indicate that the anti-inflammatory and anti-nociceptive effects of AIBP depend on cholesterol depletion from the plasma membrane and that, in the absence of an efflux mechanism, AIBP may actually promote inflammatory and cytotoxic effects.

[0228] Overall, the results of this study suggest that modulation of cholesterol content in the plasma membrane of spinal microglia has a profound effect on inflammatorhaft-derived cell signaling and subsequent gene expression of inflammatory and lipid metabolism genes, resulting in a culmination of nociceptive control under polyneuritis conditions. material and method

[0229] animal. Wild-type Abca1 fl / fl Abcg1 fl / fl , TLR4 fl / fl , Slc1a3-Cre ERT and Cx3cr1-Cre ERT2 Mice were all on a C57BL / 6 background and were either purchased from Jackson Lab (Bar Harbor, ME) or bred and weaned in-house. - / -The mice were a gift from Dr. Akira (Osaka University). fl / fl Mice were previously generated in our laboratory using ES cells derived from C57BL / 6 mice. The following mouse strains were bred in our laboratory: Apoa1bp fl / fl Cx3cr1-Cre ERT2 (AIBP-imKO), TLR4 fl / fl Cx3cr1-Cre ERT2 (TLR4-imKO), Abca1 fl / fl Abcg1 fl / fl Cx3cr1-Cre ERT2 (ABC-imKO), and Abca1 fl / fl Abcg1 fl / fl Slc1a3-Cre ERT (ABC-iaKO). All microglial conditional knockout mice used in the experiments were Cx3cr1-Cre ERT2 Mice were housed up to four per standard cage at room temperature and maintained on a 12:12-h light:dark cycle. All behavioral testing was performed during the light phase of the cycle. Food and water were both available ad libitum. All experiments were performed using male mice and in accordance with protocols approved by the University of California Institutional Animal Care and Use Committee (IACUC).

[0230] cell. BV-2 immortalized microglial cell line (Blasi et al., 1990) was cultured in Dulbecco's MEM containing 5% fetal bovine serum (FBS). Thioglycolate-elicited peritoneal macrophages were cultured in C57BL / 6 or TLR4 - / - Cells were harvested from mice and maintained in DMEM (Cellgro) supplemented with 10% heat-inactivated FBS (Cellgro) and 50 μg / mL gentamicin (Omega Scientific). HEK293 cells (RRID:CVCL_0045) were cultured in DMEM supplemented with 10% FBS and 50 μg / mL gentamicin. All cells were cultured at 37°C in a 5% CO2 atmosphere. Cell lines were used between passages 1-3.

[0231] Chemotherapy-induced peripheral neuropathy model. To develop chemotherapy-induced peripheral neuropathy (CIPN), animals were given intraperitoneal (ip) injections of cisplatin (2.3 mg / kg / injection; Spectrum Chemical MFG) on days 1 and 3. During the period of cisplatin administration, weight loss, behavioral changes, and mechanical allodynia were monitored and measured. Criteria for euthanasia were weight loss of more than 20% of body weight and irregular behavior. However, no animals required euthanasia.

[0232] Mechanical allodynia measurement. Animals were placed in bottomless cages made of clear plastic on a wire mesh surface and allowed to acclimate for at least 30 min before testing began. Tactile thresholds were measured using a series of von Frey filaments (Bioseb) ranging from 2.44 to 4.31 (0.02 to 2.00 g). Thresholds for 50% probability of withdrawal were recorded. Mechanical withdrawal thresholds were assessed using the up-down method (Chaplan et al., 1994) at input time points before treatment (baseline or day 0) and after treatment.

[0233] AIBP (Compound 7) or intrathecal delivery of saline. Mice were anesthetized using 5% isoflurane in oxygen for induction of anesthesia and 2% isoflurane in oxygen for maintenance of anesthesia. Intrathecal injections were performed according to (Hylden and Wilcox, 1980). Briefly, the lower back was shaved and disinfected, and the animals were placed in a prone position with the pelvis held between the thumb and index finger. 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 observation of a tail flick. 5 μL injections were administered at intervals of approximately 30 s. Drugs for intrathecal delivery were formulated in physiologically sterile 0.9% NaCl. Based on previous studies (Woller et al., 2018), the dose of AIBP (compound 7) for spinal delivery in these studies was 0.5 μg / 5 μL. After recovery from anesthesia, mice were assessed for normal motor coordination and muscle tone.

[0234] Intraperitoneal injection of tamoxifen for inducible Cre driver lines.In this study, we followed the Jackson Lab's tamoxifen induction protocol. Tamoxifen (Sigma-Aldrich) was dissolved in corn oil at a concentration of 10 mg / mL by shaking overnight at 37° C., wrapped in aluminum foil, and stored at 4° C. 200 μL of tamoxifen or vehicle (corn oil) was injected intraperitoneally every 24 hours for 5 consecutive days.

[0235] Ex vivo and in vitro TLR4 dimerization and lipid raft assays. The TLR4 dimerization assay uses two TLR4 antibodies for flow cytometry: MTS510, which recognizes TLR4 / MD2 as a monomer (in TLR4 units) but not as a dimer; SA15-21, which binds to any cell surface TLR4 regardless of its dimerization state (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. To assess TLR4 dimerization in vitro, BV-2 cells were preincubated with 0.2 μg / ml AIBP (compound 7) in serum-containing medium for 30 min, followed by incubation with LPS 100 ng / mL for 15 min. At the end of the incubation, cells were immediately placed on ice, washed once with PBS, and fixed with 4% formaldehyde for 10 min. Cells were then washed twice with ice-cold FACS buffer and incubated with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience) for 30 min on ice, followed by staining with PE-conjugated MTS510 antibody and APC-conjugated SA15-21 antibody (ThermoFisher and Biolegend, RRID:AB_2562503 and RRID:AB_466263, respectively) at 1:100 dilution and with CTxB-FITC (ThermoFisher) at 1:200 dilution for 30 min on ice. Cells were washed and analyzed using a FACSCanto II (BD Biosciences) flow cytometer.

[0236] For ex vivo assays, spinal cords were harvested by hydroextrusion (Kennedy et al., 2013), fixed in 4% formaldehyde, and kept on ice during processing. Single-cell suspensions were obtained from lumbar tissue using a neural tissue dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol. To remove myelin, Myelin Removal Beads II (Miltenyi Biotec) were added to the samples and incubated at 4 °C for 15 min, followed by separation on an LS column and MACS separator (Miltenyi Biotec). After isolation, cells were incubated with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience) for 30 min on ice, then stained with an antibody mix of 1:100 PerCP-Cy5.5-conjugated CD11b antibody (Biolegend, RRID:AB_893232), 1:100 rabbit anti-mouse TMEM119 antibody (Abcam, RRID:AB_2744673), PE-conjugated MTS510, APC-conjugated SA15-21 antibody (ThermoFisher, RRID:AB_2562503 and Biolegend, RRID:AB_466263, respectively) and 1:200 dilution of CTxB-FITC (ThermoFisher) for 45 min on ice, then cells were washed and stained with Alexa Fluor 4000 (Alexander, RRID:AB_40001). The cells were incubated with PECy7-conjugated anti-rabbit secondary antibody (1:250) for 30 min on ice. The cells were washed and analyzed using a FACSCanto II (BD Biosciences) flow cytometer.

[0237] For in vitro and ex vivo staining compensation, we used beads and / or single stained cells to compensate for signal overlap between channels, and used isotype controls of CD11b, MTS510 and SA15-21 antibodies with FMO to delineate gates. Data were analyzed by FlowJo (BD Bioscience, RRID:SCR_008520). From these data, we calculated the relative changes in lipid raft abundance and TLR4 dimer numbers in spinal microglia (0 dimers were randomly assigned to unstimulated or naive cells).

[0238] Immunofluorescence, confocal imaging and colocalization analysis. BV-2 cells were seeded on cover slips in 12-well plates and preincubated with 0.2 μg / ml AIBP in 5% serum-containing medium for 30 min, followed by incubation with 100 ng / ml LPS for 5 or 15 min. At the end of the incubation, cells were immediately placed on ice, washed once with PBS, and fixed with 4% formaldehyde for 10 min. Cells were washed twice with ice-cold PBS and incubated for 30 min in blocking buffer containing 5% FBS, then stained with 1:200 dilution of CTxB-Alexa555 and 1:100 dilution of mouse anti-TLR4 antibody (Abcam, RRID:AB_446735), or 1:100 rabbit anti-APOA1 antibody (Abcam) or 1:100 rabbit anti-ABCA1 (Novus Biological RRID:AB_10,000,630), washed, incubated with anti-rabbit Alexa 647-conjugated secondary antibody, and incubated with recombinant His-tagged ALOD4 and 1:100 FITC-conjugated anti-His secondary antibody (LSBio) to stain accessible cholesterol in the membrane. Cells were washed, coverslips were mounted on slides with Prolong Gold, and sealed. Slides were analyzed using a Leica SP 8 super-resolution confocal microscope equipped with Lightening deconvolution or STED.

[0239] To verify microglia-specific AIBP or ABCA1 / ABCG1 knockout, spinal cord tissues were harvested and post-fixed in 4% formaldehyde at 4°C. Tissues were then dehydrated in 30% sucrose and frozen in OCT until sectioning. Spinal cords were sliced ​​into 10 μm sections using a cryostat, and slides were stored at -20°C. Frozen sections were blocked with 2% FBS and 0.3% Triton® X100 solution, followed by incubation with 1:100 rabbit anti-AIBP antibody (kindly provided by Dr. Longhou Fang). Separate sections were stained with 1:100 rabbit anti-ABCA1 antibody or 1:100 rabbit anti-ABCG1 antibody (Novus Biological, RRID:AB_10000630 and RRID:AB_10125717) overnight at 4°C. Slides were washed and incubated with a 1:200 dilution of anti-rabbit Alexa 488 (Abcam, RRID:AB_2630356) or Alexa647-conjugated secondary antibody for 2 h, then washed three times and all sections were incubated with either Alexa488-conjugated IBA-1 antibody (Milipore-Sigma) or Alexa633-conjugated IBA1 antibody (Wako Chemicals, RRID:AB_2687911). Alternatively, 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 three times with PBS and mounted with Prolonged Gold with DAPI (Cell Signaling). Imaging of at least one slide from each animal was performed using a Leica SP8 confocal microscope with a 63X objective and Lightening deconvolution. Colocalization analysis was performed with ImageJ / FIJI (NIH, RRID:SCR_003070 / SCR_002285) using the Coloc2 tool.Thresholds, Pearson's R and Manders' coefficients above threshold, as well as masked colocalized mages, Costes P values ​​and pixel scatter plots were generated for each image. Depending on which channel represents the cell marker, tM1 or tM2 was used.

