Compositions and methods for targeting inflammatory or activated cells to treat or improve inflammatory conditions and pain.

Abstract

Providing compositions and methods for targeting inflammatory cells or activated cells to treat or improve inflammatory conditions and pain. [Solution] A method is provided for modifying the amino acid sequence and increasing the expression level of ApoA-I binding protein for treating neuropathic pain, CNS inflammation, allodynia, post-neuropathy pain, postoperative pain, chemotherapy-induced peripheral neuropathy, neurodegenerative diseases or conditions such as Alzheimer's disease, hyperalgesia, primary headaches such as migraines and cluster headaches, glaucoma or other inflammatory eye diseases, pneumonia, asthma, HIV infection, vasculitis, atherosclerosis and cardiovascular diseases.

Description

[Technical Field]

[0001] Related applications This Patent Treaty (PCT) international application claims the benefit of priority under Section 119(e) of the United States Patent Act, of U.S. Provisional Patent Application (USSN) 63 / 162,714, filed on 18 March 2021. The aforementioned application is expressly incorporated herein by reference in its entirety for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated herein by reference 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 to this invention. Technical field

[0003] The present invention generally relates to medicine, inflammation, pain management, and cell biology. In particular, alternative embodiments provide methods for modifying the structure and increasing the expression level of ApoA-I binding proteins (APOA1BP, AIBP, or AI-BP, also known as NAD(P)HX epimerase or NAXE) to treat, improve, prevent, reverse, and reduce the severity and / or duration of neuropathic pain, CNS inflammation, allodynia, post-neuropathy pain, postoperative pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced allodynia), neurodegenerative diseases or conditions such as Alzheimer's disease, hyperalgesia, primary headaches such as migraines and cluster headaches, glaucoma, pneumonia and asthma, HIV infection and its comorbidities, and / or vascular inflammation and cardiovascular diseases. Alternative embodiments provide methods for treating, improving, preventing, reversing, and reducing the severity and / or duration of neuropathic pain, allodynia, hyperalgesia, neurodegenerative diseases or conditions such as Alzheimer's disease, primary headaches such as migraines, glaucoma or other inflammatory eye diseases, pneumonia and asthma, acute respiratory distress syndrome (ARDS), sepsis, viral infections including influenza, coronavirus (e.g., COVID-19) or HIV infection, or their comorbidities, and / or vasculitis, atherosclerosis and cardiovascular diseases, comprising the administration of formulations and pharmaceutical compositions comprising structural modification and recombinantly modified APOA1BP polypeptide or protein, or peptide mimetic or synthetic APOA1BP, or a bioequivalent thereof, which is human or mammalian APOA1BP. [Background technology]

[0004] background Apolipoprotein AI-binding protein, or ApoA-I-binding protein (AIBP), also known as NAXE or NAD(P)HX epimerase, is a protein discovered during screening for proteins that physically associate with apolipoprotein AI.

[0005] The regulation of cholesterol metabolism in relation to neurodegeneration, particularly Alzheimer's disease (AD), has attracted considerable attention, partly due to the strong association between APOE gene polymorphisms and AD risk. However, the role of cholesterol regulation as a factor in the development of chronic pain remains unknown. Chemotherapy-induced peripheral neuropathy (CIPN) is one of the debilitating adverse effects of anti-cancer drug use during cancer treatment, affecting more than 50% of patients receiving 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 neurological disorders (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-related 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 adapter molecules MyD88 and TRIF, alone or in combination, attenuates and prevents hyperalgesia and allodynia in cisplatin-treated mice (Hu et al., 2018; Pevida et al., 2013; Yan et al., 2019). However, the cell types in which TLR4 activation causes allodynia remain unclear. [Overview of the project]

[0006] overview In alternative embodiments, isolated or recombinant polypeptides or chimeric polypeptides are provided, wherein the polypeptide consists of (or comprises) an ApoA-I binding protein (AIBP) amino acid sequence and the N-terminal amino acid sequence of the AIBP amino acid sequence. The N-terminal amino acid sequence of an AIBP amino acid sequence consists of at least 8 amino acids, or the N-terminal amino acid sequence of an AIBP amino acid sequence has an amino acid length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more amino acids. The N-terminal amino acid sequence of the AIBP amino acid sequence can, under relevant physiological conditions, induce unfolding of potential domains within the AIBP amino acid sequence, expose potential domains, or otherwise make them accessible in order to bind the polypeptide to TLR4. Where necessary, "relevant physiological conditions" refers to the relevant physiological conditions that the polypeptide compound experiences in vivo when it is administered to a target requiring the polypeptide compound. However, this is conditional on the N-terminal amino acid sequence of the AIBP amino acid sequence not consisting of a His tag and a protein cleavage site that, when acted upon under the aforementioned conditions, causes the His tag to disappear.

[0007] In alternative embodiments of the isolated or recombinant polypeptides, or chimeric polypeptides, provided herein, - The N-terminal amino acid sequence of the AIBP amino acid sequence consists of approximately 8 to 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 approximately 3 to 12, 8 to 20, or 10 to 40 amino acid residues are independently selected from the group consisting of arginine (R), histidine (H), and lysine (K); -The N-terminus of the N-terminal amino acid sequence of the AIBP amino acid sequence is or contains a secretory signaling amino acid sequence, which may optionally be a fibronectin secretory signaling domain, an immunoglobulin heavy chain secretory signaling domain, an immunoglobulin κ light chain secretory signaling domain, or an interleukin-2 signaling peptide secretory signaling domain (or contains these), which may optionally be MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 24): -AIBP sequences are hAIBP (coded in EQ code 6 or EQ code 5) or d24hAIBP (coded in EQ code 21 or EQ code 20); - The N-terminal amino acid sequence of the AIBP amino acid sequence consists of approximately 6, or between 5 and 40, consecutive histidine amino acid residues (e.g., HHHHHH (SEQ ID NO: 1)) at the N-terminal end of the TLR4 binding domain of the AIBP amino acid sequence; - The polypeptide has (or contains) a thrombin cleavage domain interposed between the N-terminuses of the TLR4 binding domain of the ApoA-I binding protein sequence, and the thrombin cleavage domain has one or more amino acid deletions and / or mutations within this domain to render it functionally inoperable; -The N-terminal amino acid sequence of the AIBP amino acid sequence is, MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) or MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), each having amino acid mutations in its thrombin cleavage domain to render it functionally inoperable; -The N-terminal amino acid sequence of the AIBP amino acid sequence is TETGKSKR (SEQ ID NO: 26), MDYKDHDGDYKDHDIDYKDDDDKLAAANS (Sequence ID 33), or Selected from the group consisting of MSPIDPMGHHHHHHGRRRASVAAGILVPAASPGLDGICSR (Sequence ID 7); - The AIBP amino acid sequence is (or derived from) the mammalian AIBP amino acid sequence, and, where necessary, the mammalian AIBP amino acid sequence is (or derived from) the human AIBP amino acid sequence; and / or - The human AIBP amino acid sequence is a full-length amino acid sequence of 288 amino acid residues having the NCBI reference sequence: NP_658985.2, or, if necessary, the human AIBP amino acid sequence is a human AIBP amino acid sequence having the NCBI reference sequence: NP_658985.2, with amino acids 1-24 deleted from the aforementioned AIBP amino acid sequence (or includes this).

[0008] In alternative embodiments, pharmaceutical compositions or formulations are provided comprising (or including) polypeptide compounds provided herein and at least one excipient suitable for parenteral administration (formulated for parenteral administration). In alternative embodiments, parenteral administration is by intrathecal injection or intrathecal implantation, or by intravenous or intraocular injection.

[0009] In alternative embodiments, nucleic acids are provided that consist of (or include) nucleic acid sequences encoding polypeptides provided herein.

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

[0011] Alternative embodiments provide methods and uses for treating, improving, preventing, reversing, or reducing the severity or duration of the following symptoms, or for reducing the severity of the following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, nerve or CNS inflammation includes TLR4-mediated nerve or CNS inflammation. - Allodynia, If necessary, allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary the virus is influenza or coronavirus (where coronavirus is COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary influenza A, B or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or co-occurring conditions thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, (By adding ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) to the subjects, or by increasing the level of ApoA-I binding protein), Here, the method includes: (a) A step of providing a formulation or pharmaceutical composition comprising: (i) Having a heterologous (or non-native, or non-AIBP or non-wild-type (wt), or any sequence not present in wild-type (wt)AIBP) amino-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, or wt 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 AIBP amino terminus, which are amino acid residues or peptides that are not present in AIBP or are non-native to AIBP (also referred to as AIBP variants as provided herein), If necessary, the N-terminal amino acid sequence of the AIBP amino acid sequence shall not consist of the His tag and a protein cleavage site that, when acted upon under physiological conditions (e.g., in the cellular environment or equivalent, or intracellularly), would result in the loss of the His tag. If necessary, heterologous (or non-wild-type or non-natural) amino-terminal amino acid sequences (or amino acid residues) include a peptide tag, if necessary, the peptide tag includes a multi-histidine (multi-his) tag, if necessary, the multi-his tag includes six histidine (HHHHHH (SEQ ID NO: 1)), or three, four, five, six, seven, eight, nine, ten, eleven, twelve, three, four, nine, twenty or more histidine residues. If necessary, heterologous (or non-wild-type) amino-terminal amino acid sequences include enzyme cleavage sites, and if necessary, enzyme cleavage sites include thrombin cleavage sites. If necessary, heterologous (or non-wild-type) amino-terminal amino acid sequences include secretory signals, and if necessary, secretory signals include fibronectin secretory signals (e.g., SEQ ID NO: 24), immunoglobulin heavy chain secretory signals or immunoglobulin κ light chain secretory peptides, or interleukin-2 signaling peptides. If necessary, heterologous (or non-wild-type) amino-terminal amino acid sequences include the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), Herein, all of these AIBP variants provided herein (or all AIBP amino acids, including amino-terminal sequences not present in wt AIPB, or heterologous amino-terminal peptides or amino acid residues) can, under physiological conditions, enable, expose, or make accessible the potential domain within the AIBP molecule containing amino acids 25-51 that mediates the binding of AIBP to Toll-like receptor 4 (TLR4) polypeptide, or serve that purpose (in other words, the AIBP variants provided herein have a TLR4-binding domain exposed to the extracellular environment so that the AIBP variants provided herein can bind to the TLR4 polypeptide under physiological conditions), together with recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compounds or compositions; (ii)(i) Recombinant nucleic acids encoding the APOA1BP polypeptide, If necessary, nucleic acids expressing or encoding APOA1BP polypeptide or polypeptides having APOA1BP polypeptide activity are included in the expression vehicle, vector, recombinant virus, or equivalent. If necessary, the vector or virus may be an adenovirus vector or adeno-associated virus (AAV) vector, retrovirus, lentiviral vector, herpes simplex virus, human immunodeficiency virus (HIV), or synthetic vector, or may include these. If necessary, the AAV vector may include or be: Adeno-associated virus (AAV), or adenovirus vector, AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ / 8 (trademark) (Cell Biolabs, Inc., San Diego, CA) AAV derived from rhesus macaques, or AAVrh.10hCLN2 derived from rhesus macaques. AAV capsid variant or AAV hybrid serotype, Organ-directed AAV, or cardiac-directed AAV, or cardiac-directed AAVM41 variant, Here, where necessary, the AAV is engineered to increase its efficiency in targeting specific cell types that are intolerant to wild-type (wt) AAV, and / or to improve its efficiency in infecting only the cell types of interest. If necessary, the hybrid AAV is retargeted or manipulated as a hybrid serotype by one or more modifications including: 1) transcapsidation, 2) adsorption of bispecific antibodies to the capsid surface, 3) manipulation of mosaic capsids, and / or 4) manipulation of chimeric capsids, with recombinant nucleic acids; (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), wherein the recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein is, if necessary, all or part of a human or mammalian APOA1BP, AIBP1, or AIBP2 sequence, or comprises such a 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, If necessary, a formulation or pharmaceutical composition, or a recombinant, peptide mimetic or synthetic APOA1BP, or a bioequivalent of APOA1BP, or a nucleic acid encoding APOA1BP, or a vector containing a nucleic acid encoding APOA1BP, may be supported on nanoparticles, particles, micelles, or liposomes or lipoplexes, polymerosomes, polyplexes, or dendrimers, and may further contain or express a cell or CNS permeable portion or peptide or a CNS targeting portion or peptide, as necessary, with the formulation (iii) formulation or pharmaceutical composition; (v) Providing a formulation or pharmaceutical composition comprising any formulation or pharmaceutical composition of (iii) to (iv) formulated as nanoparticles, liposomes, tablets, pills, capsules, gels, gel tablets, liquids, powders, emulsions, lotions, aerosols, sprays, lozenges, aqueous or sterile or injectable solutions, or implants (e.g., intrathecal implants); (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 preparation or pharmaceutical composition of (a)(iii) or (a)(iv) to a subject or individual in need thereof, wherein the subject or individual is a mammal, human, or animal, as necessary. This is intended to treat, improve, prevent, reverse, or reduce the severity or duration of the following symptoms, or to reduce the severity of the following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, nerve or CNS inflammation includes TLR4-mediated nerve or CNS inflammation. - Allodynia, If necessary, allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary the virus is influenza or coronavirus (where necessary coronavirus is COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary influenza A, B or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus 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 disease.

[0012] In alternative embodiments, a kit is provided comprising recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein; recombinant nucleic acid; and / or formulation or pharmaceutical composition used in the method provided herein, and optionally including instructions for carrying out the method provided herein.

[0013] In alternative embodiments, the use of the formulations or pharmaceutical compositions provided herein in the manufacture of pharmaceuticals is provided.

[0014] Alternative embodiments provide for the use of formulations or pharmaceutical compositions provided herein in the manufacture of a pharmacopoeia to treat, improve, prevent, reverse, or reduce the severity or duration of the following symptoms, or to reduce the severity of the following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, nerve or CNS inflammation includes TLR4-mediated nerve or CNS inflammation. - Allodynia, If necessary, allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary the virus is influenza or coronavirus (where coronavirus is COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary influenza A, B or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or co-occurring conditions thereof, and / or - Vascular inflammation, atherosclerosis, and cardiovascular disease.