[0240] Expression and purification of ALOD4. The pALOD4 plasmid (Gay A., 2015) was obtained from Addgene (catalog number #111026, RRID: Addgene_111026) and used to transform E. coli competent cells BL21(DE3) and Amp + Positive colonies were selected on LB plates. After induction of expression with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and lysis, His-tagged ALOD4 was purified using a Ni-NTA agarose column with imidazole elution. The protein was dialyzed against PBS and the concentration was determined. Aliquots were stored at -80°C.

[0241] Cloning and expression of wtAIBP and mutAIBP in the baculovirus / insect cell system. AIBP (compound 7) was produced in a baculovirus / insect cell system to ensure post-translational modifications and endotoxin-free preparations as described in (Choi et al., 2018; Woller et al., 2018). Human wild-type (wt) AIBP and mutant (mut) AIBP, mouse wild-type AIBP, and zebrafish wild-type AIBP (Fang et al., 2013) were cloned into the pAcHLT-C vector behind a polyhedrin promoter. The vectors contain 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 harvested to obtain baculovirus stocks. Fresh Sf9 cells were infected with AIBP-producing baculovirus, and after 3 days cell pellets were harvested, lysed, sonicated, clarified by centrifugation, and the supernatant was loaded onto a Ni-NTA agarose column and eluted with imidazole. Proteins were dialyzed against saline and concentrations were measured. Aliquots were stored at -80°C.

[0242] AIBP in spinal cord tissue (Compound 7) Pharmacokinetics of. Knockout AIBP mice were used for pharmacokinetic studies. Intrathecal injections of AIBP (2.5 μg / 5 μL) were performed as previously described (Hylden and Wilcox, 1980), and CSF was collected after 15 min, 30 min, 1 h, 4 h, or 8 h as described (Liu and Duff, 2008). Briefly, capillary tubes (0.8 × 100 mm) were pulled using a micropipette puller. Mice were anesthetized using 3% isoflurane with a mixture of 50% oxygen and 50% room air. The skin on the neck was shaved and the mouse was placed on a stereotaxic apparatus. After wiping the surgical site, a sagittal incision was made in the skin below the occipital region. Subcutaneous tissue and muscle were incised to expose the dura. The pulled capillary tube was directly punctured into the cisterna magna, and uncontaminated samples were collected. After collecting the CSF, the capillary was drained into a PCR tube containing 50 μL of 0.09% NaCl, and the mouse was then perfused with 35 mL of 0.9% NaCl. The spinal cord was flushed by hydroextrusion with 5 mL of 0.9% NaCl. Spinal cord tissue was weighed and extracted with 1 g / 10 mL of complete N-PER™ Neuronal Protein Extraction Reagent (Thermo Fisher) on ice. After 10 min of incubation on ice, samples were centrifuged (10,000 × g for 10 min at 4 °C) to pellet cellular debris, and the supernatant was diluted 1:1 with 1% BSA-TBS. Plates were coated with BE-1 anti-AIBP monoclonal antibody (5 μg / mL), incubated with spinal cord extracts or CSF samples for 3 h, and detected with rabbit polyclonal anti-AIBP antibody followed by goat anti-rabbit ALP antibody (Sigma-Aldrich, RRID:AB_258103). The plates were read as above.

[0243] FACS sorting of spinal microglia for RNA-seq.Cell suspensions from lumbar spinal cord were prepared as above, except for the fixation step. Fresh tissues were processed and blocked with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience) for 30 min, then stained with a mix of 1:50 PE-Cy7 conjugated CD11b antibody (Biolegend, RRID:AB_312799), 1:50 rabbit anti-mouse TMEM119 antibody (Abcam, RRID:AB_2744673), 1:50 PerCP-Cy5.5 conjugated CD24 antibody (Biolegend, RRID:AB_1595491), cells were then washed and incubated with (1:200) secondary Alexa488 conjugated anti-rabbit antibody (Abcam, RRID:AB_2630356) and incubated on ice for 30 min, after which cells were washed and stained with 1:50 Alexa 647 conjugated Glast1 antibody (Novus Cells were incubated with a 1:100 dilution of 5% Fibroblast Growth Hormone (Biosciences) and Life / Death Ghost Red 780 dye (Cell Signaling) for 30 min on ice. Cells were washed with sorting buffer, filtered, and then sorted into lysis buffer using a BD FACS-Aria cell sorter (BD Biosciences). Three technical replicates, each containing 400 cells from the same animal, were sorted. See Figure S2A and B for the sorting strategy and analysis of the purity of sorted microglia.

[0244] RNA-seq library preparation, sequencing and quality control.We followed the low-input bulk-seq SmartSeq2 protocol from (Rosales et al., 2018). Sorted cells were hybridized to the poly(A) tail of mRNA with oligo(dT)+ in lysis buffer containing Triton® X-100, RNase inhibitor and oligo(dT)30-VN. Reagents for PCR amplification were added, followed by reagents for reverse transcription to construct cDNA libraries (no qPCR was performed at this point). Libraries were quantified and QC was performed using Qubit double stranded high sensitivity assays plus TapeStation high sensitivity D5000 screening tape. All samples were adjusted to 1 ng cDNA to feed into the NexteraXT protocol. QC checks were performed using Qubit double stranded high sensitivity assays plus TapeStation high sensitivity D1000 screen tape. Samples were subjected to qPCR and pooled and loaded onto NovaSeq for paired-end 50 × 50 reads using the NovaSeq S1 100 cycle kit.

[0245] Splice-aware alignment of FASTQ data was performed using STAR (Dobin et al., 2013) and quality control of sequenced data and alignments was performed by FASTQC (RRID:SCR_014583), QoRTs (RRID:SCR_018665) (Hartley and Mullikin, 2015) and MultiQC tools (RRID:SCR_014982) (Ewels et al., 2016). Counting of genes associated with reads was performed using STAR (RRID:SCR_015899).

[0246] Sequencing quality control indicated good data quality (MultiQC report). Two technical replicates (Y_10 and Y_30) were removed due to suboptimal gene coverage. We analyzed differential expression using the R package, DEseq2 (RRID: SCR_015687) (Love et al., 2014). We identified a total of 18,818 genes in lumbar spinal microglia with a cutoff set at more than 10 counts per million mapped reads CPM for at least three samples. One sample showed an extremely irregular distribution in PCA compared to all other samples, clustering in the top 500 most variable genes and was therefore excluded from further analysis. We confirmed the enrichment of microglia in our samples and data using a subset of 40 microglia-specific genes reported in (Butovsky et al., 2014) as well as genes specific to neurons (Nefl), oligodendrocytes (Omg) and astrocytes (Slc6a1) (Figure S2D). DEG determination was performed by DEseq2 binomial modeling using a reduced design without condition factors, using a likelihood ratio test (LRT) including all samples across all factors to determine the main effects of cisplatin and AIBP as well as all significant genes altered by these conditions. We used the LRT model compared to a reduced design without interaction terms of condition and genotype to identify genes regulated in a genotype (ABC-imKO)-dependent manner. Significant genes were filtered using adjusted P<0.05 and 5% FDR. Determination of gene clusters by gene expression patterns of identified significant genes was performed by DESeq 2 function: degpatterns. Pairwise comparisons after LRT of experimental groups were performed with Wald test with FDR of 5%. Volcano plots include significantly different genes with absolute fold change >1.5. Pathway enrichment and GO analysis were performed using a minimum of 3 genes and P < 0.05 in metascape.org (RRID:SCR_016620) (Zhou et al., 2019).

[0247] Co-immunoprecipitation assay for TLR4 binding. Pull-down assays of eTLR4 and wtAIBP or mutAIBP in tubes were performed by mixing 1 μg of eTLR4 (Sino Biological) with AIBP in PBS containing 0.5% Triton® X-100 and incubating for 1 h at room temperature. Samples were precleared by adding Protein A / G Sepharose beads for 30 min at room temperature, followed by addition of 1 μg of BE-1 monoclonal anti-AIBP antibody and incubation for 2 h. Protein A / G Sepharose beads were added and incubated for an additional 1 h, followed by washing five times with PBS containing 0.5% Triton® X-100, and samples were immunoblotted.

[0248] HEK293 cells (RRID:CVCL_0045) were transfected with Flag-eTLR4 and Flag-AIBP (wild type or one of the mutants) constructs. Thirty-six hours after transfection, cells were harvested and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail from Sigma). Cell lysates were preincubated with Protein A / G Sepharose beads for 30 min at 4°C and immunoprecipitated with mouse anti-TLR4 antibody (Abcam) overnight at 4°C. The next day, lysates were incubated with Protein A / G beads for 1 h at 4°C. Unbound proteins were removed by washing with lysis buffer and beads were run on a Bolt Bis-Tris gel (Invitrogen). Bound AIBP was detected by immunoblotting with anti-Flag antibody (Sigma).

[0249] ELISA binding assay.To assess AIBP-TLR4 binding, 96-well plates were coated with 5 μg / ml 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 2 μg / ml biotinylated BE-1 anti-AIBP monoclonal antibody. To assess AIBP-APOA1 binding, plates were coated with BSA, wtAIBP or mutAIBP, washed, blocked, and incubated with 5 μg / ml human APOA1 (a gift from Dmitri Sviridov, Baker Heart and Diabetes Institute, Melbourne, Australia), followed by incubation with biotinylated anti-APOA1 antibody (Academy Bio-Medical Company, RRID:AB_1238781). For both assays, Neutravidin-AP was added and incubated at room temperature for 45 min, followed by LumiPhos 530 (Lumigen) and incubation for 90 min, and luminescence was measured using a luminescence plate reader (BioTek, Winooski, VT).