[0015] Alternative embodiments provide formulations, pharmaceutical compositions, or therapeutic combinations for use in methods to treat, improve, prevent, reverse, or reduce the severity or duration of the following symptoms, or to reduce the severity of the following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, nerve or CNS inflammation includes TLR4-mediated nerve or CNS inflammation. - Allodynia, If necessary, allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary the virus is influenza or coronavirus (where coronavirus is COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary influenza A, B or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or co-occurring conditions thereof, and / or - Vascular inflammation, atherosclerosis and cardiovascular disease, The formulation or therapeutic combination described herein includes the formulation or therapeutic combination provided herein. The aforementioned formulation or therapeutic combination is administered to the individual or patient in need.

[0016] In alternative embodiments, a method for exposing a potential (or hidden, unexposed, inaccessible) N-terminal TLR4 binding domain of an ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide comprises adding 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 and 50 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 to the native (or wild-type) AIBP polypeptide, 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. If necessary, the heterologous amino-terminal amino acid sequence includes a peptide tag, if necessary, the peptide tag includes a multi-histidine (multi-his) tag, if necessary, the multi-his tag includes at least six histidine (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. If necessary, heterologous amino-terminal amino acid sequences include enzymatic cleavage sites, and if necessary, enzymatic cleavage sites include thrombin cleavage sites. If necessary, heterologous amino-terminal amino acid sequences include secretory signals, and if necessary, secretory signals include fibronectin secretory signals, immunoglobulin heavy chain secretory signals, immunoglobulin κ light chain secretory peptides, or interleukin-2 signal peptides. If necessary, different amino-terminal amino acid sequences are used in the amino acid sequence. MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR(Sequence ID 2) A method including this is provided.

[0017] In an alternative embodiment, a polypeptide compound is provided, wherein the polypeptide compound comprises an ApoA-I binding protein (AIBP) amino acid sequence and the N-terminal amino acid sequence of the AIBP amino acid sequence. The N-terminal amino acid sequence of the AIBP amino acid sequence consists of at least 8 amino acids, or between 4 and 12 amino acids, or between 5 and 10 amino acids, and the N-terminal amino acid sequence of the AIBP amino acid sequence can, under relevant physiological conditions, induce unfolding of a potential domain in the AIBP amino acid sequence, expose a potential domain, or otherwise make it accessible in order to bind the polypeptide to TLR4, provided that the N-terminal amino acid sequence of the AIBP amino acid sequence does not consist of a His tag or a protein cleavage site that, when acted upon under the said physiological conditions, results in the loss of the His tag.

[0018] In an alternative embodiment, to treat, improve, prevent, reverse, or reduce the severity or duration of a TLR4-mediated disease or condition, it is administered to subjects who require it. A pharmaceutically acceptable composition comprising a polypeptide compound or nucleic acid compound provided herein, wherein the nucleic acid sequence of the nucleic acid compound encodes the amino acid sequence of the polypeptide, A method is provided by offering TLR4-mediated diseases or conditions include, but are not limited to, pro-inflammatory 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, traumatic war neurosis, post-traumatic stress syndrome (PTSS), and diseases and conditions caused by or resulting from viral infections, or their sequelae.

[0019] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims.

[0020] All publications, patents, and patent applications cited herein are incorporated herein by reference for all purposes to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. Brief explanation of the drawing

[0021] A patent or patent application file must include at least one color drawing. A copy of this patent or patent application publication containing one or more color drawings is available from the United States Patent and Trademark Office upon request and payment of the required fees.

[0022] The drawings described herein are illustrative of embodiments provided herein and are not intended to limit the scope of the invention as encompassed in the claims. [Brief explanation of the drawing]

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

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

[0025] Figures 1B-C show the lipid raft content measured by TLR4 dimerization (Figure 1B) and CTxB staining (Figure 1C) of CD11b. + / TMEM119 + The graph shows the data for the analysis of spinal cord microglia cells 24 hours after administration of saline or AIBP, i.e., on day 8 of the time course shown in Figure 1A.

[0026] Figure 1C shows images of BV-2 microglia cells incubated in complete medium with AIBP (0.2 μg / mL) or vehicle for 30 minutes, followed by incubation with LPS (100 ng / mL) for 5 minutes (left panel), with data showing Manders coefficients (colocalization analysis) with and without LPS and / or AIBP in a graph (right panel).

[0027] Figures 1E-1F show graphs illustrating the time-series AIBP levels in the CSF (Figure 1E) and lumbar spinal cord (Figure 1F). These will be discussed in detail in Example 1 below.

[0028] [Figure 2] Figures 2A-C show data illustrating gene expression in spinal microglia of CIPN mice.

[0029] Figures 2A-B show microglia (CD11b + TEMEM119 + The data shown here is from a study in which FACS selection was performed from the three groups shown in Figure 1A.

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

[0031] Figure 1B graphically shows data indicating that groups with significant DEG were clustered based on the expression profile patterns of different treatment conditions.

[0032] Figure 1C graphs data showing the upregulated (Group 1 in the right panel) and downregulated (Group 2 in the left panel) gene pathways and GO enrichment analysis induced by cisplatin treatment. Upregulated pathways are shown as "Group 1" (red) in the right panel, and downregulated pathways are shown as "Group 2" (blue) in the left panel. These will be discussed in detail in Example 1 below.

[0033] [Figure 3] Figures 3A-H show data indicating disease-related microglia (DAMs), lipid-related gene expression, and lipid droplets in spinal cord microglia of CIPN mice.

[0034] Figures 3A-3C show the same groups as in Figure 2: Figure 3A shows a volcano plot image of upward and downward regulatory genes in spinal microglia of cisplatin-treated mice compared to naive mice; Figure 3B shows a heatmap image representing disease-associated microglia (DAM) signature genes; Figure 3B shows a heatmap image of log2-normalized gene counts scaled per row, showing lipid-associated gene sets.

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

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

[0037] Figure 4A shows the pathways and gene ontology (GO) enrichment analyses of AIBP-regulated CIPN upregulatory genes (see group 3 in Figure 2B) and AIBP-regulated CIPN downregulatory genes (group 4).

[0038] Figure 4B shows the results of cisplatin / saline-treated mice (vs.) versus cisplatin / AIBP-treated mice. This shows a volcano plot of upregulated and downregulated genes in treated mice, representing differentially expressed genes (DEGs) in spinal microglia induced by itAIBP.

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

[0040] [Figure 4-2] Figure 4D shows graphs illustrating cytokine protein expression in spinal cord tissue from the WT naive group, the cisplatin / saline group, and the cisplatin / AIBP group.

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

[0042] Figure 4F graphically shows the pathways and GO enrichment analyses of all genes downregulated by AIBP.

[0043] Figure 4G shows a heatmap of non-inflammatory genes downregulated by AIBP, which is part of the peptidase inhibitor activity pathway, the most enriched pathway.

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

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

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

[0047] Figure 5C schematically shows an exemplary experimental design and timeline for tamoxifen, cisplatin, AIBP, or saline injection in mice.

[0048] Figure 5D shows a graph of the baseline (day 0) withdrawal threshold data before the initiation of cisplatin intervention.

[0049] Figure 5E shows CD11b in naive WT and ABC-imKO mice at baseline (day 0). + TMEM119 + The graph shows data illustrating TLR4 surface expression, dimerization, and lipid rafts (CTxB) in spinal cord microglia (n=5 for TLR4 surface expression and lipid raft content analysis in both groups).

[0050] [Figure 5-2]Figure 5F shows a graph of data indicating the withdrawal threshold after 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-5H graphically show data indicating the withdrawal threshold after injection of ip cisplatin and it saline or AIBP (0.5 μg / 5 μl) in TAM-induced ABC-imKO mice (Figure 5G) and non-induced (vehicle) ABC-imKO mice (Figure 5H).

[0052] Figures 5I-J show CD11b on day 8 in the groups shown in panel figures 5G and 5H. + TEMEM119 + The graphs show data illustrating TLR4 dimerization (Figure 5I) and lipid rafts (Figure 5J) in spinal cord microglia. These will be discussed in detail in Example 1 below.

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

[0054] The upper image in Figure 6A schematically shows duplicated genes and pathways induced in naive ABC-imKO microglia and shared with WT microglia from cisplatin-treated mice, indicated by dark purple lines (upper) connecting duplicated genes and light blue lines (lower) connecting duplicated enrichment pathways. The lower image in Figure 6A is a Venn diagram of upregulated genes in spinal cord microglia from WT cisplatin-treated and ABC-imKO naive mice.

[0055] Figure 6B shows an analysis of the enrichment pathways of upregulated and downregulated genes induced by ABCA1 and ABCG1 knockdown in microglia.

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

[0057] Figure 6D schematically shows the duplicated genes and pathways that are induced by cisplatin treatment in ABC-imKO microglia and shared with cisplatin-treated WT mouse microglia.

[0058] Figure 6E shows the DEG of spinal microglia in cisplatin-treated TAM-induced ABC-imKO mice compared to cisplatin-treated WT mice.

[0059] [Figure 6-2] Figures 6F-6G show heatmaps of DEG upregulatory genes (Figure 6F) or downregulatory genes (Figure 6G) in ABC-imKO microglia under either naive or cisplatin conditions. These will be discussed in detail in Example 1 below.

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

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

[0062] Figure 7B schematically shows a volcano plot of upregulated and downregulated genes in CIPN treated with AIBP, comparing the effects of AIBP in ABC-imKO mice and WT mice.

[0063] Figure 7C schematically shows a heatmap of the number of log2-normalized inflammatory genes altered in an ABC-dependent manner by AIBP (depressive regulation by AIBP in WT microglia, but upregulation by AIBP in ABC-imKO).

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

[0065] Figure 7E schematically shows a heatmap of non-inflammatory genes that are ABC-dependently regulated by AIBP.

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

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

[0068] Figure 8A schematically shows an exemplary experimental design and timeline: tamoxifen; cisplatin; AIBP; and / or saline are injected.

[0069] Figure 8B graphically shows the baseline (day 0 in Figure 8A) withdrawal thresholds before the start of cisplatin intervention.

[0070] Figure 8C graphically shows the withdrawal thresholds tested before (naive, day - 7 in the panel A timeline) and after (TAM, day 0) tamoxifen injection regimen in WT and Cx3cr1-Cre ERT2 (without floxed gene) mice.

[0071] Figures 8D - F graphically show the withdrawal thresholds after i.p. cisplatin and i.t. saline or AIBP injection in (Figure 8D) TAM-induced AIBP-imKO mice; non-induced (vehicle) AIBP-imKO mice (Figure 8E); and globally AIBP knockout mice bred in-house (Figure 8F).

[0072] Figure 8G graphically shows the withdrawal thresholds in WT and tamoxifen-induced TLR4-imKO mice after cisplatin injection. These will be discussed in detail in Example 1 below.

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

[0074] Figure 9A schematically shows human AIBP having a signal peptide, amino acids (aa)1-24, previously uncharacterized N-terminal domains (aa25-51), and YjeF_N domains (aa52-288).

[0075] Figure 9B shows images of PAGE isolation of a flag-tagged deletion mutant of human AIBP co-expressed with a 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 images of PAGE isolation of his-tagged human (hu), mouse (mo), and zebrafish (zf) AIBPs, all lacking the signal peptide, which were expressed in baculovirus / insect cell lines, conjugated to eTLR4-His in vitro, followed by IP with anti-TLR4 antibody and IB with anti-His antibody.

[0077] Figures 9D–H show data from three independent experiments illustrating the 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), blotting, and quantification of eTLR4 with wtAIBP or mutAIBP in vitro, including anti-AIBP antibodies.

[0078] Figure 9D shows PAGE isolation (left image) where plates coated with eTLR4 were incubated with wtAIBP or mutAIBP and ELISA was performed. The right image shows a graph of the TLR4 / AIBP levels in wt and mu AIBP.

[0079] Figure 9E graphically shows the binding of wt or mut AIBP (or no AIBP) to AIBP in immobilized eTLR4 when ELISA was performed using plates coated with BSA, wtAIBP, or mutAIBP and incubated with APOA1.

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

[0081] Figure 9H shows APOA1 bound to AIBP using confocal imaging, illustrating the binding of wtAIBP and mutAIBP to BV-2 microglia cells that were unstimulated or treated with LPS for 15 minutes. These will be discussed in detail in Example 1 below.

[0082] [Figure 10-1] Figures 10A-G show data indicating that intrathecal delivery of AIBP lacking a TLR4 binding domain cannot alleviate CIPN allodynia.

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

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

[0085] Figure 10D graphically shows the withdrawal thresholds in WT mice in response to ip cisplatin, followed by itwtAIBP, mutAIBP, or saline.

[0086] Figures 10E-F show CD11b derived from the lumbar spinal cord of mice on day 21 from the experimental group shown in panel figure 10D. + / TMEM119 + The graphs show TLR4 dimerization (Figure 10E) and lipid rafts (Figure 10F) in microglia.

[0087] [Figure 10-2] Figure 10G schematically shows the effects of cisplatin-induced tissue injury (injury-associated molecular pattern (DAMP)) and chemotherapy-induced peripheral neuropathy (CIPN) using AIBP treatment on microglial gene expression and lipid droplet accumulation, with black dots in the plasma membrane and ER representing cholesterol. This will be discussed in detail in Example 1 below.

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

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

[0090] [Figure 13] Figure 13 schematically shows TLR4 binding of various exemplary manipulated forms of AIBP: all proteins were expressed and purified from baculovirus / insect cell lines: His-d24AIBP: corresponding to the amino acid sequence shown in Figure 12, the amino acid sequence is shown in the orange box for the “cleavable His tag” sequence, all other figures show various modifications and corresponding changes to the amino acid sequence introduced into the AIBP molecule, the green “N-terminal domain” box represents amino acid sequences 25-51 of native AIBP, the right column shows the results of co-immunoprecipitation experiments of AIBP variants with the recombinant extradomain of TLR4, for His-24 AIBP: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), for “cleaved His-d24 AIBP”: GSPGLDGICSR (SEQ ID NO: 9), for “5xD mut His-d24 AIBP”: MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (Sequence ID 19), GSDGDDGDDDR (Sequence ID 10) for "Severed 5xD His-d24 AIBP", MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGICSR (Sequence ID 11) for "2xD mut His-d24 AIBP", and GSPGLDGICSR (Sequence ID 9) for "Severed His-d24 AIBP".

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

[0092] [Figure 15] TLR4 affinity: Various AIBP constructs for optimizing the structure for baculovirus / insect cell expression systems are schematically shown: 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": For "3xFLAG": MAGILVPRGSPGLDGICSR (SEQ ID NO: 18) For "5XD": GSDGDDGDDDR (SEQ ID NO: 11).

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

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

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

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

[0097] Figures 17C-D graphically show flow cytometry analysis of CD11b+DRG macrophage cells 24 hours after treatment with it saline or AIBP, with TLR4 dimerization (Figure 17C) and lipid raft content measured by CTxB staining (Figure 17D). These will be discussed in Example 1 below.

[0098] [Figure 18-1] Figures 18A-E (or Figure S2, or Supplementary Figure 2) illustrate the FACS sorting strategy for spinal cord microglia, quality control for RNA-seq, and phenotypic regulation.

[0099] Figure 18A shows the sorting strategies 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, as well as CD11b and TMEM119.

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

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

[0102] Figures 18D-E show heatmaps of CIPN suppressor genes upregulated by AIBP (group 4) (Figure 18D) and CIPN-inducing genes downregulated by AIBP (group 3) (Figure 18E) in wild-type mice. These will be discussed in Example 1 below. [Figure 18-2] Same as above.

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

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

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

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

[0107] Figure 19D shows the DAPI, GFAP, ABCA1, merge, and COLOC masks with and without tamoxifen. These will be discussed in more detail in Example 1 below.

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

[0109] Figures 20A and 20B show graphs of data obtained when wild littermates bred in-house as controls for ABC-imKO mice were administered a tamoxifen regimen (TAM, 200 μL / day, 10 mg / mL, for 5 consecutive days), followed by (Figure 20A) injecting AIBP (0.5 μg / 5 μL) or saline (5 μL) via it, and administering itLPS (0.1 μg / 5 μL) 2 hours later; and (Figure 20B) administering cisplatin (2.3 mg / Kg) via ip injection on days 1 and 3, followed by injecting AIBP (0.5 μg / 5 μL) or saline (5 μL) via it on day 7.

[0110] Figure 20C shows, in graph form, the data obtained on day 7 when ABC-imKO mice were injected with TAM, then cisplatin as described above, and subsequently it saline (5 μL), AIBP (0.5 μg / 5 μL), or hp-β-CD (0.25 mg / 5 μL).

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

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

[0113] [Figure 20-3]Figure 20E shows heatmaps of pathway enrichment for cisplatin upregulatory genes in WT and ABC-imKO microglia, using cutoff P<0.05, enrichment >1.5, and minimal duplication of three genes within the pathway. These 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, demonstrating that the BE-1 monoclonal antibody has similar affinity for wtAIBP and mutAIBP.

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

[0116] Figure 21B graphically shows data from a sandwich ELISA using BE-1 as the capture antibody in a microtiter plate, and dose-response curves for wtAIBP and mutAIBP were detected using rabbit polyclonal anti-AIBP antibody. These will be discussed in more detail in Example 1 below.

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

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

[0119] Figure 22B shows a graph of APOA1BP / HPRT1 mRNA in non-asthmatic and asthmatic samples.

[0120] Figure 22C shows a graph of AIBP expression in the bronchial epithelium. These will be discussed in more detail in Example 3 below.

[0121] [Figure 23-1] Figures 23A-F graphically show that compound 7 reduces airway hyperresponsiveness and eosinophilic pneumonia in HDM models of asthma in female and male mice, as will be discussed in more detail in Example 3 below. [Figure 23-2] Same as above.

[0122] [Figure 24] Figures 24A-M show that AIPB reduces retinal neurodegeneration in D2 glaucoma mice, as will be discussed in more detail in Example 4 below.

[0123] [Figure 25] Figures 25A-D show that AIPB reduces retinal neurodegeneration and improves visual function in a microbead-induced hypertension mouse model, as will be discussed in more detail in Example 4 below.

[0124] [Figure 26] Figures 26A-B show that AIPB reduces retinal neurodegeneration in a mouse neural crash model, as will be discussed in more detail in Example 4 below.

[0125] Similar reference numerals in various drawings indicate the same elements. [Modes for carrying out the invention]

[0126] Next, various exemplary embodiments provided herein will be referenced in detail, examples of which are shown in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of the aspects and embodiments of the invention and should not be construed as a limitation on the scope of the invention.

[0127] Detailed explanation Alternative embodiments provide compositions and methods for modulating or manipulating a protein (APOA1BP, AIBP, or AI-BP) including modification of the amino acid sequence, addition, maintenance, enhancement, or upmodulation of the expression of the recombinant ApoA-I binding protein (APOA1BP, AIBP, or AI-BP), using pharmaceutical compounds and formulations including nucleic acids, polypeptides, and gene and polypeptide delivery vehicles, as well as kits comprising all or some components for carrying out these compositions and methods. Alternative embodiments also provide compositions and methods for altering the 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 that target and / or have the ability to penetrate the blood-brain barrier, as well as nucleic acid (gene) delivery vehicles such as vectors and viruses, including adeno-associated virus (AAV) delivery vehicles contained within AIBP-expressing nucleic acids; and compositions and methods for directly delivering either AIBP polypeptide or AIBP-expressing nucleic acid via intrathecal (it) administration.

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

[0129] The inventors also demonstrate that AIBP deletion mutants lacking the TLR4 binding domain do not reverse neuropathic pain in a mouse model of chemotherapy-induced peripheral neuropathy. AIBP binding to TLR4 is important because this innate immune receptor is highly expressed in inflammatory cells, concentrates in lipid rafts on the cell surface, and mediates the inflammatory response. Enlarged / clustered lipid rafts with increased TLR4 content and evidence of TLR4 dimerization are called "inflamma rafts." By binding to TLR4, AIBP targets inflammatory cells, disrupts inflamma rafts, and inhibits inflammation, spinal nerve inflammation, and neuropathic pain. Its effects are applicable to many inflammatory disease conditions mediated by TLR4.

[0130] The inventors also found that in native AIBP, the N-terminal TLR4-binding domain is latent, 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, for example, in the recombinant form of AIBP provided herein, 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 includes an amino acid sequence derived from a commercially available pAcHLT-C vector (BD Biosciences).

[0132] The TLR4 receptor localizes and dimerizes in membrane lipid rafts. Enlarged, cholesterol-rich lipid rafts containing activating receptors and adapter molecules (referred to here as inflamma rafts (Miller et al., 2020)) function as organizing platforms for initiating inflammatory signaling and cellular responses. Regulation of cholesterol content within the plasma membrane can influence inflamma 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, inflamma rafts modulate the activation of numerous other receptors and components of signaling pathways, as outlined in Miller et al., 2020. Therefore, we hypothesized that CIPN is associated with altered cholesterol dynamics in spinal cord microglia, leading to inflamma raft formation and persistent neuroinflammation in the spinal cord.

[0133] To test this hypothesis, the inventors measured spinal cord microglia lipid rafts and TLR4 dimerization in CIPN mice. To manipulate cholesterol dynamics, the inventors used mice with induced microglia-specific knockdown of cholesterol transporters Abca1 and Abcg1, administered intrathecally by apoAI-binding protein (AIBP), an effective multiplier for cholesterol removal from several cell types (Choi et al., 2018; Fang et al., 2013; Woller et al., 2018). The inventors demonstrate that AIBP induces cholesterol redistribution in the microglia membrane and enhances the colocalization of accessible cholesterol and cholesterol transporter ABCA1. This redistribution depletes cholesterol from the plasma membrane and sets the conditions for inflammatory rafts to revert to physiological lipid rafts. Microglia-specific Abca1 / Abcg1 knockdown induced pain in naive mice, preventing AIBP from reversing CIPN allodynia, highlighting the importance of microglial cholesterol homeostasis in the development of neuropathic pain. Furthermore, characterization of CIPN-related gene expression changes in microglia suggests cholesterol metabolic disorders. Recombinant AIBP sequence

[0134] In an alternative embodiment, an engineered protein sequence is disclosed comprising an ApoA-I binding protein (AIBP) amino acid sequence and the N-terminal amino acid sequence of the AIBP amino acid sequence. Here, the N-terminal amino acid sequence of the AIBP amino acid sequence includes a peptide tag, the peptide tag includes a multi-histidine (multi-his) tag, and in particular, the multi-his tag includes six consecutive histidine residues (HHHHHH (SEQ ID NO: 1)).

[0135] In other embodiments, the heterologous amino-terminal amino acid sequence includes the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), in which the thrombin cleavage site is non-functional due to a mutation in the thrombin cleavage site.

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

[0137] In alternative embodiments, a method is provided for in vivo administration of 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 10 and 100 amino acids, or between about 20 and 80 amino acids, or between about 30 and 50 amino acids, or any heterologous amino acid sequence sufficient to cause unfolding and exposure of the potential (or hidden, unexposed) N-terminal TLR4 binding domain of the AIBP polypeptide.

[0138] In alternative embodiments, mouse AIBPs are used, for example, mouse AIBPs having the sequence encoded by SEQ ID NO: 3 and / or the amino acid sequence of SEQ ID NO: 4, which may optionally be supplemented with a fibronectin secretion signal (italic) at the N-terminus and / or a His tag (underlined) at the C-terminus (i.e., further included); the product is abbreviated as FIB-mAIBP-His. Sequence ID 3: [ka] Sequence ID 4: [ka]

[0139] In alternative embodiments, variants of the human AIBP (hAIBP) polypeptide provided herein (e.g., human AIBP having heterologous amino acid sequences that expose a TLR4 (or otherwise potential) binding site), or nucleic acids encoding variant AIBP provided herein, are administered to patients or individuals in need of administration, or used to manufacture formulations or pharmaceuticals, or used to create vectors or expression vehicles for administration, or included in kits provided herein, and the AIBP variant may include or be encoded by: Human AIBP-coded nucleic acid (cDNA) (SEQ ID NO: 5) [ka] [ka] Human AIBP polypeptide (SEQ ID NO: 6) [ka]

[0140] In some embodiments, modified hAIBPs are used that retain the TLR4 binding domain and replace the N-terminal residue with a native signal peptide, for example, the hAIBP contains amino acids 25-288 of the hAIBP sequence known as d24hAIBP (coding nucleic acid): [ka] Here, the corresponding d24hAIBP polypeptide is: [ka] That is the case.

[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 (or further included) with a fibronectin secretion signal (italic); the product is abbreviated as FIB-d24hAIBP and named Compound 1: Human FIB-d24hAIBP (compound 1)-coding 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 modification is as follows: MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (Sequence ID 24).

[0143] In one embodiment, a secretory signal is added to ensure potent secretion of AIBP. For example, a fibronectin secretory signal is added to the N-terminus of AIBP (see the italicized sequences of SEQ ID NOs: 3 and 4). Alternatively, a nucleic acid encoding a secretory signal is added to the AIBP coding sequence. In alternative embodiments, the secretory signal may be a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin κ light chain secretory peptide (see, e.g., PLoS One. 2015;10(2):e0116878), or an interleukin-2 signal peptide (see, e.g., J. Gene Med. 2005 Mar;7(3):354-65).

[0144] In alternative embodiments, the polypeptide coding sequence is functionally linked to a promoter, such as a constitutive, inducible, tissue-specific (e.g., nerve or brain tissue-specific) or ubiquitous promoter, or other transcription activator.

[0145] In other embodiments, the product from 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 (sequence number 26).

[0146] In other embodiments, the sequence of the AIBP polypeptide is modified at its C-terminus to incorporate further peptide fragments. This is 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 has a His tag (FIB-d24 AIBP-His) and an N-terminal modification: LRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (Sequence ID 29) This is d24hAIBP, which includes this.

[0148] In another embodiment, compound (compound 4) is obtained by post-translational modification of the signal peptide: [ka] [ka] Here, the hAIBP fragment is d24hAIBP-His, and the N-terminal modification is TETGKSKR (sequence number 26).

[0149] In other embodiments, the polypeptide coding sequence is functionally linked to a promoter, such as a constitutive, inducible, tissue-specific (e.g., nerve or brain tissue-specific) or ubiquitous promoter or other transcription activator.

[0150] In other embodiments, full-length human AIBP is modified at its N-terminus. Such modifications facilitate TLR4 binding, for example, nucleic acid (cDNA) encoding compound 5:

Chem.

Chem.

[0151] In other embodiments, the hAIBP sequence that retains the potential TLR4 binding domain is modified at its N-terminus. Examples of sequences include the DNA and peptide sequences of amino acids 25 - 288 of hAIBP (d24hAIBP): (Nucleic acid sequence encoding compound 6):

Chem.

Chem.

Chem.

Chem.

[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 and 100 amino acids, or between about 8 and 40 amino acids, or between about 30 and 50 amino acids, or any heterologous amino acid sequence sufficient to cause unfolding and exposure of the potential (or hidden, unexposed) N-terminal TLR4 binding domain of the AIBP polypeptide.