[0250] Flow cytometry assay for AIBP cell binding. BV-2 microglial cells were stimulated or not with 100ng / mL LPS for 15 min, blocked with Tris-buffered saline (TBS) containing 1% BSA for 60 min on ice, and incubated with either 2μg / mL BSA or 2μg / mL AIBP for 2 h on ice. Cells were fixed and incubated with 1μg / mL FITC-conjugated anti-His antibody (LSBio) for 1 h at 4°C, and analyzed using a FACSCanto II (BD Biosciences) flow cytometer and FlowJo software (RRID:SCR_008520).

[0251] Cytokine measurement in spinal tissue by ELISA.Levels of IL-6 (DY406), IL-1β (DY401), MCP-1 (DY479), and MIP2 (DY452) in spinal cord lysates were measured using Mouse DuoSet ELISA (R&D Systems) according to the manufacturer's instructions.

[0252] Statistical analysis. For non-RNAseq datasets, results were analyzed using Student's t-test (for differences between two groups), one-way ANOVA (for multiple groups), or two-way ANOVA with Bonferroni post-hoc test (for multiple group time course experiments) using GraphPad Prism (RRID:SCR_002798). Differences between groups with P<0.05 were considered statistically significant. Figure legend

[0253] FIG. 1 demonstrates reversal of pain behavior and reduction of activated TLR4 dimers associated with pro-inflammatory lipid rafts (Inflammarafts) by wild-type (wt) AIBP protein in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN).

[0254] Figure 1. Chemotherapy-induced peripheral neuropathy alters TLR4 dimerization and lipid rafts in spinal microglia: reversal by AIBP. A, Withdrawal thresholds in WT mice in response to ip cisplatin (2 injections of 2.3 mg / kg / day) followed by a single dose of it saline (5 μl) or AIBP (compound 7) (0.5 μg / 5 μl). Naive mice received no injections. Data from two independent experiments (n=6 per group). B-C, CD11b indicating TLR4 dimerization. + / TMEM119 +Analysis of spinal microglial cells (B) and lipid raft content (C) measured by CTxB staining 24 h after it saline or AIBP, i.e., day 8 of the time course shown in A. Data from three independent experiments (n = 9 / group for TLR4 dimerization, n = 12 for lipid raft staining). D, BV-2 microglial cells were incubated with AIBP (compound 7) (0.2 μg / mL) or vehicle in complete medium for 30 min, followed by incubation with LPS (100 ng / mL) for 5 min. Scale bar, 5 μm. Bar graphs show Manders' tM1 coefficients. E-F, Male APOA1BP - / - Pharmacokinetics of itAIBP (2.5 μg / 5 μL) in the CSF (E) and lumbar spinal cord (F) of mice (n = 5). * , P < 0.05; ** , P < 0.01; *** , P<0.001. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in grouped analyses; one-way ANOVA with Tukey post-hoc test for multiple comparisons of three groups and imaging quantification.

[0255] FIG. 2 compares gene signature changes in naive mice, mice treated with the chemotherapeutic drug cisplatin, and mice treated with cisplatin and wild-type (wt) AIBP protein.

[0256] Figure 2. Gene expression in spinal microglia of CIPN mice. A–B, microglia (CD11b + TEMEM119 +) were FACS sorted from three groups shown in Figure 1A: WT naive, or injected with cisplatin (days 1 and 3), followed by it saline (5 μL) on day 7, or AIBP (compound 7) (0.5 μg / 5 μL) on day 8, terminated on day 8, and subjected to RNA-seq; n=3 biological replicates (mice) for naive and cisplatin / saline, n=2 for cisplatin / AIBP (each biological replicate was short-combined from three technical replicates from the same animal). A, Heatmap of DEGs across all samples (all technical replicates are shown in columns). Significant (adjusted P<0.01) up- or down-regulated genes showing main effects tested by LRT (likelihood ratio test). B, Groups of significant DEGs clustered based on their expression profile patterns in different treatment conditions. C, Pathway and GO enrichment analysis of upregulated (group 1 in panel 2B) and downregulated (group 2) genes induced by cisplatin treatment using adjusted P < 0.05 and absolute fold change > 1.5 and a minimum overlap of three genes within a pathway. Upregulated pathways are shown in red and downregulated pathways in blue.

[0257] Figure 3 compares differences in disease-associated microglia (DAM) gene expression signatures and lipid droplets in chemotherapy-treated mice versus naïve and wtAIBP-treated CIPN mice.

[0258] Figure 3. DAM- and lipid-related gene expression and lipid droplets in spinal microglia from CIPN mice.A–C, Same groups as in Fig. 2. A, Volcano plots of up- and down-regulated genes in spinal microglia from cisplatin-treated and naive mice. Cutoffs of adjusted P < 0.05 and absolute fold change > 1.5 are represented by light green dots. B, Heatmap representing disease-associated microglia (DAM) signature genes. C, Heatmap of log2-normalized gene counts scaled per row showing lipid-associated 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. 1A; n = 5 fields of 5 mice per group from 2 independent experiments. Scale bar, 20 μm. Mean ± SEM; * , P < 0.05 compared with the naïve group, tested by one-way ANOVA with Tukey's test for multiple comparisons in grouping analysis.

[0259] FIG. 4 summarizes the changes in gene expression in CIPN mice treated with wtAIBP protein.

[0260] Figure 4. Gene expression in spinal microglia of CIPN mice: AIBP (Compound 7) The effect.Experimental conditions and analysis are the same as in Figure 1; n = 2–3 biological replicates per group (each biological replicate was summarized from three technical replicates). A, Pathway and GO enrichment analysis of CIPN upregulated genes (group 3 in Figure 2B) downregulated by AIBP (compound 7) and CIPN downregulated genes (group 4) upregulated by AIBP (compound 7) using adjusted P < 0.05 and absolute fold change > 1.5 and minimum overlap of three genes in the pathway. Upregulated pathways are shown in red and downregulated pathways in blue. B, DEGs in spinal microglia induced by itAIBP. Adjusted P < 0.05 and Benjamini-Hochberg FDR < 5% were represented in volcano plots of upregulated and downregulated genes in mice treated with cisplatin / AIBP vs. cisplatin / saline. Cutoffs adjusted P < 0.05 and absolute fold change > 1.5 are indicated by light green dots. C, Heatmap of inflammatory genes in group 3 that are upregulated in CIPN and downregulated by AIBP. D, Cytokine protein expression in spinal tissues from WT naive, cisplatin / saline and cisplatin / AIBP groups; n=5 per group. E, Heatmap of inflammatory genes that are not induced by cisplatin but are 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 minimum overlap of 3 genes in the 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 ± SEM; * P<0.05 comparing the naïve group with the cisplatin / it saline group.

[0261] FIG. 5 demonstrates that cholesterol transporters ABCA1 and ABCG1 are required for AIBP-mediated reversal of pain in a mouse model of CIPN.

[0262] Figure 5. ABCA1 and ABCG1 expression in microglia regulates nociception and AIBP allodynia in a mouse model of CIPN. (Compound 7) Required for mediated reversal. A-B, BV-2 cells were incubated with AIBP (compound 7) (0.2 μg / mL) or vehicle in complete medium for 30 min, followed by incubation with LPS (100 ng / mL) for 5 min. Colocalization of accessible cholesterol in lipid rafts with ABCA1 (A) and APOA1 (B). Scale bar, 7 μm. Bar graphs show Manders' tM1 coefficient. C, Experimental design and timeline: Tamoxifen (TAM, 10 mg / mL, 200 μL / day), Cisplatin (2.3 mg / Kg), AIBP (compound 7) (0.5 μg / 5 μl) or saline (5 μl). D, Baseline (day 0) withdrawal thresholds before the start of cisplatin intervention. Data from three independent experiments (n=8 for vehicle-treated ABC-imKO mice, n=16 for TAM-treated ABC-imKO mice, n=16 for littermate Abca1 treated with TAM). fl / fl Abcg1 fl / fl (n = 15 for no-Cre[WT] mice). E, CD11b in naive WT and ABC-imKO mice at baseline (day 0). + TMEM119 + TLR4 surface expression and dimerization and lipid rafts (CTxB) in spinal microglia (n=5 for TLR4 surface expression and lipid raft content analysis for both groups, n=8 for WT and n=9 for ABC-imKO for TLR4 dimerization). F. Withdrawal threshold after it saline or AIBP (compound 7) (0.5 μg / 5 μl) followed by it LPS (0.1 μg / 5 μl) in TAM-induced ABC-imKO mice (n=4 per group). G-H, Withdrawal threshold after ip cisplatin and it saline or AIBP (0.5 μg / 5 μl) injection in TAM-induced ABC-imKO (G) and non-induced (vehicle) ABC-imKO (H) mice (n=6 per group); CD11b at day 8 in groups shown in panels G and H + TEMEM119 +Data from two independent experiments I–J, TLR4 dimerization (I) and lipid rafts (J) in spinal microglia. Mean ± SEM from two independent experiments (n=7–8). * , P < 0.05; *** , P<0.001. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in time-course analyses; t-test for two groups, and one-way ANOVA with Tukey post-hoc test for multiple comparisons of more than two groups.

[0263] FIG. 6 characterizes gene expression in ABC gene knockout mice.

[0264] Figure 6. Gene expression in spinal microglia of ABC-imKO mice. Microglia (CD11b + TEMEM119 +) were FACS sorted from three groups of ABC-imKO mice: naïve, or cisplatin-injected (days 1 and 3) followed by it saline (5 μL) on day 7, or AIBP (compound 7) (0.5 μg / 5 μL) injection on day 8; n=3 biological replicates (each biological replicate was abbreviated from three technical replicates). RNA-seq datasets from ABC-imKO and WT (not littermates) mice acquired in the same experiment. A, Top: Overlapping genes and pathways induced in naïve ABC-imKO microglia and shared with WT microglia from cisplatin-treated mice are indicated by purple lines connecting overlapping genes and blue lines connecting overlapping enriched pathways. Bottom: Venn diagram of genes upregulated in spinal microglia from WT cisplatin and ABC-imKO naïve mice. B. Enrichment pathway analysis of up- and downregulated genes induced by ABCA1 and ABCG1 knockdown in microglia using cutoff P<0.05, enrichment>1.5 and minimum overlap of three genes in the pathway. C, DEGs in naive spinal microglia of TAM-induced ABC-imKO mice. Adjusted P<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 of cisplatin-treated mice. 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 in either naive or cisplatin conditions.