[0153] In an alternative embodiment, the N-terminal sequence of the amino acid contains 3 to 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 their bioactive properties, for example by removing putative peptide cleavage sites. Examples of sequences are represented as follows: (Nucleic acid sequence encoding Compound 8):

Chemical formula

Chemical formula

[0159] Alternative embodiments provide pharmaceutical formulations or compositions comprising nucleic acids and polypeptides for carrying out methods and uses provided herein for modulating neuropathic pain, the methods comprising upmodulating the expression of recombinant ApoA-I binding protein (APOA1BP, AIBP, or AI-BP). Alternative embodiments also provide pharmaceutical formulations or compositions for use in vivo, in vitro, or ex vivo methods for treating, preventing, reversing, and / or improving neuropathic pain. In alternative embodiments, pharmaceutical compositions and formulations used to carry out the methods and uses provided herein, comprising recombinant APOA1BP nucleic acid and polypeptide or resulting in increased expression or activity of recombinant APOA1BP nucleic acid and polypeptide, are administered to individuals in need of administration in an amount sufficient to treat, prevent, reverse and / or improve, for example, neuropathic pain, neurodegenerative diseases or conditions, optionally chronic or progressive neurodegenerative diseases, optionally Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy, optionally traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), optionally glaucoma or other inflammatory eye diseases, optionally pneumonia and asthma, optionally HIV infection or its comorbidities, and / or vasculitis, atherosclerosis and cardiovascular disease. In alternative embodiments, pharmaceutical compositions and formulations used to carry out the methods and uses provided herein, comprising recombinant APOA1BP nucleic acid and polypeptide or resulting in increased expression or activity of recombinant APOA1BP nucleic acid and polypeptide, are administered to individuals in need of administration in an amount sufficient to prevent or reduce the intensity and / or frequency of neuropathic pain or neurodegenerative disease or condition.

[0160] In alternative embodiments, the pharmaceutical compositions used to carry out the methods and uses provided herein may be administered parenterally, topically, or orally, or by topical administration such as aerosol or transdermally. The pharmaceutical compositions may be formulated in any manner and administered in various unit dosage forms depending on the condition or disease and its severity, the general medical condition of each patient, and the preferred method of administration obtained. Further details regarding the techniques for formulation and administration are adequately described in the scientific literature and patent documents, for example, Remington's Pharmaceutical Sciences Please refer to the latest edition from Maack Publishing Co., Easton PA ("Remington's").

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

[0162] In alternative embodiments, the formulations and pharmaceutical compositions used to carry out the methods and uses provided herein may include solutions of compositions (including peptide mimes, racemic mixtures or racemic compounds, isomers, stereoisomers, derivatives and / or analogs of compounds) placed in or dissolved in pharmaceutically acceptable carriers, for example, acceptable vehicles and solvents that can be used include water and Ringer's solution and isotonic sodium chloride. Furthermore, sterile fixative oils can be used as solvents or suspension media. For this purpose, any fixative oil can be used, including synthetic monoglycerides or diglycerides, or fatty acids such as oleic acid. In one embodiment, the solutions and formulations used to carry out the methods and uses provided herein can be manufactured to be sterile and free from generally undesirable substances. In one embodiment, these solutions and formulations are sterilized by conventional well-known sterilization techniques.

[0163] The solutions and formulations used to carry out the methods and uses provided herein may include pH adjusters and buffers, toxicity modifiers, and other auxiliary substances necessary to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the active agent in these formulations can vary widely and may be selected primarily based on fluid volume, viscosity, etc., according to the specific mode of administration chosen (in vivo, in vitro, or ex vivo) and the desired outcome.

[0164] The methods and uses provided herein, and the compositions and formulations used to carry out such methods and uses, may be delivered by the use of liposomes. By using liposomes, particularly by using liposomes whose surface carries target cell-specific ligands (e.g., damaged or diseased neurons or CNS tissue) or otherwise preferentially directed to specific tissue or organ types, the delivery of active agents to target cells in in vivo, in vitro, or ex vivo applications can be concentrated. Nanoparticles, nanolipid particles, and liposomes

[0165] For example, nanoparticles, nanolipid particles, vesicles, and liposome membranes containing compounds used to carry out the methods and uses provided herein for delivering compositions comprising recombinant APOA1BP nucleic acid and polypeptide 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 containing cell surface polypeptides, for example, to target a desired cell type or organ, such as nerve cells or the CNS.

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

[0167] Liposomes can be produced by any method, including, for example, a method for generating liposomes by encapsulating an active agent (e.g., recombinant APOA1BP nucleic acid and polypeptide), as described by Park et al., U.S. Patent Application Publication No. 20070042031, which comprises 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 area to produce a liposome solution, wherein liposomes encapsulating the active agent are generated substantially instantaneously when the organic lipid solution is mixed with the aqueous solution; and then immediately mixing the liposome solution with a buffer to produce a diluted liposome solution.

[0168] In one embodiment, a liposome composition used to carry out the methods and uses provided herein comprises, for example, a substituted ammonium and / or polyanion to target the delivery of a compound (e.g., recombinant APOA1BP nucleic acid and polypeptide) to a desired cell type (e.g., endothelial cells, nerve cells, or any tissue or region requiring it, e.g., CNS), as described in U.S. Patent Application Publication No. 20070110798.

[0169] For example, as described in U.S. Patent Application Publication 20070077286, nanoparticles are provided that contain a compound in the form of nanoparticles (e.g., secondary nanoparticles) containing an active agent (e.g., recombinant APOA1BP nucleic acid and polypeptide used to carry out the methods provided herein). In one embodiment, nanoparticles are provided that contain a lipid-soluble active agent or a lipid-soluble water-soluble active agent that acts with a divalent or trivalent metal salt.

[0170] In one embodiment, for example, as described in U.S. Patent Application Publication No. 20050136121, a solid lipid suspension can be used to formulate the composition used to carry out the methods and uses provided herein and deliver it in vivo to mammalian cells, e.g., the central nervous system (CNS). Modification of delivery vehicle and modification of AIBP

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

[0172] In alternative embodiments, recombinant AIBP peptides or polypeptides, or nanoparticles, liposomes, etc., containing AIBP (e.g., including or having recombinant APOA1BP nucleic acid or polypeptide used to carry out the methods provided herein) are directly or indirectly linked or conjugated to blood-brain barrier (BBB) ​​targeted agents, such as transferrin, insulin, leptin, insulin-like growth factor, cationic peptides, lectins, receptor-associated proteins (RAPs) (39kD chaperones localized to the endoplasmic reticulum and Golgi, lipoprotein receptor-associated protein (LRP) receptor family ligands), apolipoprotein B-100-derived peptides, antibodies against transferrin receptors (e.g., peptide-mimicking monoclonal antibodies), antibodies against insulin receptors (e.g., peptide-mimicking monoclonal antibodies), antibodies against insulin-like growth factor receptors (e.g., peptide-mimicking monoclonal antibodies), antibodies against leptin receptors (e.g., peptide-mimicking monoclonal antibodies), etc. In alternative embodiments, any protocol or modification, such as AIBP peptides or polypeptides, or AIBP-containing nanoparticles, liposomes, etc., that facilitate BBB passage, can be used, for example, as described in U.S. Patent Application Publication No. 20050142141;20050042227. For example, any protocol, such as direct intracranial injection, transient BBB permeabilization treatment, and / or modification, such as AIBP peptides or polypeptides, or AIBP-containing nanoparticles, liposomes, etc., can be used to enhance CNS or brain delivery of the composition 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 to deliver the composition (e.g., recombinant APOA1BP nucleic acid and polypeptide) to the CNS or brain in vivo, for example. For example, a delivery vehicle comprising a polycation, cationic polymer and / or cationic peptide, such as a polyethyleneimine derivative, can be used, as described in U.S. Patent Application Publication No. 20060083737. In one embodiment, the delivery vehicle is a transdextrin cell engineered to express or overexpress endogenous or exogenous AIBP and then secrete it.

[0174] In one embodiment, the dried polypeptide-surfactant complex is used to formulate a composition used to carry out the method provided herein, for example, as described in U.S. Patent Application Publication No. 20040151766.

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

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

[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, the nucleic acids, vectors, or recombinant viruses are designed for in vivo use or for CNS delivery and expression.

[0178] In alternative embodiments, compositions and methods are provided for the delivery and controlled expression of recombinant AIBP coding nucleic acids or genes, or expression vehicles (e.g., vectors, recombinant viruses, etc.) containing (or having contained therein) recombinant AIBP coding nucleic acids or genes, as a result of the AIBP protein being released into the bloodstream or systemic circulation and producing beneficial effects in the body, for example, in the CNS, brain, or other targets.

[0179] In alternative embodiments, a method is provided for easily and efficiently turning AIBP-expressing nucleic acid or gene expression on and off to ensure targeted treatment and optimal safety.

[0180] In alternative embodiments, recombinant AIBP proteins or proteins expressed by one or more AIBP-expressing nucleic acids or genes have beneficial or desirable effects (e.g., therapeutic or preventive) on tissues or organs, such as the brain, CNS, or other targets, even if they are secreted into the blood or systemic circulation at a distance (e.g., anatomically distant) from their one or more sites of action.

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

[0182] In alternative embodiments, this “peripheral” delivery mode, such as expression vehicles, vectors, or recombinant viruses injected via IM or IV, can avoid the problems encountered when genes or nucleic acids are directly expressed in the organ itself (e.g., the brain or CNS). Sustained secretion of AIBP in the bloodstream or systemic circulation also avoids the difficulties and costs associated with administering proteins by infusion.

[0183] In alternative embodiments, recombinant viruses (e.g., long-acting viruses or viral vectors), or vectors, or expression vectors, etc., may be administered, for example, to an outpatient, for example, in a hospital setting, by systemic intravenous (e.g., IV) injection, intramuscular (IM) injection, inhalation, or by particulate gun particle delivery systems (e.g., so-called "gene guns"). In alternative embodiments, after several days or weeks (e.g., four weeks), an individual, patient, or subject is administered (e.g., by inhalation, injection, or swallowing) a chemical or pharmaceutical that induces AIBP-expressing nucleic acid or gene expression. For example, an oral antibiotic that activates gene expression (e.g., doxycycline or rapamycin) is administered once daily (or more or less frequently). In alternative embodiments, after "activation" or induction of expression of a nucleic acid or gene (e.g., by an inducible promoter), the AIBP protein is synthesized and released into the circulation of the subject (e.g., into the blood), and subsequently has a desirable physiological effect that benefits the individual or patient, such as a therapeutic or prophylactic effect (e.g., beneficial to the function of the heart, kidneys, or lungs). If the physician or subject wishes to discontinue the AIBP treatment, the subject simply discontinues taking the activating chemical or drug, such as an antibiotic. Alternative embodiments include the use of an "expression cassette" that includes or has, for example, a structural gene or transcript in a host (e.g., encoding the AIBP protein) that is compatible with such a nucleotide sequence used to carry out the method provided herein, such as an AIBP-expressing nucleic acid that can affect the expression of the nucleic acid. The expression cassette may include at least one promoter operably linked to a polypeptide coding sequence or an inhibitory sequence; further, in one embodiment, it may include other sequences, such as a transcription termination signal. Further factors necessary or useful for making the expression, such as enhancers, may also be used.

[0184] In alternative embodiments, the expression cassette may also include plasmids, expression vectors, recombinant viruses, and recombinant “naked DNA” vectors of any form. In alternative embodiments, the “vector” may include nucleic acids that can infect, transfect, transiently or permanently transduce cells. In alternative embodiments, the vector may be naked nucleic acids or nucleic acids complexed with proteins or lipids. In alternative embodiments, the vector may include viral or bacterial nucleic acids and / or proteins and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.). In alternative embodiments, the vector may include, but is not limited to, replicons (e.g., RNA replicons, bacteriophages) to which DNA fragments can be attached and replicated. Thus, the vector may include, but is not limited to, RNA, autonomously self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Patent No. 5,217,879), and may include both expression plasmids and non-expression plasmids. Alternatively, the vector can be stably replicated by the cell during mitosis as an autonomous structure, or it can be incorporated into the host genome.

[0185] In alternative embodiments, “promoter” includes all sequences capable of driving the transcription of a coding sequence in a cell, such as a mammalian cell, such as a muscle, nerve, or brain cell. Promoters used in the constructs provided herein include cis-acting transcriptional regulatory elements and regulatory sequences involved in regulating or modulating the timing and / or rate of transcription of nucleic acids, such as AIBP coding nucleic acids. For example, a promoter may be a cis-acting transcriptional regulatory element, which may include enhancers, promoters, transcriptional terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to perform transcription (e.g., switching on / off, regulating, modulating).

[0186] In alternative embodiments, a “constitutive” promoter may be a promoter that continuously drives expression under most environmental conditions and developmental or cellular differentiation conditions. In alternative embodiments, an “inducible” or “regulatory” promoter may direct the expression of a nucleic acid, such as an AIBP-coding nucleic acid, under the influence of environmental conditions, administered chemicals, or developmental conditions. Gene therapy and gene delivery vehicles

[0187] In alternative embodiments, the method of the present invention involves the use of a nucleic acid (e.g., a gene or polypeptide encoding recombinant AIBP coding nucleic acid) delivery system for delivering a nucleic acid or gene, or an AIBP-expressing nucleic acid, transcript, or message payload to one or more cells in vitro, ex vivo, or in vivo, for example, 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 includes: adeno-associated virus (AAV), lentiviral vector, or adenovirus vector; AAV serotype AAV5, AAV6, AAV8, or AAV9; rhesus macaque-derived AAV, or rhesus macaque-derived AAV AAVrh.10hCLN2; organ-targeting AAV, or neuronal-targeting AAV; and / or AAV capsid variant or AAV hybrid serotype. In alternative embodiments, the AAV is engineered to increase its efficiency in targeting specific cell types that are intolerant to wild-type (wt) AAV, and / or to improve its efficiency in infecting only the cell types of interest. In alternative embodiments, hybrid AAVs are retargeted or manipulated as hybrid serotypes by one or more modifications, including 1) transcapsidization, 2) adsorption of bispecific antibodies to the capsid surface, 3) manipulation of mosaic capsids, and / or 4) manipulation of chimeric capsids. Methods for manipulating adeno-associated virus (AAV) capsids to increase the efficiency of targeting specific cell types that are intolerant to wild-type (wt) virus, and to improve the efficiency of infecting only the cell types of interest, are well known in the art. For example, Wu et al., Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006 Jul 7; Choi, et al., Curr. Gene See Ther. 2005 Jun;5(3):299-310.

[0189] For example, in an alternative embodiment, an AIBP coding nucleic acid payload is delivered for expression in the CNS using serotypes AAV-8, AAV-9, AAV-DJ, or AAV-DJ / 8® (Cell Biolabs, Inc., San Diego, CA), which show increased uptake into brain tissue in vivo. In an alternative embodiment, the following serotypes or variants thereof are used to target specific tissues. [Table 1]

[0190] In alternative embodiments, rhesus macaque-derived AAV AAVrh.10hCLN2 or its equivalent may be used, and rhesus macaque-derived AAV may not be inhibited by any pre-existing immune response in humans; see, for example, Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct;23(5):324-35, Epub 2012 Nov 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct 17; these teach that direct administration of AAVrh.10hCLN2 to the CNS of rats and non-human primates at doses scalable to humans yields an acceptable safety profile and mediates significant payload expression in the CNS.

[0191] Adeno-associated virus (AAV) is a common infectious agent in primates, and therefore healthy primates possess a large pool of AAV-specific neutralizing antibodies (NAbs) that inhibit AAV-mediated gene transfer therapy strategies. Therefore, the methods provided herein may include screening patient candidates for AAV-specific NAbs prior to treatment, particularly with the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy; see, for example, Sun, et al., J.Immunol.Methods. 2013 Jan 31;387(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 may be administered for prophylactic and / or therapeutic purposes. In therapeutic uses, the compositions are administered to subjects already suffering from a disease, condition, infection, or deficiency in an amount sufficient to cure, alleviate, or partially cessate the clinical signs of a disease, condition, infection, or disease and its complications, including, for example, neuropathic pain ("therapeutic effective dose"). For example, in alternative embodiments, pharmaceutical compositions and formulations comprising recombinant APOA1BP nucleic acid or polypeptide provided herein may be used for neuropathic pain, pro-inflammatory neuropathic pain, inflammation of nerves or CNS, allodynia, post-neuropathy pain or neuropathic pain, postoperative pain or neuropathic pain, chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced allodynia), neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, and Alzheimer's disease as needed. It is administered to individuals in a dose sufficient to treat, prevent, reverse and / or improve chronic traumatic encephalopathy (CTE) or related tauopathies, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), migraine, hyperalgesia, glaucoma or other inflammatory eye diseases as needed, pneumonia and asthma as needed, HIV infection or its comorbidities as needed, and / or vasculitis, atherosclerosis and cardiovascular disease as needed.

[0193] The amount of pharmaceutical composition sufficient to achieve this is defined as the "therapeutic dose." The effective dosage schedule and amount for this use, i.e., the "dosage regimen," depends on various factors, including the stage of the disease or condition, the severity of the disease or condition, the patient's overall health status, the patient's physical condition, and age. The mode of administration is also considered when calculating a patient's dosage regimen.

[0194] In alternative embodiments, a viral vector such as an adenovirus or AAV vector is administered to an individual that requires administration, and in alternative embodiments, the dosage administered to a human is approximately 2 × 10⁻¹⁶ per kg of body weight.12 Vector genome (vg / kg), or approximately 10 per kg of body weight 10 ~10 14 Between vector genomes (vg / kg), or about 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15The doses include vg / kg or higher, which can be administered as a single dose or multiple doses as needed. In alternative embodiments, these doses are administered orally, intramedically, intravenously, or intrathecally. In alternative embodiments, the vector is delivered as a formulation or pharmaceutical, for example, the vector is contained in nanoparticles, particles, micelles, or liposomes or lipoplexes, polymerosomes, polyplexes, or dendrimers. In alternative embodiments, these doses are administered once daily, once weekly, or in any variant thereof as needed, to maintain the in vivo expression level of recombinant AIBP, which can be monitored by actually measuring AIBP expression or by monitoring therapeutic effects, such as pain relief. The drug regimen also takes into account pharmacokinetic parameters well known in the art, namely the absorption rate, bioavailability, metabolism, and clearance of the active drug (see, for example, Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest Remington's (mentioned above)). With the latest technology, clinicians can determine drug regimens for individual patients, active drugs, and the disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidelines for determining that drug regimens administered by implementing the methods provided herein, i.e., dose schedules and dosage levels, are accurate and appropriate.

[0195] Depending on the dosage and frequency required and tolerated by the patient, the formulation may be administered as a single or multiple dose. The formulation must provide a sufficient amount of the active agent to effectively treat, prevent or improve the condition, disease, or symptom described herein. For example, the daily dose of another exemplary pharmaceutical formulation for oral administration of the composition used to carry out the methods provided herein is between about 0.1 to 0.5 to about 20, 50, 100 or 1000 ug per kilogram of body weight per day or more. In alternative embodiments, dosages of about 1 mg to about 4 mg per kg of body weight per patient per day are used. Lower dosages can be used in the bloodstream, intracavitary, or intraorganic in contrast to oral administration. Substantially higher dosages can be used for topical or oral administration, or by powder, spray, or inhalation. Practical methods for preparing formulations that can be administered parenterally or non-parenterally are known or obvious to those skilled in the art and are described in detail in publications such as Remington's mentioned above.

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

[0197] In alternative embodiments, bioequivalents of compounds used to carry out the methods provided herein, such as recombinant APOA1BP activity polypeptides, are also provided. These bioequivalents used to carry out the methods provided herein include, for example, bioequivalents of recombinant APOA1BP nucleic acids and polypeptides, which in alternative embodiments may involve the substitution of one or more substituents and / or groups having substantially similar physical or chemical properties to substituents and / or groups having substantially similar physical or chemical properties to those of the compounds used to carry out the methods or uses provided herein. In one embodiment, the purpose of replacing one bioequivalent with another is to improve the desired biological or physical properties of the compound without causing a significant change in its chemical structure.

[0198] For example, in one embodiment, one or more hydrogen atoms are replaced with one or more fluorine atoms, for example, at a site of metabolic oxidation; this can prevent metabolism (catabolism) from occurring. Because 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 with blocked metabolic pathways may have a longer half-life or lower toxicity. A device for directly delivering therapeutic drugs to the CNS or brain.

[0199] In alternative embodiments, pharmaceutical compositions and formulations comprising nanoparticles and liposomes used to carry out 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, U.S. Patent Application Publication 20080140056 describes a catheter that advances rostrally in the lumen of the spinal canal for direct delivery of pharmaceuticals and formulations to the brain. Implantable infusion devices can also be used. For example, a catheter for delivering fluid from an infusion device to the brain may be inserted subcutaneously from the abdomen to the patient's skull, and the catheter may access the individual's brain through a drilled hole. Alternatively, the catheter may be implanted to deliver the drug into the spinal canal within the patient's spinal column. Flexible guide catheters having a distal end for introduction under the patient's skull and a proximal end remaining outside the patient's body can also be used. See, for example, U.S. Patent Application Publication 20060129126.

[0200] In alternative embodiments, the pharmaceutical compositions and formulations used to carry out the methods provided herein are delivered by direct delivery of the pharmaceutical compositions and formulations comprising nanoparticles and liposomes, or by direct transplantation of AIBP-expressing cells into the brain using, for example, cell transplantation cannulas, syringes, etc., as described in U.S. Patent Application Publication No. 20080132878; or elongated medical insertion devices, etc., as described in U.S. Patent No. 7,343,205; or surgical cannulas, etc., as described in U.S. Patent No. 4,899,729. Transplantation cannulas, syringes, etc., can also be used for the direct injection of liquids, such as fluid suspensions.

[0201] In alternative embodiments, the pharmaceutical compositions and formulations used to carry out the methods provided herein are delivered together with a tracer detectable, for example, by magnetic resonance imaging (MRI) and / or radiocomputed 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 target tissue, for example, as described in U.S. Patent No. 7,371,225. Kit and instructions

[0202] For example, a kit is provided comprising a composition (including a device described herein) and / or instructions for carrying out a method provided herein for treating, improving or preventing neuropathic pain. Thus, kits, cells, vectors, etc., can also be provided. In alternative embodiments, a kit is provided comprising a composition used to carry out a method provided herein, or a composition, pharmaceutical composition, or formulation provided herein, and optionally including instructions for its use.

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

[0204] Examples Example 1: Demonstrated efficacy in an exemplary method for treating pain This embodiment describes and demonstrates exemplary embodiments and the effectiveness of methods provided herein for treating or improving neuropathic pain, including, for example, allodynia and TLR4-mediated inflammatory neuropathic pain.

[0205] Neuroinflammation is a major component in the transition to and persistence of neuropathic pain. Spinal neuroinflammation involves activation of TLR4 localized in hypertrophied cholesterol-enriched lipid rafts called inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia resulted in inflammaraft formation and induced tactile allodynia in naive mice. ApoA-I binding protein (AIBP) promoted cholesterol depletion from inflammarafts and reversed neuropathic pain in a chemotherapy-induced peripheral neuropathy (CIPN) model in wild-type mice. However, AIBP (compound 7) failed to reverse allodynia in mice with ABCA1 / ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. AIBP mutants lacking the TLR4 binding domain did not bind to microglia and did not reverse CIPN allodynia. The sustained therapeutic effect of a single dose of AIBP (compound 7) in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and a reduction in lipid droplet accumulation in microglia. These results suggest a cholesterol-driven mechanism of neuropathic pain regulation by controlling TLR4 inflammaflox 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 lipid raft formation in spinal microglia, suggesting altered membrane cholesterol dynamics and increased TLR4 dimerization (Figures 1B and 1C). Intrathecal AIBP (compound 7) reversed CIPN-associated allodynia in spinal microglia and normalized lipid raft and TLR4 dimer levels (Figures 1A-C). These data suggest that TLR4 receptor dimerization, the first step in the activation of the TLR4 inflammatory cascade, occurs in microglial lipid rafts, as demonstrated in other cell types (Cheng et al., 2012; Zhu et al., 2010). This idea was supported by in vitro experiments in which the localization of TLR4 in lipid rafts was significantly increased in LPS-treated BV-2 microglia cells, 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 determined to be Tlr4 - / - The findings were verified using mouse-derived cells (Figures S1A and B). Since macrophages in the dorsal root ganglia (DRG) also express TLR4 in the nociceptive response, the inventors evaluated their TLR4 dimerization and lipid raft content. However, DRG CD11b + No significant changes were observed in myeloid cells at this stage (Figures S1C and D). CSF and AIBP in the spinal cord (Compound 7) Short-term exposure

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

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

[0209] Using pairwise comparisons of the CIPN and naive groups after LRT analysis, we also observed downregulation of Cx3cr1, P2ry12, and Tmem119 homeostasis markers (Figure 3A and B), which are phenotypes associated with the transition to neurodegenerative disease-associated microglia (DAMs) (Masuda et al., 2019; Nugent et al., 2020; Prinz et al., 2019). A subset of microglial DAM signature genes revealed a DAM signature characterized by decreased homeostasis genes and increased inflammatory and cholesterol metabolism genes. The regulation of phagocytic TAM receptors Tyrobp, Axl, and Mertk partially mimicked DAMs characteristic of neurodegenerative diseases in CIPN microglia, except that we observed downregulation of Trem2, and no significant effect on its partner receptor gene, Tyrobp (Figure 3B). This suggests a decrease in phagocytic phenotypes associated with changes in 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, including the gene encoding the lipid droplet protein PLIN2, were downregulated by AIBP (compound 7) (Figures 3B and 3C). PLIN2 immunohistochemistry validated RNA-seq results, showing increased number and size of lipid droplets in spinal microglia of cisplatin-treated mice, as well as a reversal of this effect by AIBP (compound 7) (Figures 3D-H). AIBP (Compound 7) Selective reversal of CIPN-induced changes in inflammatory gene expression.

[0211] Analysis of the gene group upregulated by CIPN and reversed by AIBP (group 3 in Figures 2B and S2F) revealed enrichment of inflammatory responses, leukocyte chemotaxis, and neutrophil degranulation pathways (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 interaction factor gene Tril (Figures 4B and C). Examining cytokine protein expression in spinal cord tissue, the inventors 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) shows enrichment in the TLR4 signaling pathway, along with cytokine-cytokine receptor interactions, protein kinase A and C and MAPK regulatory pathways, receptor-mediated endocytosis, and other membrane signaling pathways (Figure 4F). Calcium and membrane potential regulation was also downregulated, and enrichment of the peptidase inhibitor pathway shows the effect of AIBP (compound 7) on recently reported pain-related peptidase inhibitor-related genes such as Pi16 and the α-synuclein gene Snca, which interact with the lipid membrane and regulate vesicular transport and neurotransmitter release (Figure 4G).

[0212] Differential expression analysis of microglia from AIBP (compound 7)-treated mice in a CIPN model 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-related protein Dgka (Figure 4H). Overall, these results suggest that the AIBP (compound 7)-induced reversal of microglial reprogramming induced in CIPN mice is involved in the regulation of lipid metabolism and the transport of lipid droplets and / or extracellular receptors from the membrane. AIBP (Compound 7) This method cannot reverse allodynia in mice with ABCA1 / ABCG1-deficient microglia.

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

[0214] Next, the inventors created tamoxifen-induced microglia-specific ABCA1 and ABCG1 double knockout mice (ABC-imKO, Figure 5C) and found that ABCA1 and ABCG1 knockdown was associated with spinal IBA1 + Observed in microglia, 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 without stimulation (Day 0) (Figure 5D). These results add to the evidence that impaired cholesterol transport in microglia leads to a facilitated state. In fact, we observed that spinal cord 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 in WT mice, itAIBP (compound 7) failed to prevent itLPS-induced mechanical allodynia in ABC-imKO mice (Figures 5F and S5A). Next, we induced CIPN in ABC-imKO mice using cisplatin and observed a further rapid onset of allodynia. Furthermore, while 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 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 ABCA1 or ABCG1 expression, reduced allodynia in ABC-imKO mice (Figure S4C). Naive ABC-imKO mice had significantly higher TLR4 dimerization and lipid raft abundance than naive WT mice, and these were not significantly altered by either cisplatin or AIBP in ABC-imKO microglia (Figures 5I and J). These results support the idea that AIBP requires cholesterol transporters to alter inflamma raft and TLR4 dimerization dynamics in microglia and reverse allodynia. ABCA1 / ABCG1 deficiency reprograms microglia to adopt a CIPN-like phenotype.