[0265] FIG. 7: Comparison of gene expression in wild-type and ABC knockout mice treated with the AIBP protein provided herein (compound 7).

[0266] Figure 7. Microglial reprogramming by AIBP depends on ABCA1 / ABCG1 expression.A, Venn diagram comparing the effect of AIBP treatment on gene expression in cisplatin-induced CIPN in WT and ABC-imKO mice. B, Volcano plot representation of up- and down-regulated genes by AIBP treatment in CIPN comparing AIBP effects in ABC-imKO and WT mice. Cutoffs of adjusted P<0.05 and absolute fold change>1.5 are indicated by light green dots. C, Heatmap of normalized log2 gene counts of inflammatory genes altered by AIBP in an ABC-dependent manner (down-regulated by AIBP in WT microglia but up-regulated by AIBP in ABC-imKO). D, Heatmap of cholesterol synthesis and LXR-related genes comparing cisplatin and AIBP effects 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 genes upregulated by AIBP in ABC-imKO microglia using a cutoff P < 0.05, enrichment > 1.5 and a minimum overlap of three genes within a pathway.

[0267] FIG. 8 demonstrates that knockout of either AIBP or TLR4 contributes to pain behavior (nociception).

[0268] Figure 8. Microglial endogenous AIBP and TLR4 are important in nociception. A, Experimental design and timeline. Tamoxifen (TAM, 10 mg / mL, 200 μL / day); Cisplatin (2.3 mg / kg / day); AIBP (compound 7) (0.5 μg / 5 μl); Saline (5 μl). B, Baseline (day 0 in A) withdrawal thresholds before the start of cisplatin intervention. Mean ± SEM (n=15 for vehicle-treated, n=16 for TAM-treated AIBP-imKO mice, n=16 for TAM-treated littermate Apoa1bp fl / fl (n = 8 for no-Cre[WT] mice). C, WT and Cx3cr1-Cre ERT2(without floxed genes) mice were tested for withdrawal thresholds before (naive, day -7 of panel A timeline) and after (TAM, day 0) a tamoxifen injection regimen (10 mg / mL, 200 µL / day, 5 days). (n=5 per group; one animal was found dead in the WT+TAM group). No statistical differences were found. D-F, Withdrawal thresholds after ip cisplatin and it saline or AIBP (compound 7) injections in TAM-induced AIBP-imKO mice (C; n=6-7, data from two independent experiments), non-induced (vehicle) AIBP-imKO mice (D; n=4-5, data from two independent experiments), and in-house bred whole-body AIBP knockout mice (E; n=4 per group). G, Withdrawal thresholds in WT and tamoxifen-induced TLR4-imKO mice after cisplatin injection (n=4 for in-house bred wild type and n=7 for TLR4-imKO mice). Mean ± SEM * , P < 0.05; ** , P<0.01. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in grouped analyses; one-way ANOVA with Tukey post-hoc test for multiple comparisons of >2 groups.

[0269] Figure 9: Identifying sequence motifs that contribute to the binding of AIBP to TLR4. Figure 9. Identification of domains in the AIBP molecule involved in TLR4 binding.A, Human AIBP: signal peptide (aa1-24), a previously uncharacterized N-terminal domain (aa25-51), and the YjeF_N domain (aa52-288). B. Flag-tagged deletion mutants of human AIBP were co-expressed with Flag-tagged TLR4 ectodomain (eTLR4) in HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-TLR4 antibody and immunoblotted (IB) with anti-Flag antibody. C, His-tagged human (hu), mouse (mo), and zebrafish (zf) AIBPs lacking all signal peptides, expressed in the baculovirus / insect cell system, were combined in vitro with eTLR4-His, followed by IP with anti-TLR4 antibody and IB with anti-His antibody. D–H, Binding of His-tagged wild-type (wt, 25–288aa) and deletion mutant (mut, 52–288aa) human AIBP to eTLR4, APOA1 and microglia. IP of eTLR4 and wtAIBP or mutAIBP in vitro with anti-AIBP antibody; blot and quantification from three independent experiments (D). ELISA (E) with plates coated with eTLR4 and incubated with wtAIBP or mutAIBP (n=3). ELISA (F) with plates coated with BSA, wtAIBP or mutAIBP and incubated with APOA1. Flow cytometry (n=6) (F) and confocal imaging (G) showing binding of wtAIBP and mutAIBP (2 μg / mL) to BV-2 microglial cells unstimulated or treated with LPS (100 ng / mL) for 15 min. Detection with anti-His antibody (flow) and anti-TLR4 antibody (imaging). Scale bar, 10 μm. Mean ± SEM *** , P < 0.001; ** , P < 0.01; * , P<0.05; ns, not significant. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in time-course analyses; t-test for two groups and one-way ANOVA with Tukey post-hoc test for multiple comparisons of more than two groups.

[0270] FIG. 10 demonstrates that mutant AIBP that does not bind to TLR4 does not reverse pain behavior in CIPN mice, suggesting a model for AIBP modulation of TLR-mediated pain.

[0271] Figure 10. Intrathecal delivery of AIBP lacking the TLR4-binding domain fails to reduce CIPN allodynia. A-B, TLR4 dimerization (A) and lipid rafts (B) in BV-2 cells pretreated with wtAIBP or mutAIBP (0.2 μg / mL) and stimulated with 100 ng / mL LPS for 15 min. Mean ± SEM (n = 7 for control, n = 5 for mutAIBP, n = 9 for LPS, and n = 8 for wtAIBP in TLR4 dimerization analysis; n = 8 for control, n = 13 for mutAIBP, LPS, and wtAIBP in lipid raft analysis; data from two independent experiments). C, Withdrawal thresholds in WT mice treated with itAIBP (0.5 μg / 5 μL) or saline (5 μL) followed by itLPS (0.1 μg / 5 μL); n = 5 per group. D, Withdrawal thresholds in WT mice in response to i.p. cisplatin (2.3 mg / kg / day) followed by it.wtAIBP (0.5 μg / 5 μL), mutAIBP (0.5 μg / 5 μL) or saline (5 μL). Naive mice received no injections (n ​​= 7 for naive group, n = 8 for wtAIBP and mutAIBP groups, n = 9 for it.saline group; data from two independent experiments). E–F, CD11b from lumbar spinal cord of mice in the experimental group shown in panel D on day 21. + / TMEM119 + TLR4 dimerization (E) and lipid rafts (F) in microglia (n=7–9; data from two independent experiments). * , P < 0.05; ** , P < 0.01; *** , P<0.005. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in time-course analysis; and one-way ANOVA with Tukey post-hoc test for multiple comparisons of groups greater than 2. G, Effect of CIPN and AIBP (compound 7) treatment on microglial gene expression and lipid droplet accumulation. Black dots in the plasma membrane and ER represent cholesterol.

[0272] Figure 11 postulates a model for the exposure of the TLR4-binding site of AIBP in modified AIBP sequences: Figure 11. Model for unfolding or exposing the cryptic N-terminal domain in the AIBP molecule. This figure summarizes the results of the experiments shown in Figures 12-14, which demonstrate that in native AIBP, the N-terminal domain (green) is hidden, cryptic, or not sufficiently exposed to mediate AIBP binding to TLR4 (top panel). Extending the N-terminus with additional amino acids (orange) alters the AIBP conformation, making the N-terminal domain (green) of AIBP accessible for TLR4 binding (bottom panel). Figure 12. An example of the amino acid sequence of an exemplary engineered AIBP provided herein. The amino acid sequence of the extended AIBP molecule is represented in the bottom panel of Figure 11. Blue letters, amino acids of the native AIBP sequence; green box, TLR4-binding sequence (amino acids 25-51 of the human AIBP sequence); black letters and red box, additional amino acids. FIG. 13 demonstrates TLR4 binding of specific modified AIBP sequences derived from a baculovirus expression system.

[0273] Figure 13. TLR4 binding of various engineered forms of AIBP. All proteins were expressed and purified from the baculovirus / insect cell system. The top diagram, representing His-d24AIBP, corresponds to the amino acid sequence shown in Figure 12. The amino acid sequence below the top diagram shows the sequence of the orange boxed "cleavable His tag". All other diagrams show the various modifications and corresponding changes in the amino acid sequence that were introduced into the AIBP molecule. The green "N-terminal domain" box represents the amino acid 25-51 sequence of native AIBP. The right column shows the results of co-immunoprecipitation experiments of AIBP variants with recombinant ectodomain of TLR4. Figure 14 demonstrates TLR4 binding of specific modified AIBP sequences from a mammalian expression system.

[0274] Figure 14. TLR4 binding of various engineered forms of AIBP, cont. 1. All proteins were co-expressed with full-length TLR4 in mammalian systems. SS, secretion signal, corresponds to amino acids 1-24 in the human AIBP sequence. The right column shows the results of co-immunoprecipitation of AIBP variants with cell lysates and TLR4.

[0275] Figure 15. Various AIBP constructs for optimizing the structure of TLR4 affinity: Baculovirus / insect cell expression system.

[0276] FIG. 16 confirms that N-terminal modifications to AIBP are required for TLR4 binding in an E. coli expression system: Figure 16. TLR4 binding of various engineered forms of AIBP, cont. 2. All proteins were expressed and purified from E. coli. The right column shows the results of co-immunoprecipitation experiments of AIBP variants with recombinant ectodomain of TLR4. There was no TLR4 binding with AIBP variants d24AIBP-his or d51AIBP-his.