[0216] To understand the effects of cholesterol transport on transcriptional changes induced by CIPN and AIBP, we analyzed differential gene expression in ABC-imKO microglia. We identified 121 genes that were significantly altered across two genotypes and three experimental conditions (Figure S4D). In spinal cord microglia of naive ABC-imKO mice not loaded with cisplatin, most upregulated genes and enriched pathways overlapped with upregulated genes induced by cisplatin in WT mice (Figures 6A and B). Among the enriched pathways in naive ABC-imKO mice, we identified interferon-responsive, inflammatory, complement-activated, and arachidonic acid metabolic 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, similar to the changes induced by cisplatin in WT mice (Figure 6C and F). 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 mice, pathways such as phagosomes, actin dynamics for phagocytic cup formation, and cell cycle pathways were not enriched in ABC-imKO microglia from cisplatin-treated mice (Figure 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-related Fcrls and Cybb(NOX2) (Figure 6F and G). In the absence of cholesterol transporters, cisplatin did not induce cholesterol synthesis pathway genes and downregulated Ch25h and Dhcr24, suggesting that the presence of excess free cholesterol favors the accumulation of desmosterol, an LXR agonist and a key regulator of the 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 in the nociceptive process in ABC-imKO mice. AIBP (Compound 7) Microglia reprogramming by this method depends on the expression of ABCA1 and ABCG1.

[0218] To understand the differential effect of AIBP (compound 7) in WT mice and ABC-imKO mice, we compared the upregulatory and downregulatory genes induced by AIBP treatment in both genotypes (Figures 7A and B). The effect of AIBP (compound 7) on gene regulation differed significantly, with only a few common genes being downregulated in both genotypes (Figure 7A). In the absence of cholesterol transport mechanisms, AIBP failed to regulate inflammatory genes and instead induced their expression (Figures 7B and 7C). The induction of inflammatory genes correlated with an increase in other cholesterol biosynthesis genes, including Dhcr24 and Srebf2, which were downregulated in WT microglia (Figure 7D). This suggests that the decrease in desmosterol and the increase in cholesterol content regulate the expression of inflammatory genes in microglia. Consistent with this, induction of LXR-regulated genes such as Apoe, Apoc1, and Pparg was observed in microglia of ABC-imKO mice but not in WT mice (Figure 7D). Compared to WT, other non-inflammatory genes that were regulated in the opposite direction by AIBP (compound 7) in ABC-imKO included the endopeptidase activity genes Pi16 and Capn11, as well as the AMPA receptor and synaptic modulator Arc (Figure 7E). In ABC-imKO microglia, AIBP (compound 7) upregulated cholesterol metabolic pathways, cytokine release and chemokine signaling regulation, kinase and endopeptidase activity, and lamellar pseudopod and fibrous organizing pathways (Figure 7F). Most of these pathways were downregulated by AIBP in WT microglia (Figure 4F). In summary, these data indicate that the reprogramming of microglial gene expression induced by AIBP (compound 7) is dependent on cholesterol homeostasis regulated by cholesterol transporters ABCA1 and ABCG1. Microglia AIBP and TLR4 modulate nociception.

[0219] Since the above experiments suggested that AIBP (compound 7) regulates cholesterol homeostasis and activation of microglial TLR4 in nociception, we investigated whether microglia-specific knockout of AIBP or TLR4 affects CIPN allodynia. We generated tamoxifen-induced microglia-specific Apoa1bp knockout mice and Tlr4 knockout mice (AIBP-imKO and TLR4-imKO, Figure 8A). Knockdown of endogenous AIBP in microglia induced mechanical allodynia even before loading the mice with cisplatin (Day 0, Figure 8B). This suggests that AIBP plays a role in maintaining microglial homeostatic function in mechanonosensitivity. Control Cx3cr1-Cre lacking the floxed gene ERT2 In mice, tamoxifen injection did not induce mechanical allodynia (Figure 8C). Following cisplatin loading, microglia AIBP knockdown resulted in a faster decrease in the mechanical threshold compared to control mice (comparing day 2 in Figure 8D with day 6 in Figure 8E) and showed higher sensitization in microglia 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 (Figures 8C and 8D). In systemic Apoa1bp knockout mice, we observed no basal allodynia compared to WT mice, and itAIBP rescued CIPN-induced allodynia (Figure 8F). In contrast to AIBP-imKO or ABC-imKO mice, TLR4-imKO mice were protected from the rapid onset of cisplatin-induced allodynia and exhibited delayed, mild allodynia (Figure 8G), suggesting a role for TLR4 expression in microglia in mediating pain sensitization. Identification of the AIBP domain 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 evaluate the previously reported in vivo importance of AIBP-TLR4 binding (Woller et al., 2018). Here, we took a different approach and created AIBP mutants that did not bind to TLR4. To elucidate which domain of AIBP is involved in binding to TLR4, we began by mutating amino acids predicted from the crystal structure of the YjeF_N domain of AIBP (Figure 9A) involved in protein-protein interactions (Jha et al., 2008), but these mutants retained TLR4 binding properties (not shown). Next, we developed a series of deletion mutants of AIBP that scanned the full length of the protein and tested them in a pull-down assay using the TLR4 external domain (eTLR4) (Figure 9B). These experiments suggested that the N-terminal domain of amino acids 25-51, located behind the aa 1-24 signal peptide, is involved in eTLR4 binding (Figures 9A and 9B). The aa 25-51 N-terminal domain was not structured in the publicly available crystal structure of mouse AIBP (Jha et al., 2008). Both human and mouse AIBP contain homologous aa 25-51 N-terminal domains, but zebrafish AIBP does not. In fact, unlike human and mouse AIBP, zebrafish AIBP did not bind to human eTLR4 (Figure 9C). For further experiments, we expressed and purified wtAIBP (aa 25-288) lacking the signal peptide and mutAIBP (aa 52-288) lacking both the signal peptide and the N-terminal domain from baculovirus / insect cell lines.

[0221] Unlike wtAIBP, mutAIBP did not bind to eTLR4 in either a pull-down assay (Figure 9D) or an ELISA using an eTLR4-coated plate, nor did it bind to bound AIBP in detection using a 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 both wtAIBP and mutAIBP (Figure S5B). The binding of mutAIBP to APOA1 was unchanged compared to wtAIBP (Figure 9F). In cell culture experiments, wtAIBP bound to LPS-stimulated BV-2 microglia, but mutAIBP did not (Figures 9G and 9H). The increased wtAIBP binding in response to LPS can be explained by the recruitment of TLR4 to the cell surface and its localization to inflammaft (Yvan-Charvet et al., 2008; Zhang et al., 2018; Zhu et al., 2010). In summary, these results suggest a role for the aa 25-51 N-terminal domain of AIBP in TLR4 binding. AIBP lacking a TLR4 binding domain cannot alleviate CIPN allodynia.

[0222] Unlike wtAIBP, mutAIBP, which lacks a TLR4 binding site, was unable to inhibit LPS-induced TLR4 dimerization in BV-2 microglia (Figure 10A), but retained its overall ability to reduce lipid rafts (Figure 10B). Next, we tested the hypothesis that this TLR4 targeting mediates the therapeutic effect of AIBP. Mice administered with saline or mutAIBP before itLPS developed allodynia rapidly and to a similar degree, whereas itwtAIBP prevented LPS-induced mechanical allodynia (Figure 10C). In the CIPN mouse model, itwtAIBP reversed established allodynia, and the sustained therapeutic effect lasted for at least 14 days (Figure 10D). However, itmutAIBP induced only a modest transient reversal of the mechanical threshold, failing to reach naive or baseline levels and lasting only 2-3 days (Figure 10D). Mice were sacrificed on day 21 and their lumbar spines were analyzed. Notably, even at this late stage, cisplatin-induced polyneuritis continued to be associated with increased TLR4 dimerization and lipid rafts in spinal microglia, which were significantly reduced by itwtAIBP but not by mutAIBP (Figures 10E and F), similar to the effect observed on day 8 (Figures 1B and C). These results support the hypothesis that AIBP targeting of TLR4 inframa rafts mediates the majority of the therapeutic effect of AIBP in the mouse model of CIPN. Consideration

[0223] In this study, we report a novel mechanism of selective cholesterol depletion from inflammaft, hosted by TLR4, in spinal microglia as a new level of modulation of neuropathic pain in chemotherapy-induced peripheral neuropathy (Figure 10G) and possibly other neurological disorders. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia induced spontaneous allodynia in naive mice exhibiting similarities to the cisplatin effect. Importantly, the absence of ABCA1 and ABCG1 expression in microglia reversed LPS or cisplatin-induced allodynia or abolished the ability of AIBP to reduce inflammaft and TLR4 dimerization in spinal microglia. This unique 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 the unique ability to destroy inframalaft in activated cells (singular It possesses the ability, but has little effect on physiological lipid rafts in quiescent cells. No. The inventors have proposed that this is due to the binding of AIBP 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, the inventors identified the N-terminal domain of AIBP as the binding site for TLR4 and demonstrated the crucial role of this domain in enabling AIBP binding to activated microglia and its therapeutic effect in CIPN. Based on this, the inventors propose that AIBP is a selective treatment directed towards inflammaft, in contrast to the non-selective cholesterol removal brought about by cyclodextrins, APOA1 and APOA1-mimicking peptides or LXR agonists. Mutant human AIBP lacking the N-terminal domain still binds to APOA1, and wild-type zebrafish AIBP, which naturally lacks this N-terminal domain, still increases cholesterol efflux from endothelial cells, regulates angiogenesis, and modulates the emergence of hematopoietic stem cells and progenitor cells from hematopoietic endothelial cells (Fang et al., 2013; Gu et al., 2019). This suggests that there are different, TLR4-independent mechanisms in the interaction between AIBP and endothelial cells.

[0225] Intrathecal delivery of AIBP exhibited a sustained therapeutic effect in a mouse model of CIPN, observed over a long period of 10 weeks in our previous study (Woller et al., 2018) and over 2 weeks in this study. This contrasts sharply with the short-term exposure to itAIBP. AIBP peaked at 30 minutes and was almost completely eliminated from both CSF and lumbar spinal tissue by 4 hours. The dissociation between exposure and therapeutic effect suggests a disease-modifying effect of AIBP. A decrease in CTxB binding and a reduction in the proportion of TLR4 dimers in spinal microglia were observed 24 hours and even 2 weeks after a single itAIBP injection, indicating sustained disruption of inflammaft by AIBP, in contrast to their sustained presence in the microglia of CIPN mice injected with saline. In addition to its targeted effect on TLR4 inflammaft, the disease-modifying effect of AIBP may involve reprogramming of gene expression profiles in spinal microglia. AIBP reversed the expression of only 3% of all genes affected by CIPN in spinal cord microglia, but AIBP significantly reduced the levels of inflammatory gene expression and inflammatory cytokines in spinal cord tissue induced by cisplatin regimens. These included genes encoding cytokines and chemokines described as having 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, cisplatin regimens induced transcriptional changes similar to the gene signatures of disease-related 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 have recently been described as being associated with aging and neurodegeneration (Marschallinger et al., 2020; Nugent et al., 2020). Homeostatic genes downregulated during the transition of microglia to these pathological phenotypes (Masuda et al., 2019; Nugent et al., 2020; Prinz et al., 2019) were also downregulated in microglia of CIPN mice. The CIPN-induced downregulation of microglial Abca1 and Abcg1 expression is an important factor for understanding the effects of AIBP. AIBP (compound 7) did not reverse the reduction of Abca1 or Abcg1 mRNA associated with CIPN, but its ability to stabilize the ABCA1 protein and promote cholesterol efflux (Zhang et al., 2016) may be sufficient to normalize cholesterol metabolism in microglia. The effect of AIBP (compound 7) on allodynia was replicated, albeit transiently, by itAPOA1 or LXR agonists (Woller et al., 2018). Furthermore, a negative association has been found between ABCA1 single nucleotide variants and quality of life scores in patients with painful bone metastases (Furfari et al., 2017). However, we cannot rule out other mechanisms unrelated to the reversal of a subset of CIPN-affected genes that AIBP (compound 7) may reprogram microglia to confer a protective phenotype in a pain-enhanced state.

[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, even upregulating some of them, and upregulating the 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 efflux mechanisms, AIBP may actually promote inflammatory and cytotoxic effects.

[0228] Overall, the results of this study suggest that the regulation of cholesterol content in the plasma membrane of spinal microglia significantly influences inflammaraft-derived cellular signaling and subsequent gene expression of inflammatory and lipid metabolism genes, ultimately leading to peak control of nociceptives under conditions of polyneuropathy. material and method

[0229] animal. Wild-type Abca1 fl / fl Abcg1 fl / fl , TLR4 fl / fl Slc1a3-Cre ERT and Cx3cr1-Cre ERT2 All mice were purchased from Jackson Lab (Bar Harbor, ME) or bred and weaned in-house, using a C57BL / 6 background. TLR4 - / -The mouse was donated by Dr. Akira (Osaka University). Apoa1bp fl / fl The mice were previously generated in our laboratory using ES cells derived from C57BL / 6 mice. The following mouse strains were crossed 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 microglia conditional knockout mice used in the experiment were treated with Cx3cr1-Cre to avoid the generation of Cx3cr1 knockout. ERT2 Each mouse possessed only one allele. Mice were housed at room temperature, up to four per standard cage, and maintained in a 12:12 light-dark cycle. All behavioral tests were conducted during the light phase of the cycle. Both food and water were freely available. All experiments were conducted using male mice, following protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California.

[0230] cell. BV-2 immortalized microglia cell line (Blasi et al., 1990) was cultured in Dulbecco's MEM containing 5% fetal bovine serum (FBS). Thioglycolic acid-induced peritoneal macrophages were cultured in C57BL / 6 or TLR4. - / - Cells were isolated from mice and maintained in DMEM (Cellgro) supplemented with 10% thermally 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 and 3.

[0231] A model of chemotherapy-induced peripheral neuropathy. To induce chemotherapy-induced peripheral neuropathy (CIPN), cisplatin (2.3 mg / kg / injection; Spectrum Chemical MFG) was administered intraperitoneally (ip) on days 1 and 3. During cisplatin administration, weight loss, behavioral changes, and mechanical allodynia were monitored and measured. Euthanasia criteria were weight loss exceeding 20% ​​of body weight and irritable behavior. However, no animals required euthanasia.