[0277] Figure 17A-D (or Figure S1 or Supplementary Figure 1) provides validation of the specificity of the TLR4 antibodies used for flow cytometry and microscopy and also shows TLR4 dimerization and lipid rafts measured in dorsal root ganglion macrophages:

[0278] FIG. 17A graphically depicts flow cytometry of single cell suspensions from the spinal cord of WT (left image) and Tlr4− / − mice (right image) showing TLR4-APC and TLR4 / MD2-PE antibody staining of CD11b+ (PercP-Cy5.5) / TMEM199+ (Pe-Cy7) microglia:

[0279] FIG. 17B shows confocal images of peritoneal-induced macrophages from WT and Tlr4− / − mice co-stained with F4 / 80-FITC and TLR4-647 antibodies; scale bar, 5 μm; and,

[0280] Figures 17C-D graphically depict flow cytometry analysis of CD11b+ DRG macrophage cells showing TLR4 dimerization (Figure 17C) and lipid raft content measured by CTxB staining (Figure 17D) 24 hours after it saline or AIBP, i.e., day 8 of the time course shown in Figure 17A; data from two independent experiments (n=5 for control and AIBP groups, n=9 for cisplatin it saline group).

[0281] Figure 18A-E (or Figure S2, or Supplementary Figure 2) shows the FACS sorting strategy of spinal microglia, quality control and phenotypic control for RNA-seq:

[0282] FIG. 18A shows the sorting strategy for lumbar CD11b+TMEM119+ 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 CD11b and TMEM119;

[0283] FIG. 18B shows flow cytometry analysis of sorted microglia measuring the purity of sorted cells, including TMEM119 and CD11b, SSC-A and GLAST1, and SSC-1 and CD24, and the absence of GLAST1+ astrocytes or CD24+ neurons;

[0284] Figure 18C shows microglia lineage analysis using a heatmap of microglia-specific genes. Log+1 of normalized counts from all samples was calculated for 40 microglia-specific genes described in Butovsky et al., 2014, as well as three genes that are specifically expressed at low levels in microglia but at high levels in neurons (Nefl), oligodendrocytes (Omg), and astrocytes (Slc6a1);

[0285] Figures 18D-E show heat maps of CIPN-suppressed genes upregulated by AIBP (group 4) (Figure 18D) and CIPN-induced genes downregulated by AIBP (group 3) in wild-type mice (Figure 18E); Log2-normalized gene counts scaled by rows and columns represent all technical replicates of three biological samples.

[0286] Figure 19A-D (or Figure S3 or Supplementary Figure 3) provides immunohistochemical validation of conditional knockout of ABCA1 and ABCG1 in spinal microglia of tamoxifen-induced ABC-imKO mice:

[0287] IHC of spinal cord cryosections from vehicle and tamoxifen-induced ABC-imKO mice shows ABCA1 and ABCG1 staining and colocalization with IBA1 (microglia), NeuN (neurons) and GFAP (astroglia). Slides were mounted with PrologGold with DAPI. Confocal images were acquired with a 63x objective and analyzed for colocalization with ImageJ software. Colocalization masks and Pearson's R values, Mander's colocalization coefficients above threshold and randomized Costes P values ​​were calculated as described in Methods for at least one slide for each animal in the experiment. Representative images and values ​​shown correspond to one animal per condition. Scale bar, 50 μm.

[0288] Figure 20A-E (or Figure S4 or Supplementary Figure 4) shows tactile allodynia data for tamoxifen-treated WT mice in itLPS and CIPN experiments. We also provide additional RNA-seq data for ABC-imKO-dependent genes, and cisplatin effects on ABC-imKO versus WT mice. As controls for ABC-imKO mice, in-house bred WT littermate mice were subjected to a tamoxifen regimen (TAM, 200 μL / day, 10 mg / mL, for 5 consecutive days) followed by (Figure 20A) injection of itAIBP (0.5 μg / 5 μL) or saline (5 μL) followed 2 h later by itLPS (0.1 μg / 5 μL) (n=4 for it saline, n=5 for itAIBP); (Figure 20B) ip injection of cisplatin (2.3 mg / Kg) on ​​days 1 and 3 followed by it injection of AIBP (0.5 μg / 5 μL) or saline (5 μL) on day 7 (n=4 per group). Tactile allodynia (withdrawal threshold) was measured using von Frey filaments. Mean ± SEM * , P<0.05. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in time-course analyses. C, ABC-imKO mice were injected with TAM as above, then cisplatin, followed by it saline (5 μL), AIBP (0.5 μg / 5 μL) or hp-β-CD (0.25 mg / 5 μL) on day 7. Tactile thresholds 24 h after it injection are shown. Mean ± SEM (n = 3–4 / group). **, P<0.01. One-way ANOVA with Dunnett's multiple comparison test. Figure 20D, Heatmap of differentially regulated genes across all conditions (naive, induced by cisplatin / saline or cisplatin / AIBP) regulated by the ABC-imKO scheme. All significant genes (condition:genotype) obtained from a likelihood ratio test using a reduced model without interaction terms. Log2 normalized gene counts scaled by rows and columns represent all technical replicates of 2-3 biological samples from each group. Figure 20E, Heatmap of pathway enrichment of cisplatin-upregulated genes in WT and ABC-imKO microglia using cutoffs P<0.05, enrichment >1.5 and a minimum overlap of 3 genes within a pathway. Heatmap represents common and specific pathways enriched by cisplatin in both genotypes.

[0289] Figure 21 (or Figure 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. Figure 21A: IHC of spinal cord cryosections from vehicle and tamoxifen-induced AIBP-imKO mice showing colocalization of AIBP staining with IBA1 (microglia), NeuN (neurons) and GFAP (astrocytes). Slides were mounted with PrologGold containing DAPI. Confocal images were acquired with a 63x objective and analyzed for colocalization with ImageJ software. Colocalization masks and Pearson's R values, Mander's colocalization coefficients and randomized Costes' P values ​​were calculated as described in the methods for at least one slide for each animal in the experiment. Representative images and values ​​shown correspond to one animal per condition. Scale bar, 50 µm. Figure 21B: Sandwich ELISA using BE-1 as a capture antibody in a microtiter plate. A rabbit polyclonal anti-AIBP antibody was used to detect dose-response curves for wtAIBP and mutAIBP. No statistical difference was found for BE-1 affinity for wtAIBP and mutAIBP using two-way ANOVA with Bonferroni post-hoc test for multiple comparisons. Example 2. Structural determinants of AIBP binding to TLR4

[0290] This example summarizes the results of pull-down experiments to test the binding of different AIBP variants expressed in insect, mammalian or bacterial systems to the ectodomain of TLR4. The results of this example are unexpected in that the depictions of activity in Figure 13 and Figure 14 demonstrate that not all N-terminal modifications expose the TLR4 binding domain. As an example, the predicted cleavage products of the N-terminal His tag that contains the cleavage site do not demonstrate TLR4 binding. These unexpected data suggest that N-terminal modifications to AIBP polypeptides have specific amino acid composition requirements.

[0291] Pull-down assays were performed using compounds provided herein. Compounds 3, 7, 8 or 9 and other constructs described in Figures 13 and 14 were purified from either baculovirus (BD Bioscience) or CHO (ExpiCHO, Expression Systems, ThermoFisher) cell expression systems and incubated with TLR4 protein (Sino biological). Pull-down was performed using anti-AIBP antibodies as described. TLR4 bound to AIBP was detected by Western blot using anti-his antibody (both modified AIBP and TLR4 have his tag). Detailed experimental information on the pull-down method is provided in Example 1.

[0292] In an alternative assay, modified AIBP and TLR4 were transfected into HEK293 cells. For this study, transfected Flag-AIBP (exemplified for compounds 5 and 6) and Flag-TLR4-his constructs were expressed, and transfected cells were harvested and lysed. Cell lysates were co-immunoprecipitated with anti-TLR4 antibody, and then immunoblotted with anti-flag antibody. Detailed experimental information on the pull-down method is provided in Example 1. Example 3: Efficacy demonstrated in an exemplary model: Asthma Decreased expression of AIBP in bronchial epithelial cells from asthmatic patients:

[0293] Apolipoprotein AI-binding protein (AIBP; gene name APOA1BP or NAXE) is a secreted protein (1) that promotes the removal of excess cholesterol from activated cells, including primary alveolar macrophages, endothelial cells, and microglia (2-4). We have demonstrated that pulmonary surfactant can function as a cholesterol receptor when incubated with alveolar macrophages (4). In addition, ApoA-I is found in bronchoalveolar lavage fluid (BALF) (5). These findings suggest that cholesterol efflux occurs not only in blood and tissues, but also in lung air spaces. AIBP binds to surfactant protein B and enhances cholesterol efflux from alveolar macrophages to surfactant (4). This normalizes lipid raft content in the plasma membrane, reduces inflammatory signaling in alveolar macrophages, and reduces the expression of inflammatory cytokines. In response to inhalation-induced LPS lung injury, AIBP is secreted into BALF (4). Additionally, AIBP promotes mitophagy, helps maintain mitochondrial function, and reduces oxidative stress in macrophages. (6) The hypothesis that AIBP expression helps protect against inflammation means that increasing AIBP levels in the lungs could have therapeutic effects.

[0294] Because of the broad anti-inflammatory protection afforded by AIBP in the lung ( 4 ) and other tissues ( 3 , 7 ), in this study we investigated whether pulmonary expression of endogenous AIBP has an impact in asthmatics and whether inhaled AIBP can reduce lung inflammation and alleviate airway hyperresponsiveness in a mouse model of asthma.

[0295] Immunohistochemistry of postmortem human lung tissue obtained from nonasthmatic subjects revealed a pattern of predominant AIBP protein expression in bronchial epithelial cells. Interestingly, AIBP expression was significantly decreased in bronchial epithelial cells of postmortem lungs of subjects with asthma (see Figure 22A). Furthermore, primary bronchial epithelial cells isolated from postmortem lungs of asthmatic subjects had significantly lower APOA1BP mRNA expression compared to nonasthmatic subjects (see Figure 21D). Similar to human asthma, expression of endogenous AIBP in bronchial epithelium was significantly decreased in house dust mite (HDM)-challenged mice compared to control mice administered intranasal PBS (see Figure 22C). This pattern of decreased AIBP expression in bronchial epithelium after acute HDM challenge of mice was not observed in a mouse acute lung injury model (4). Furthermore, the lung cell types expressing the highest levels of AIBP differed in the two models, with the highest AIBP expression in acute lung injury observed in recruited inflammatory cells (i.e., neutrophils and macrophages).(4) In contrast, the main recruited inflammatory cells after acute HDM challenge (i.e., eosinophils) did not express high levels of AIBP.

[0296] Because endogenous AIBP expression is reduced in asthma (Figure 21), and administration of AIBP as a recombinant protein or via adeno-associated viral delivery has resulted in anti-inflammatory and protective effects in neuroinflammation and neuropathic pain (7), vascular inflammation and atherosclerosis (3), and acute lung injury (4), we tested whether intranasal delivery of recombinant AIBP (compound 7) would have therapeutic effects in a mouse model of asthma.

[0297] Compound 7 was administered 2 hours prior to administration of HDM.

[0298] Intranasal administration of HDM to female mice four times weekly induces pulmonary eosinophilic inflammation and airway hyperresponsiveness (AHR) to methacholine challenge (8). Two doses of compound 7, 2.5 and 25 μg, or vehicle (PBS) were administered weekly to 8-week-old C57BL / 6J female and male mice by intranasal instillation 2 h prior to intranasal HDM. Intranasal compound 7 produced no obvious adverse effects. As expected, HDMI-challenged female mice pretreated with PBS developed AHR. In contrast, pretreatment with compound 7 dose-dependently reduced HDM-induced AHR, with the 25 μg dose resulting in a near-complete inhibition of AHR (Figure 23A), and also produced a dose-dependent reduction in HDM-induced lung and BAL eosinophilia (Figure 23B-C). In this model, HDM does not result in significant changes in the numbers of alveolar macrophages or neutrophils. (8) Similar results were obtained with compound 7 in male mice (Figures 23D-F).

[0299] Taken together, our studies demonstrate that AIBP expression is significantly decreased in human bronchial epithelial cells from asthmatic patients compared to nonasthmatic patients, and also in bronchial epithelial cells after HDM challenge in a mouse model of asthma. This result is consistent with the observation of reduced ApoA-I levels in BALF from asthmatic patients compared to nonasthmatic patients (5). Since airway epithelial and bronchial inflammation are major components of asthma leading to airway smooth muscle contraction, airway obstruction, and asthma exacerbation (9), restoring the levels of AIBP, which has anti-inflammatory properties, may represent a novel therapeutic strategy for asthma. Our results with intranasal administration of compound 7, which shows therapeutic effects in an acute HDM mouse model of asthma, support this proposition. Since inhaled corticosteroids (ICS) are the cornerstone of treatment for moderate / severe asthma, further testing in preclinical models and then human subjects with asthma is needed to determine whether compound 7 has an additive anti-inflammatory effect on asthma control when combined with ICS and / or is an alternative anti-inflammatory to ICS in asthmatic subjects who do not respond well to ICS or have side effects from ICS. Materials and Methods for Example 3 Human lung specimens

[0300] Postmortem human lungs from asthmatic and nonasthmatic subjects were procured by the Arkansas Regional Organ Recovery Agency and the National Disease Research Interchange and delivered to the Lung Cell Biology Laboratory at the Arkansas Children's Research Institute. Immunohistochemistry was performed at the University of California, San Diego. Subjects were classified as asthmatic if they had a physician diagnosis of asthma documented in their hospital medical records and were using asthma medications at the time of death. Subjects were classified as nonasthmatic if they did not have a physician diagnosis of asthma and no asthma medication use was documented in their hospital medical records at the time of death. The acquisition of deceased donor tissue was reviewed by the University of Arkansas for Medical Sciences Institutional Review Board and determined not to be human subjects research. The study was approved by the University of California, San Diego Human Research Protections program. Human bronchial epithelial cells

[0301] Primary bronchial epithelial cells were isolated from the bronchi of postmortem lungs. Briefly, the bronchi were dissected and the interior of each bronchus was scraped with a Cell Lifter (Corning, Inc.) to obtain bronchial epithelial cells. Bronchial epithelial cells were harvested and cultured in CnT-17 medium (Cellntec, Bern, Switzerland). These primary bronchial epithelial cells were >95% pure as assessed by E-cadherin expression by flow cytometry. Immunohistochemistry of human and mouse lungs

[0302] Paraffin-embedded lung sections were stained using a cocktail of mouse anti-human and anti-mouse AIBP monoclonal antibodies A7 and BE-1 (6, 7) developed in our laboratory, mixed in a 1:2 ratio. Mouse and human AIBP are highly homologous, so both antibodies recognize mouse and human proteins. 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 μm length of bronchial basement membrane in human specimens. AIBP expression in mouse lungs was measured using the mean gray value tool in Image J (NIH), and values ​​in the cytosol of bronchial epithelium of bronchioles with inner diameters of 150–200 μm were normalized to values ​​in adjacent alveoli. The operator was blinded to the identity of the samples. Quantification of APOA1BP mRNA

[0303] To quantify APOA1BP mRNA in human bronchial epithelial cells from asthmatic and nonasthmatic patients, total RNA from each cell sample was processed for RT-qPCR as previously described (8). Briefly, samples were treated with RNA-STAT-60 (TelTest) and reverse transcribed with oligo-dT and the SuperScript II kit (Life Technologies). qPCR was performed using TaqMan PCR Master Mix and TaqMan primers for human APOA1BP (Hs.PT.58.22278956, Integrated DNA Technologies, Coralville, IA). The relative amount of APOA1BP mRNA was normalized to the relative amount of the housekeeping gene hypoxanthine phosphoribosyltransferase-1 (HPRT1). Preparation of compound 7

[0304] Briefly, compound 7 was expressed in a baculovirus / insect cell system to ensure post-translational 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 >90% pure with no detectable aggregates (HPLC-SEC) and less than 0.2EU / mg residual endotoxin. Storage stability testing of compound 7 for up to 6 months at -80°C or 1 week at 4°C did not indicate any loss in its potency or purity. Acute HDM mouse model of asthma

[0305] All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. Eight-week-old wild-type C57BL / 6J mice (male and female) were administered 100 μg of intranasal HDM (Dermatophagoides pteronyssinus) extract (Greer Laboratories) on days 0, 7, 14, and 21 as previously described (8). Two hours before each HDM administration, 50 μl of either PBS control or a 2.5 μg or 25 μg dose of compound 7 solution was administered intranasally. Control groups received intranasal PBS instead of HDM. On day 24, airway hyperresponsiveness to methacholine was assessed as described (8), and mice were sacrificed to collect BAL and lungs. BAL was collected by lavage with 1 mL of PBS via the tracheal catheter, centrifuged, and the pellet was resuspended in 1 mL of PBS. After determining the total cell counts in the BAL, the number of differential cells was quantified on Wright-Giemsa-stained slides (8). Lung eosinophil counts were quantified in the peribronchial space of paraffin-embedded lung sections stained with anti-mouse major basic protein (MBP) rabbit polyclonal antibody (kindly provided by the Mayo Medical Education and Research Foundation). Results are expressed as the number of positively stained peribronchial cells per bronchiolar with an internal diameter of 150–200 μm. At least five bronchioles were counted on each slide. The operator was blinded to the identity of the samples.

[0306] statistics: All results are presented as mean ± SEM. A statistical software package (GraphPad Prism) was used for analysis. The Mann-Whitney test was used for analysis of two groups. When comparing more than two groups, two-way or one-way ANOVA with post-hoc Tukey's multiple comparison test was used. A P value of less than 0.05 was considered statistically significant. Figure legend for Example 3

[0307] Figure 22. Decreased AIBP expression in bronchial epithelium. A. Postmortem lung specimens from non-asthmatic and asthmatic human subjects stained with anti-AIBP antibody. Quantification of AIBP positive bronchial epithelium (n=6). B. APOA1BP mRNA in bronchial epithelial cells isolated from non-asthmatic (n=9) and asthmatic (n=11) subjects normalized to HPRT1. C, Lungs from mice administered vehicle or 100 μg HDM intranasally four times weekly stained with anti-AIBP antibody. Quantification of AIBP staining in bronchial epithelium (n=8). Mean ± SEM; * , P < 0.05; *** , P<0.001.

[0308] Figure 23. RFT1081 reduces airway hyperresponsiveness and eosinophilic lung inflammation in an acute HDM model of asthma in female and male mice. Female (A-C) and male (D-F) C57BL / 6J mice received four weekly intranasal instillations of 2.5 μg or 25.0 μg RFT1081 or PBS. Two hours later, mice received intranasal instillations of 100 μg HDM or vehicle. Three days after the last challenge, mice were tested for airway resistance to methacholine (A, D) and lungs (B, E) and BAL (C, F) were harvested for analysis as indicated. Mean ± SEM; n = 8 mice per group. * , P < 0.05; ** , P < 0.01; **** , P < 0.001; *** , P<0.0001 Example 4: Efficacy demonstrated in exemplary methods: Glaucoma

[0309] AAV-AIBP protects retinal ganglion cells and their axons and improves visual function in experimental glaucoma.

[0310] Glaucoma DBA / 2J(D2) mouse model. The advantage of using the genetic D2 model together with age-matched non-glaucomatous control D2-Gpnmb+ mice is that it can recapitulate the chronic IOP elevation of human glaucoma, with retinal pathology developing at approximately 9-10 months of age (1, 2). We recognize that, like other animal models, D2 has limitations because in these mice, glaucoma-like pathology develops secondary to anterior segment abnormalities with adhesions and pigment dispersion (1, 2). In preliminary studies, we have demonstrated that Apoa1bp - / - A significant increase in cholesterol content in the retina of D2 mice was observed compared to that of WT mice (see FIG. 24A), suggesting that AIBP deficiency induces excessive cholesterol accumulation in the retina. Notably, in 10-month-old glaucomatous D2 mice, the inventors found that D2-Gpnmb + We also found that the cholesterol content of the retina was significantly elevated compared to that of glaucomatous D2 mice (Figure 24B). Therefore, we tested whether overexpression of AIBP by in vivo delivery of AAV-AIBP could reverse excess cholesterol accumulation, protect RGCs and their axons, and preserve the central visual pathway in glaucomatous D2 mice. The AAV serotype we used in our mouse model was AAV-DJ / 8, which expressed mouse AIBP with a fibronectin signal peptide ensuring robust protein secretion. We intravitreally injected AAV-Null or AAV-AIBP at 5 months of age and analyzed tissue samples (retina, optic nerve head, and brain) at 10 months of age. We assessed RGC and their axonal survival by staining for RNA-binding proteins with multiple splicing (RBPMS) and neurofilament 68 (NF 68) and preservation of the central visual pathway by cholera toxin subunit B (CTB) labeling of the superior colliculus (SC), which represents the entire retinal projection via active uptake and transport. 3、4After AAV-AIBP injection, AIBP protein expression was detected in the retina at 10 months of age (Figure 24E). AAV-AIBP, but not AAV-Null, significantly reduced cholesterol content (Figures 24C and 24D) and protected RGCs in the central and peripheral regions of glaucomatous D2 retinas (Figures 24G and 24H). Furthermore, we observed a significant improvement in CTB transport to the SC (Figures 24J-M), suggesting that AAV-AIBP helped maintain the structural and functional integrity of the optic nerve.