[0232] Mechanical allodynia measurement. Animals were placed in transparent, bottomless plastic cages on a wire mesh surface and allowed to acclimate for at least 30 minutes before the start of the experiment. Tactile thresholds were measured using a series of von Frey filaments (Bioseb) ranging from 2.44 to 4.31 g (0.02 to 2.00 g). The threshold for a 50% detachment probability was recorded. Mechanical detachment thresholds were assessed using the up-down method (Chaplan et al., 1994) at pre-treatment (baseline or day 0) and post-treatment input points.

[0233] AIBP (Compound 7) Alternatively, intrathecal delivery of saline solution. Mice were anesthetized using 5% isoflurane in oxygen for induction and 2% isoflurane in oxygen for maintenance. Intrathecal injection was performed according to (Hylden and Wilcox, 1980). Briefly, the lumbar region 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 along the midline between the L5 and L6 vertebrae. Successful entry was assessed by observing tail flick. 5 μL injections were administered at approximately 30-second intervals. The drug for intrathecal delivery was formulated with physiologically sterile 0.9% NaCl. Based on previous studies (Woller et al., 2018), the dose of AIBP (compound 7) for vertebral delivery in these studies was 0.5 μg / 5 μL. After recovery from anesthesia, mice were evaluated for normal motor coordination and muscle tone.

[0234] Intraperitoneal injection of tamoxifen for inducible Cre driver strains.In this study, the inventors followed the Jackson Lab 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 administered 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 recognizes TLR4 / MD2 as monomers (in TLR4 units) but not as dimers; SA15-21 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 bound to ganglioside GM1. To evaluate TLR4 dimerization in vitro, BV-2 cells were pre-incubated for 30 minutes with 0.2 μg / ml AIBP (compound 7) in serum-containing medium, followed by incubation with 100 ng / mL LPS for 15 minutes. At the end of incubation, the cells were immediately placed on ice, washed once with PBS, and fixed with 4% formaldehyde for 10 minutes. The cells were then washed twice with ice-cold FACS buffer and incubated on ice for 30 minutes with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience). They were then stained on ice for 30 minutes with 1:100 dilutions of PE-conjugate MTS510 antibody and APC-conjugate SA15-21 antibody (ThermoFisher and Biolegend, RRID:AB_2562503 and RRID:AB_466263, respectively) with 1:200 dilution of CTxB-FITC (ThermoFisher). The cells were washed and analyzed using a FACSCanto II (BD Biosciences) flow cytometer.

[0236] For ex vivo assays, spinal cord was collected by hydroextrusion (Kennedy et al., 2013), fixed with 4% formaldehyde, and kept on ice during processing. A nerve tissue dissociation kit (Miltenyi Biotec) was used according to the manufacturer's protocol to obtain a single-cell suspension from lumbar spine tissue. To remove myelin, Myelin Removal Beads II (Miltenyi Biotec) were added to the sample, incubated at 4°C for 15 minutes, and then separated using an LS column and MACS separator (Miltenyi Biotec). After isolation, cells were incubated on ice for 30 minutes with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience), then stained on ice for 45 minutes with an antibody mix of 1:100 PerCP-Cy5.5 conjugate CD11b antibody (Biolegend, RRID: AB_893232), 1:100 rabbit anti-mouse TMEM119 antibody (Abcam, RRID: AB_2744673), PE conjugate MTS510, APC conjugate SA15-21 antibody (ThermoFisher, RRID: AB_2562503 and Biolegend, RRID: AB_466263, respectively), and 1:200 dilution CTxB-FITC (ThermoFisher), then washed and Alexa. Cells were incubated on ice for 30 minutes with PECy7 conjugate anti-rabbit secondary antibody (1:250). Cells were washed and analyzed using a FACSCanto II (BD Biosciences) flow cytometer.

[0237] For in vitro and ex vivo staining correction, the inventors corrected signal overlap between channels using beads and / or single-stained cells and depicted the gate using isotype controls of CD11b, MTS510, and SA15-21 antibodies along with FMO. The data were analyzed by FlowJo (BD Bioscience, RRID: SCR_008520). From these data, the inventors calculated the relative changes in the abundance of lipid rafts and the number of TLR4 dimers in spinal cord microglia (0 dimers were randomly assigned to unstimulated or naive cells).

[0238] Immunofluorescence, confocal imaging, and colocalization analysis. BV-2 cells were seeded onto coverslips in 12-well plates and pre-incubated for 30 minutes with 0.2 μg / ml AIBP in 5% serum medium, followed by incubation with 100 ng / mL LPS for 5 or 15 minutes. At the end of incubation, the cells were immediately placed on ice, washed once with PBS, and fixed with 4% formaldehyde for 10 minutes. Cells were washed twice with ice-cold PBS and incubated for 30 minutes in blocking buffer containing 5% FBS. They were then stained with 1:200 dilution CTxB-Alexa555 and 1:100 dilution mouse anti-TLR4 antibody (Abcam, RRID: AB_446735), or 1:100 dilution rabbit anti-APOA1 antibody (Abcam) or 1:100 dilution rabbit anti-ABCA1 (Novus Biological RRID: AB_10,000,630), washed, incubated with anti-rabbit Alexa 647 conjugate secondary antibody, and incubated with recombinant His-tagged ALOD4 and 1:100 FITC conjugate 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 with Lightning deconvolution or STED.

[0239] To validate microglia-specific AIBP or ABCA1 / ABCG1 knockout, spinal cord tissue was collected and post-fixed in 4% formaldehyde at 4°C. The tissue was then dehydrated with 30% sucrose and frozen by OCT until sectioning. The spinal cord was sliced ​​into 10 μm sections using a cryostat, and the slides were stored at -20°C. The frozen sections were blocked with 2% FBS and 0.3% Triton® X100 solution and incubated with 1:100 rabbit anti-AIBP antibody (courtesy of Dr. Longhou Fang). Separate sections were stained overnight at 4°C with 1:100 rabbit anti-ABCA1 antibody or 1:100 rabbit anti-ABCG1 antibody (Novus Biological, RRID:AB_10000630 and RRID:AB_10125717). The slides were washed and incubated for 2 hours with a 1:200 dilution of anti-rabbit Alexa 488 (Abcam, RRID: AB_2630356) or Alexa 647 conjugate secondary antibody, then washed three times. All sections were incubated with either Alexa 488 conjugate IBA-1 antibody (Milipore-Sigma) or Alexa 633 conjugate IBA1 antibody (Wako Chemicals, RRID: AB_2687911). Alternatively, the slides were incubated with either 1:100 Alexa 488 conjugate anti-NeuN antibody (Cell Signaling, RRID: AB_2799470) or 1:100 Alexa 488 conjugate anti-GFAP antibody (Cell Signaling, RRID: AB_2263284). The slides were washed three times with PBS and mounted on Prolonged Gold containing DAPI (Cell Signaling). Images of at least one slide from each animal were acquired using a Leica SP8 confocal microscope with a 63X objective lens and Lightening deconvolution. Colocalization analysis was performed in ImageJ / FIJI (NIH, RRID: SCR_003070 / SCR_002285) using the Coloc2 tool.For each image, a threshold, Pearson's R-coefficient and Manders coefficients exceeding the threshold, as well as masked colocalized images, Costes P-values, and pixel scatter plots were generated. tM1 or tM2 was used depending on which channel represented the cell marker.

[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 competent E. coli cells BL21 (DE3). + Positive colonies were selected using LB plates. After induction of expression with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and lysis, His-tagged ALOD4 was purified using an imidazole-eluting Ni-NTA agarose column. The protein was dialyzed against PBS and its concentration was measured. Aliquots were stored at -80°C.

[0241] Cloning and expression of wtAIBP and mutAIBP in baculovirus / insect cell lines. AIBP (compound 7) was generated in a baculovirus / insect cell system to ensure post-translational modification and endotoxin-free preparation, 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 vector contained an N-terminal His tag to enable purification and detection. Insect Sf9 cells were transfected with BestBac baculovirus DNA (Expression Systems) and the AIBP vector. After 4–5 days, the supernatant was collected to obtain a baculovirus stock. Fresh Sf9 cells were infected with AIBP-producing baculovirus. After 3 days, the cell pellet was collected, lysed, sonicated, and clarified by centrifugation. The supernatant was loaded onto a Ni-NTA agarose column and eluted with imidazole. Proteins were dialyzed against saline and their concentrations were measured. Aliquots were stored at -80°C.

[0242] AIBP in spinal cord tissue (Compound 7) Pharmacokinetics. Knockout AIBP mice were used for pharmacokinetic studies. Intrathecal injection of AIBP (2.5 μg / 5 μL) was performed as previously described (Hylden and Wilcox, 1980), and CSF was collected at 15 minutes, 30 minutes, 1 hour, 4 hours, or 8 hours later as described (Liu and Duff, 2008). Briefly, a capillary tube (0.8 × 100 mm) was drawn using a micropipette puller. Mice were anesthetized using 3% isoflurane with a mixture of 50% oxygen and 50% room air. The skin of the neck was shaved, and the mice were placed on a stereotactic device. After wiping the surgical site, a sagittal incision was made in the skin below the back of the head. The subcutaneous tissue and muscle were incised to expose the dura mater. The drawn capillary tube was directly punctured into the cisterna magna to collect an uncontaminated sample. After collecting CSF, the capillaries were poured into a PCR tube containing 50 μL of 0.09% NaCl, and the mice were then perfused with 35 mL of 0.9% NaCl. The spinal cord was washed with hydroextrusion using 5 mL of 0.9% NaCl. The spinal cord tissue was weighed and extracted on ice with 1 g / 10 mL of complete N-PER™ neuron protein extraction reagent (Thermo Fisher). After incubation on ice for 10 minutes, the samples were centrifuged (10,000 × g for 10 minutes at 4°C) to pellet the cell 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) and incubated with spinal cord extract or CSF sample for 3 hours, followed by detection using rabbit polyclonal anti-AIBP antibody, and then goat anti-rabbit ALP antibody (Sigma-Aldrich, RRID: AB_258103). The plate was read in the same way as above.

[0243] FACS sorting of spinal cord microglia for RNA-seq.A cell suspension from the lumbar spinal cord was prepared in the same manner as described above, except for the fixation step. Fresh tissue was processed and blocked for 30 minutes with 2% normal mouse serum containing anti-CD16 / CD32 antibody (FcγR blocker, BD Bioscience), then stained with a mix of 1:50 PE-Cy7 conjugate CD11b antibody (Biolegend, RRID: AB_312799), 1:50 rabbit anti-mouse TMEM119 antibody (Abcam, RRID: AB_2744673), and 1:50 PerCP-Cy5.5 conjugate CD24 antibody (Biolegend, RRID: AB_1595491), then the cells were washed and incubated with (1:200) secondary Alexa488 conjugate anti-rabbit antibody (Abcam, RRID: AB_2630356) on ice for 30 minutes, then the cells were washed and stained with 1:50 Alexa 647 conjugate Glast1 antibody (Novus Cells were incubated on ice for 30 minutes with a 1:100 dilution of Life / Death Ghost Red 780 dye (Cell Signaling). After washing with sorting buffer and filtering, the cells were sorted into lysis buffer using a BD FACS-Aria cell sorter (BD Biosciences). Three technical replicas, each containing 400 cells from the same animal, were sorted. See Figures S2A and S2B for sorting strategies and analysis of sorted microglia purity.

[0244] Preparation, sequencing, and quality control of RNA-seq libraries.The inventors followed the low-input bulk seq SmartSeq2 protocol (Rosales et al., 2018). Cells sorted into lysis buffer containing Triton® X-100, an RNase inhibitor, and oligo(dT)30-VN were hybridized to the mRNA poly(A) tail with oligo(dT)+. After adding reagents for PCR amplification, cDNA libraries were constructed by adding reagents for reverse transcription (qPCR was not performed at this point). Libraries were quantified and QC performed using a Qubit double-strand high-sensitivity assay in addition to a TapeStation high-sensitivity D5000 screening tape. All samples were prepared to 1 ng of cDNA for ingestion in the NexteraXT protocol. QC checks were performed using a Qubit double-strand high-sensitivity assay in addition to a TapeStation high-sensitivity D1000 screen tape. Samples were subjected to qPCR and pooling and loaded into NovaSeq for paired-end 50×50 reads using a NovaSeq S1 100 cycle kit.

[0245] Splice recognition alignment of FASTQ data was performed using STAR (Dobin et al., 2013), and quality control of sequenced data and alignments was performed using FASTQC (RRID:SCR_014583), QoRTs (RRID:SCR_018665) (Hartley and Mullikin, 2015), and the MultiQC tool (RRID:SCR_014982) (Ewels et al., 2016). Gene counts associated with reads were performed using STAR (RRID:SCR_015899).

[0246] Sequence quality control demonstrated good data quality (MultiQC report). Two technical replicas (Y_10 and Y_30) were removed due to suboptimal gene coverage. The inventors analyzed differential expression using the R package, DEseq2 (RRID: SCR_015687) (Love et al., 2014). For at least three samples, the inventors identified a total of 18,818 genes in lumbar spinal microglia using a cutoff set to more than 10 counts per 1 million mapped reads CPM. One sample was excluded from further analysis because it showed an extremely irregular distribution in PCA compared to all other samples and clustered to the top 500 most variable genes. The inventors confirmed microglia enrichment in their samples and data using a subset of 40 microglia-specific genes reported by Butovsky et al. (2014) and 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 and 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. The inventors used an LRT model compared to a reduced design without condition and genotype interaction terms to identify genes regulated in a genotype (ABC-imKO)-dependent manner. Significant genes were filtered using adjusted P<0.05 and 5% FDR. Gene clustering by gene expression patterns of identified significant genes was performed by the DESeq2 function:degpatterns. Pairwise comparisons after LRT were performed in the experimental groups using the Wald test, with an FDR of 5%. The volcano plots included genes that were significantly different with an absolute magnification change of more than 1.5. Pathway enrichment and GO analysis were performed using metascape.org (RRID: SCR_016620) with a minimum of 3 genes and P<0.05 (Zhou et al., 2019).