[0311] Microbead-induced ocular hypertension model. Recently, we successfully developed a mouse model of microbead-induced ocular hypertension, which showed significant loss of RGCs 6 weeks after treatment in 4-month-old C57BL / 6J mice (Figure 25). To further verify the protective effect of AAV-AIBP on RGCs and visual function in vivo, we intravitreally injected AAV-Null or AAV-AIBP 3 weeks before microbead injection. AAV-AIBP significantly reduced RGC death (see Figure 25C) and, importantly, improved visual dysfunction (see Figure 25D).

[0312] Optic nerve crush (ONC) model. ONCs serve as a useful model of traumatic optic neuropathy as well as glaucomatous injury, as they similarly induce RGC death and degeneration. 5 We intravitreously injected AAV-Null or AAV-AIBP 3 weeks before ONC, and then evaluated RGC survival by RBPMS staining 1 week after ONC. Overexpression of AIBP was found to protect RGCs from ONC damage (Figure 26).

[0313] Taken together, these findings demonstrate that AAV-delivered AIBP expression reduces cholesterol content, protects RGCs and their axons, inhibits microglial activation (not shown), and preserves visual function in three different in vivo models of glaucomatous neurodegeneration. Legend for figures of Example 4

[0314] FIG. 24. AAV-AIBP attenuates retinal neurodegeneration in glaucomatous DBA / 2J(D2) mice. A and B, Apoa1bp - / - Mouse. Filipin staining for cholesterol (A). Quantification of filipin intensity in the inner retina (B). C-M, Glaucoma D2 mice. Filipin staining for cholesterol (C). Quantification of filipin intensity in the inner retina (D). Confirmation of AIBP expression in the retina by immunoblotting with anti-His antibody (E). IOP measurement (F). RBPMS (green) positive RGCs in the peripheral retinal region (G). Quantitative analysis of RGC survival in the central and peripheral retina (H). NF68 (green) positive axons in the glial layer (I). CTB labeling (red) and Brn3a (green) in the retina (J). CTB labeling in the SC (K and L). Quantification of CTB intensity in the SC (M). Mean ± SEM; n = 5-8 retinas. * P < 0.05, ** P < 0.01, *** P<0.001, **** P<0.0001 (one-way ANOVA, Turkey's multiple comparison test). Scale bars: 20 μm (A and C) and 50 μm (G and I).

[0315] Figure 25. AAV-AIBP attenuates retinal neurodegeneration and improves visual function in a microbead-induced hypertensive mouse model. A, Time course of IOP in microbead-injected eyes. B, Representative images of the peripheral region of the retina by TUJ1 staining 6 weeks after microbead injection. C, Quantitative analysis of RGC survival in the central region of the retina. D, Visual function measured by PERG analysis. Mean ± SM; n = 5–8 retinas. * P < 0.05, *** P < 0.001 and **** P<0.0001 (one-way ANOVA, Tukey's multiple comparison test). Bar: 50 μm.

[0316] Figure 26. AAV-AIBP alleviates retinal neurodegeneration and mouse optic nerve crush model. A, Representative images of RBPMS-positive RGCs in the central region of the retina after ONC injury; B, Quantitative analysis of RGC survival in the central and peripheral regions of the retina. Mean ± SEM; n = 5-8 retinas. * P < 0.05, ** P < 0.01, **** P<0.0001 (one-way ANOVA, Tukey's multiple comparison test). Bar: 50 μm. References for Example 1 [ka] [ka] [ka] [ka] [ka] References for Example 3 [ka] References for Example 4 [ka]

[0317] Several embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. An isolated or recombinant polypeptide, comprising an ApoA-I binding protein (AIBP) amino acid sequence and an amino acid sequence N-terminal to the AIBP amino acid sequence, the amino acid sequence N-terminal to the AIBP amino acid sequence is at least 8 amino acids long, 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 long; the amino acid sequence N-terminal to the AIBP amino acid sequence is capable of inducing unfolding of, exposing or otherwise making accessible a cryptic domain in the AIBP amino acid sequence for binding of the polypeptide to TLR4 under relevant physiological conditions; Optionally, "relevant physiological conditions" refers to the relevant physiological conditions experienced by said polypeptide compound in vivo when provided to a subject in need thereof by administration; provided that the amino acid sequence N-terminal to the AIBP amino acid sequence does not consist of a His tag and a proteolytic cleavage site that results in the loss of the His tag when acted upon under said conditions.

2. 2. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence is composed of 8 to 40 consecutive amino acid residues, between 3 and 12 of which are independently selected from the group consisting of arginine (R), histidine (H) and lysine (K).

3. 2. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence is a secretion signal amino acid sequence.

4. 4. The isolated or recombinant polypeptide of claim 3, wherein the secretory signal amino acid sequence is a fibronectin secretory signal domain, an immunoglobulin heavy chain secretory signal domain, an immunoglobulin kappa light chain secretory signal domain, or an interleukin-2 signal peptide secretory signal domain.

5. 5. The isolated or recombinant polypeptide of claim 4, wherein the fibronectin secretory signal domain is MLRGPGPGRLLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 24).

6. 2. The isolated or recombinant polypeptide of claim 1, wherein the AIBP sequence is hAIBP (SEQ ID NO: 6) or d24hAIBP (SEQ ID NO: 8).

7. 2. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence N-terminal to the AIBP amino acid sequence is composed of six consecutive histidine amino acid residues (HHHHHH; SEQ ID NO: 1) N-terminal to the TLR4 binding domain of the AIBP amino acid sequence.

8. 8. The isolated or recombinant polypeptide of claim 7, wherein the polypeptide has a thrombin cleavage domain interposed between the N-terminus of the TLR4 binding domain of the ApoA-I binding protein sequence, the thrombin cleavage domain having one or more amino acid deletions and / or mutations within said domain thereby rendering it functionally inoperable.

9. The amino acid sequence on the N-terminal side of the AIBP amino acid sequence is 2. The isolated or recombinant polypeptide of claim 1, wherein the polypeptide is MSPIDPMGHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) or MSPIDPMGHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 39), each having an amino acid mutation in its thrombin cleavage domain thereby rendering it functionally inoperable.

10. The amino acid sequence on the N-terminal side of the AIBP amino acid sequence is TETGKSKR (SEQ ID NO:26), MDYKDHDGDYKDHDIDYKDDDDKLAAANS (SEQ ID NO: 33), and MSPIDPMGHHHHHGRRRASVAAGILVPAASPGLDGICSR (SEQ ID NO: 44) 2. The isolated or recombinant polypeptide of claim 1, selected from the group consisting of:

11. 11. The isolated or recombinant polypeptide of claim 10, wherein the AIBP amino acid sequence is an AIBP amino acid sequence of a mammalian AIBP amino acid sequence.

12. 12. The isolated or recombinant polypeptide of claim 11, wherein the mammalian AIBP amino acid sequence is the AIBP amino acid sequence of a human AIBP amino acid sequence.

13. 13. The isolated or recombinant polypeptide of claim 12, wherein the human AIBP amino acid sequence is a full-length amino acid sequence of 288 amino acid residues comprising the NCBI Reference Sequence: NP_658985.

2.

14. 13. The isolated or recombinant polypeptide of claim 12, wherein the human AIBP amino acid sequence comprises the NCBI Reference Sequence: NP_658985.2, and wherein amino acids 1 to 24 are deleted from the AIBP amino acid sequence.

15. 15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 14 and at least one excipient suitable for parenteral administration.

16. 16. The pharmaceutical composition of claim 15, wherein parenteral administration is by intrathecal injection or intrathecal implant.

17. A nucleic acid compound comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 14.

18. An expression vector comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 14.

19. The expression vector of claim 18 , wherein the expression vector is a recombinant adenovirus.

20. A method for treating or preventing the alleviation of symptoms of ApoA-I in a subject by administering an ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) or increasing the level of an ApoA-I binding protein: -neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain comprises Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - nerve or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. - Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the post-nerve or tissue injury pain or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection. - post-operative or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g. cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post traumatic stress disorder, traumatic war neurosis or post traumatic stress syndrome (PTSS), - primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - glaucoma or other inflammatory eye diseases, - pulmonary inflammation and asthma, - acute respiratory distress syndrome (ARDS), - sepsis, a viral infection, optionally said virus being influenza or a coronavirus (optionally said coronavirus is COVID-19) or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), V), Cytomegalovirus (CMV), Rubivirus or Rubella virus, Enterovirus, viral meningitis, Rhinovirus, Varicella-Zoster virus or Chickenpox virus, Orthopoxvirus or Variola or Smallpox virus, Epstein-Barr virus (EBV), Adenovirus, Hantavirus, Flaviviridae or Dengue virus, Zika virus, or Chikungunya virus infection, or comorbidities thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, 2. A formulation or pharmaceutical composition for treating, ameliorating, preventing, reversing or reducing the severity or duration of a symptom of, or reducing the severity of, said symptom, comprising The formulation or pharmaceutical composition comprises: (i) a recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition having a heterologous amino terminal amino acid sequence of at least about 10 amino acids, or between about 5-20 amino acids, or between about 10-100 amino acids, or between about 20-80 amino acids, or between about 30-50 amino acids, or having 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 at the amino terminus of said AIBP, which amino acid residues or peptides are not present in wt AIBP or are non-native (to AIBP) (also referred to as AIBP variants as provided herein); Optionally, the heterologous amino terminal amino acid sequence comprises a peptide tag, optionally wherein the peptide tag comprises a multi-histidine (multi-his) tag, optionally wherein the multi-his tag comprises 6 histidines (HHHHHH (SEQ ID NO: 1)), or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more histidine residues; Optionally, the heterologous amino terminal amino acid sequence comprises an enzymatic cleavage site, and optionally, the enzymatic cleavage site comprises a thrombin cleavage site; Optionally, the heterologous amino terminal amino acid sequence comprises a secretory signal, optionally, the secretory signal comprises a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin kappa light chain secretory peptide, or an interleukin-2 signal peptide; Optionally, the heterologous amino terminal amino acid sequence comprises the amino acid sequence MSPIDPMGHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO:2); said variant being capable of unfolding or exposing or making accessible a cryptic domain in said AIBP molecule comprising amino acids 25-51 that mediates the binding of AIBP to TLR4; and (ii) a recombinant nucleic acid encoding the APOA1BP polypeptide of (i), Optionally, a nucleic acid expressing or encoding an APOA1BP polypeptide or a polypeptide having APOA1BP polypeptide activity is included in an expression vehicle, vector, recombinant virus, or equivalent, Optionally, the vector or virus is or comprises an adenoviral or adeno-associated viral (AAV) vector, a retrovirus, a lentiviral vector, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector; Optionally, the AAV vector comprises or is: Adeno-associated virus (AAV), or adenovirus vectors; AAV serotypes or variants AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ / 8™ (Cell Biolabs, Inc., San Diego, CA) Diego, CA) A rhesus monkey-derived AAV, or the rhesus monkey-derived AAV AAVrh.10hCLN2, AAV capsid mutants or AAV hybrid serotypes, an organotropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41 mutant; wherein, optionally, the AAV has been engineered to increase the efficiency of targeting a particular cell type that is non-permissive to wild-type (wt) AAV and / or to improve the efficiency of infecting only a cell type of interest; Optionally, the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications including: 1) transencapsidation, 2) adsorption of bispecific antibodies to the capsid surface, 3) engineering of a mosaic capsid, and / or 4) engineering of a chimeric capsid; and (iii) any of the formulations or pharmaceutical compositions of (i)-(ii), wherein the recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein is or comprises all or a portion of a human or mammalian APOA1BP, or AIBP1 or AIBP2 sequence; (iv) A formulation or pharmaceutical composition of any of (i)-(iii) formulated for in vivo administration; or formulated for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, Optionally, said formulation or pharmaceutical composition, or said recombinant, peptidomimetic or synthetic APOA1BP, or biological equivalent of APOA1BP, or a nucleic acid encoding said APOA1BP, or a vector containing a nucleic acid encoding said APOA1BP, is carried in a nanoparticle, particle, micelle, or liposome or lipoplex, polymersome, polyplex, or dendrimer, which may, optionally, further comprise or express a cell or CNS penetrating moiety or peptide, or a CNS targeting moiety or peptide; or (v) any of (i)-(iv) formulations or pharmaceutical compositions formulated as nanoparticles, liposomes, tablets, pills, capsules, gels, geltabs, liquids, powders, emulsions, lotions, aerosols, sprays, lozenges, aqueous or sterile or injectable solutions, or implants (e.g., intrathecal implants); Optionally, the subject is a human or an animal. Formulation or pharmaceutical composition.

21. 21. A kit comprising a recombinant or isolated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15 or 16, or a formulation or pharmaceutical composition according to claim 20.

22. 21. Use of a recombinant or isolated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15 or 16, or a formulation or pharmaceutical composition used according to claim 20, in the manufacture of a medicament.

23. The recombinant or isolated polypeptide according to any one of claims 1 to 14, the pharmaceutical composition according to claim 15 or 16, the formulation or pharmaceutical composition according to claim 20 for use in the following conditions: -neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain comprises Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - nerve or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. - Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the post-nerve or tissue injury pain or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection. - post-operative or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g. cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post traumatic stress disorder, traumatic war neurosis or post traumatic stress syndrome (PTSS), - primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - glaucoma or other inflammatory eye diseases, - pulmonary inflammation and asthma, - acute respiratory distress syndrome (ARDS), - sepsis, a viral infection, optionally said virus being influenza or a coronavirus (optionally said coronavirus is COVID-19) or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), V), Cytomegalovirus (CMV), Rubivirus or Rubella virus, Enterovirus, viral meningitis, Rhinovirus, Varicella-Zoster virus or Chickenpox virus, Orthopoxvirus or Variola or Smallpox virus, Epstein-Barr virus (EBV), Adenovirus, Hantavirus, Flaviviridae or Dengue virus, Zika virus, or Chikungunya virus infection, or comorbidities thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease 20. Use in the manufacture of a medicament for treating, ameliorating, preventing, reversing or reducing the severity or duration of said symptoms, or for reducing the severity of said symptoms.

24. Symptoms of: -neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain comprises Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - nerve or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. - Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the post-nerve or tissue injury pain or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection. - post-operative or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g. cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post traumatic stress disorder, traumatic war neurosis or post traumatic stress syndrome (PTSS), - primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - glaucoma or other inflammatory eye diseases, - pulmonary inflammation and asthma, - acute respiratory distress syndrome (ARDS), - sepsis, a viral infection, optionally said virus being influenza or a coronavirus (optionally said coronavirus is COVID-19) or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), V), Cytomegalovirus (CMV), Rubivirus or Rubella virus, Enterovirus, viral meningitis, Rhinovirus, Varicella-Zoster virus or Chickenpox virus, Orthopoxvirus or Variola or Smallpox virus, Epstein-Barr virus (EBV), Adenovirus, Hantavirus, Flaviviridae or Dengue virus, Zika virus, or Chikungunya virus infection, or comorbidities thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, 20. A formulation, pharmaceutical composition or therapeutic combination for use in a method for treating, ameliorating, preventing, reversing or reducing the severity or duration of a symptom of, or for reducing the severity of, a symptom of, The formulation or therapeutic combination comprises a recombinant or isolated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15 or 16, or a formulation or pharmaceutical composition according to claim 20, A formulation, pharmaceutical composition or therapeutic combination, characterized in that said formulation or therapeutic combination is administered to an individual or patient in need thereof.

25. 1. A method for exposing a latent (or hidden, unexposed, inaccessible) N-terminal TLR4-binding domain of an ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide, comprising adding to a native AIBP polypeptide a heterologous amino-terminal amino acid sequence of at least about 10 amino acids, or between about 5-50 amino acids, or between about 10-100 amino acids, or adding about 20-80 amino acids, or between about 30-50 amino acids, or adding 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 to the amino terminus that are not present in wt AIBP or are non-native (non-AIBP) amino acid residues or peptides; Optionally, the heterologous amino terminal amino acid sequence comprises a peptide tag, optionally wherein the peptide tag comprises a multi-histidine (multi-his) tag, optionally wherein the multi-his tag comprises at least six histidines (HHHHHH (SEQ ID NO:1)), or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more histidine residues; Optionally, the heterologous amino terminal amino acid sequence comprises an enzymatic cleavage site, and optionally, the enzymatic cleavage site comprises a thrombin cleavage site; Optionally, the heterologous amino terminal amino acid sequence comprises a secretory signal, optionally comprising a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin kappa light chain secretory peptide, or an interleukin-2 signal peptide; Optionally, the heterologous amino terminal amino acid sequence is MSPIDPMGHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) The method includes:

26. A composition comprising a recombinant or isolated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15 or 16, or a formulation or pharmaceutical composition according to claim 20, for use as a pharmaceutical.

27. ​​Symptoms of: -neuropathic pain, - Inflammation-induced neuropathic pain, Optionally, the inflammation-induced neuropathic pain comprises Toll-like receptor 4 (TLR4)-mediated inflammation-induced neuropathic pain. - nerve or CNS inflammation, Optionally, the neuronal or CNS inflammation comprises TLR4-mediated neuronal or CNS inflammation. - Allodynia, Optionally, the allodynia comprises TLR4-mediated allodynia. - pain following nerve or tissue injury or neuropathic pain, Optionally, the post-nerve or tissue injury pain or neuropathic pain is caused or triggered by, or is a sequela of, trauma, chemotherapy, arthritis, diabetes, or viral infection. - post-operative or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g. cisplatin-induced CIPN or allodynia), - a neurodegenerative disease or condition, optionally a chronic or progressive neurodegenerative disease or condition, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, traumatic brain injury (TBI), post traumatic stress disorder, traumatic war neurosis or post traumatic stress syndrome (PTSS), - primary headache, migraine or cluster headache as appropriate, - Hyperalgesia, - glaucoma or other inflammatory eye diseases, - pulmonary inflammation and asthma, - acute respiratory distress syndrome (ARDS), - sepsis, a viral infection, optionally said virus being influenza or a coronavirus (optionally said coronavirus is COVID-19) or a human immunodeficiency virus (HIV) or a virus causing an HIV infection, (optionally influenza A, B or C), or a hepatitis virus, Rous sarcoma virus (RSV), Paramyxoviridae or measles virus, Paramyxovirus or mumps virus, herpes simplex virus (HSV), V), Cytomegalovirus (CMV), Rubivirus or Rubella virus, Enterovirus, viral meningitis, Rhinovirus, Varicella-Zoster virus or Chickenpox virus, Orthopoxvirus or Variola or Smallpox virus, Epstein-Barr virus (EBV), Adenovirus, Hantavirus, Flaviviridae or Dengue virus, Zika virus, or Chikungunya virus infection, or comorbidities thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease 21. A composition comprising a recombinant or isolated polypeptide according to any one of claims 1 to 14, a pharmaceutical composition according to claim 15 or 16, or a formulation or pharmaceutical composition for use in claim 20 for treating, ameliorating, preventing, reversing or reducing the severity or duration of a symptom of, or reducing the severity of, said symptom.

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