[0247] Co-immunoprecipitation assay for TLR4 binding. Pull-down assays of eTLR4 and wtAIBP or mutAIBP in test tubes using 0.5% Triton® Immunoblotting was performed by mixing 1 μg of eTLR4 (Sino Biological) with AIBP in PBS containing X-100 and incubating at room temperature for 1 hour. Samples were pre-clarified by adding Protein A / G Sepharose beads at room temperature for 30 minutes, followed by the addition of 1 μg of BE-1 monoclonal anti-AIBP antibody and incubation for 2 hours. Protein A / G Sepharose beads were added, incubated for another 1 hour, and then washed five times with PBS containing 0.5% Triton® X-100 before immunoblotting of the samples.

[0248] HEK293 cells (RRID:CVCL_0045) were transfected with Flag-eTLR4 and Flag-AIBP (wild-type or one of the mutants) constructs. 36 hours after transfection, cells were harvested and lysed in 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 a Sigma protease inhibitor cocktail). Cell lysates were pre-incubated with Protein A / G Sepharose beads at 4°C for 30 minutes and immunoprecipitated overnight at 4°C with mouse anti-TLR4 antibody (Abcam). The following day, the lysates were incubated with Protein A / G beads at 4°C for 1 hour. Unbound proteins were removed by washing with lysis buffer, and the beads were flowed on a Bolt Bis-Tris gel (Invitrogen). The bound AIBP was detected by immunoblotting with an anti-Flag antibody (Sigma).

[0249] ELISA binding assay.To evaluate 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 evaluate AIBP-APOA1 binding, plates were coated with BSA, wtAIBP, or mutAIBP, washed, blocked, incubated with 5 μg / ml human APOA1 (donated by Dmitri Sviridov, Baker Heart and Diabetes Institute, Melbourne, Australia), and followed by incubation with biotinylated anti-APOA1 antibody (Academy Bio-Medical Company, RRID: AB_1238781). In both assays, neutraavidin-AP was added and incubated at room temperature for 45 minutes, followed by the addition of LumiPhos 530 (Lumigen) and incubation for 90 minutes. Luminescence was then measured using a luminescence plate reader (BioTek, Winooski, Vermont).

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

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

[0252] Statistical analysis. For datasets other than RNAseq datasets, results were analyzed using GraphPad Prism (RRID: SCR_002798) with 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 time-course experiments with multiple groups). Differences between groups with P<0.05 were considered statistically significant. Legend for the figure

[0253] Figure 1 demonstrates the reversal of pain behavior and the 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 threshold in WT mice in response to ip cisplatin (2.3 mg / kg / day, administered twice), followed by a single dose of it saline (5 μl) or AIBP (compound 7) (0.5 μg / 5 μl). Naive mice did not receive injections. Data from two independent experiments (n=6 per group). B-C. CD11b showing TLR4 dimerization. + / TMEM119 +Analysis of spinal cord microglia cells (B) and lipid raft content measured by CTxB staining 24 hours after in saline or AIBP, i.e., on day 8 of the time course shown in A (C). Data from 3 independent experiments (n=9 / group for TLR4 dimerization, n=12 for lipid raft staining). D, BV-2 microglia cells incubated with AIBP (compound 7) (0.2 μg / mL) or vehicle in complete medium for 30 minutes, followed by incubation with LPS (100 ng / mL) for 5 minutes. Scale bar, 5 μm. Bar graph shows Manders' tM1 coefficient. E-F, male APOA1BP - / - Pharmacokinetics of itAIBP (2.5 μg / 5 μL) in mouse CSF (E) and lumbar spinal cord (F) (n=5). * P<0.05; ** P<0.01; *** P<0.001. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons in group analysis; one-way ANOVA with Tukey post-hoc test for multiple comparisons of three groups and imaging quantification.

[0255] Figure 2 compares the changes in gene signatures between naive mice, mice treated with the chemotherapeutic agent 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 +FACS sorting was performed from the three groups shown in Figure 1A: WT naive mice were injected with cisplatin (on days 1 and 3), followed by administration of 5 μL of saline or AIBP (compound 7) (0.5 μg / 5 μL) on day 7, and the procedure was completed on day 8 for RNA-seq; n=3 biological replicas (mice) for naive and cisplatin / saline mice, and n=2 for cisplatin / AIBP (each biological replica was a short summary from three technical replicas from the same animal). A. Heatmap of DEGs across all samples (all technical replicas are shown in columns). Significant (adjusted P<0.01) upregulatory genes showing the main effect, tested by LRT (likelihood ratio test). Log2 relative expression, B. Groups of significant DEGs clustered based on expression profile patterns under different treatment conditions. C, pathway and GO enrichment analysis of cisplatin-treated upregulatory genes (group 1 in panel 2B) and downregulatory genes (group 2) using adjusted P<0.05 and absolute magnification change>1.5 and minimal duplication of three genes within the pathway. Upregulatory pathways are shown in red, and downregulatory pathways are shown in blue.

[0257] Figure 3 compares the differences in disease-related microglia (DAM) gene expression signatures and lipid droplets between chemotherapy-treated mice and naive mice and CIPN mice treated with wtAIBP.

[0258] Figure 3. DAM and lipid-related gene expression and lipid droplets in spinal microglia of CIPN mice.A-C, same group as in Figure 2. A, Volcano plot of upregulated and downregulated genes in spinal microglia of cisplatin-treated and naive mice. Adjusted P<0.05 and absolute magnification change>1.5 cutoffs are shown as light green dots. B, Heatmap representing disease-associated microglia (DAM) signature genes. C, Heatmap of log2-normalized gene counts scaled per row showing lipid-related gene sets. D-H, Lipid droplet accumulation in spinal microglia measured by PLIN2 immunostaining in spinal sections co-stained with IBA1 and DAPI. Experimental conditions were the same as in Figure 1A; n=5 fields of view with 5 mice per group from two independent experiments. Scale bar, 20 μm. Mean ± SEM; * Compared to the naive group, P<0.05 was observed, and the results were tested by one-way ANOVA with Tukey's test for multiple comparisons in group analysis.

[0259] Figure 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 were the same as in Figure 1; n=2-3 biological replicas per group (each biological replica was abbreviated from three technical replicas). A. Pathway and GO enrichment analysis of AIBP (compound 7) downregulated CIPN upregulatory genes (group 3 in Figure 2B) and AIBP (compound 7) downregulated CIPN downregulatory genes (group 4) using adjusted P<0.05 and absolute digit change>1.5 and minimal duplication of three genes within the pathway. Upregulated pathways are shown in red, and downregulated pathways are shown in blue. B. DEG of spinal cord microglia induced by itAIBP. Adjusted P<0.05 and Benjamini-Hochberg FDR<5% are represented in volcano plots of upregulated and downregulated genes in mice treated with cisplatin / AIBP versus cisplatin / saline. Cutoff adjusted P<0.05 and absolute digit change>1.5 are shown as light green dots. C. Heatmap of inflammatory genes in group 3 that are upregulated by CIPN and downregulated by AIBP. D. Cytokine protein expression in spinal tissue 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 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 magnification change>1.5 and minimum duplication of three genes in the pathway. G. Heatmap of non-inflammatory genes downregulated by AIBP (compound 7) in the most enriched pathway: peptidase inhibitor activity pathway. H. Heatmap of genes whose downregulation in CIPN is reversed by AIBP (compound 7). Mean ± SEM; * P<0.05 was observed when comparing the naive group with the cisplatin / it saline group.

[0261] Figure 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. Expression of ABCA1 and ABCG1 in microglia controls nociception and AIBP in a mouse model of CIPN and allodynia. (Compound 7) It is necessary for mediation reversal. A-B. BV-2 cells were incubated in complete medium with AIBP (compound 7) (0.2 μg / mL) or vehicle for 30 minutes, followed by incubation with LPS (100 ng / mL) for 5 minutes. Co-localization of accessible cholesterol with ABCA1 (A) and APOA1 (B) in lipid rafts. Scale bar, 7 μm. Bar graph shows 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. Withdrawal threshold at baseline (day 0) before initiation of cisplatin intervention. Data from three independent experiments (n=8 for vehicle-treated ABC-imKO mice, n=16 for TAM-treated ABC-imKO mice, and TAM-treated littermates Abca1 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, dimerization, and lipid rafts (CTxB) in spinal cord 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 itLPS (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) mice and non-induced (vehicle) ABC-imKO (H) mice (n=6 per group); CD11b on day 8 in the groups shown in panels G and H. + TEMEM119 +Data from two independent experiments I–J in spinal cord microglia: TLR4 dimerization (I) and lipid rafts (J). 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-series analysis; t-test for two groups; and one-way ANOVA with Tukey post-hoc test for multiple comparisons of groups greater than two.

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

[0264] Figure 6. Gene expression in spinal microglia of ABC-imKO mice. Microglia (CD11b + TEMEM119 +FACS sorting was performed from three groups of ABC-imKO mice: naive or cisplatin injection (days 1 and 3), followed by injection of it saline (5 μL) or AIBP (compound 7) (0.5 μg / 5 μL) on day 7, and completion on day 8; n=3 biological replications (each biological replication was a short summary from three technical replicates). RNA-seq datasets from ABC-imKO mice and WT (not littermates) mice were obtained in the same experiment. A, top: Duplication genes and pathways induced in naive ABC-imKO microglia and shared with WT microglia in cisplatin-treated mice are shown by purple lines connecting duplicate genes and blue lines connecting duplicate enrichment pathways. Bottom: Venn diagram of upregulated genes in spinal cord microglia from WT cisplatin and ABC-imKO naive mice. B. Enrichment pathway analysis of upregulated and downregulated genes induced by ABCA1 and ABCG1 knockdown in microglia, using cutoff P<0.05, enrichment >1.5, and minimal duplication of three genes within the pathway. C. DEG in naive spinal microglia of TAM-induced ABC-imKO mice. Adjusted P<0.05 and Benjamini-Hochberg FDR<5%. D. Duplication genes and pathways induced by cisplatin treatment in ABC-imKO microglia and shared with WT microglia of cisplatin-treated mice. E. DEG 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. Heatmaps of upregulated (F) or downregulated (G) DEG in ABC-imKO microglia under either naive or cisplatin conditions.

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

[0266] Figure 7. Microglia reprogramming by AIBP is dependent on ABCA1 / ABCG1 expression.A. Venn diagram comparing the effects of AIBP treatment on gene expression in cisplatin-induced CIPN in WT mice and ABC-imKO mice. B. Volcano plot showing upregulation and downregulation of genes in CIPN by AIBP treatment, comparing the effects of AIBP in ABC-imKO mice and WT mice. Adjusted cutoffs of P<0.05 and absolute magnification change >1.5 are shown as light green dots. C. Heatmap of normalized log2 gene counts of inflammatory genes altered in an ABC-dependent manner by AIBP (downregulation by AIBP in WT microglia, but upregulation by AIBP in ABC-imKO). D. Heatmap of cholesterol synthesis and LXR-related genes comparing the effects of cisplatin and AIBP in wild-type and ABC-imKO. E. Heatmap of non-inflammatory genes regulated by AIBP in an ABC-dependent manner. Analysis of the AIBP-mediated upregulated gene enrichment pathway in ABC-imKO microglia using F. cutoff P<0.05, enrichment >1.5, and minimal duplication of three genes within the pathway.

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

[0268] Figure 8. Endogenous AIBP and TLR4 in microglia 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 withdrawal threshold before initiation of cisplatin intervention (Day 0 in A). Mean ± SEM (n=15 for vehicle treatment, n=16 for TAM-treated AIBP-imKO mice, Apoa1bp in littermates treated with TAM) fl / fl For no-Cre[WT] mice, n=8). C, WT and Cx3cr1-Cre ERT2(floxed gene-free) 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 to have died in the WT+TAM group). No statistical differences were observed. 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 facility-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 facility-bred wild-type, 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 group analysis; one-way ANOVA with Tukey post-hoc test for multiple comparisons of two groups.

[0269] Figure 9: Identify sequence motifs that contribute to the binding of AIBP and TLR4. Figure 9. Identification of domains in the AIBP molecule involved in TLR4 binding.A. Human AIBP: signal peptide (aa1-24), previously uncharacterized N-terminal domain (aa25-51), and YjeF_N domain (aa52-288). B. Flag-tagged deletion mutants of human AIBP were co-expressed with flag-tagged TLR4 external domain (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) AIBP lacking all signal peptides, expressed in baculovirus / insect cell lines, were combined in vitro with eTLR4-His, followed by IP containing anti-TLR4 antibody and IB containing 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 using plates coated with eTLR4 and incubated with wtAIBP or mutAIBP (n=3) (E). ELISA using plates coated with BSA, wtAIBP, or mutAIBP and incubated with APOA1 (F). 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 minutes. Detection by anti-His antibody (flow) and anti-TLR4 antibody (imaging). Scale bar, 10 μm. Average ± 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 longitudinal analysis; t-test for two groups; and one-way ANOVA with Tukey post-hoc test for multiple comparisons of groups greater than two.

[0270] Figure 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 a TLR4 binding domain cannot alleviate 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 minutes. Mean ± SEM (in TLR4 dimerization analysis, n=7 for the control group, n=5 for the mutAIBP group, n=9 for the LPS group, and n=8 for the wtAIBP group; in lipid raft analysis, n=8 for the control group, and n=13 for the mutAIBP, LPS, and wtAIBP groups; data from two independent experiments). C, withdrawal threshold in WT mice administered 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 threshold in WT mice responding to ip cisplatin (2.3 mg / kg / day), followed by it wt AIBP (0.5 μg / 5 μL), mut AIBP (0.5 μg / 5 μL), or saline (5 μL). Naive mice did not receive injections (n=7 for the naive group, n=8 for the wt AIBP and mut AIBP groups, n=9 for the it saline group; data from two independent experiments). E-F. CD11b from the lumbar spinal cord of mice from the experimental groups 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 by Bonferroni post-hoc test for multiple comparisons in time-series analysis; and one-way ANOVA by Tukey post-hoc test for multiple comparisons of groups greater than 2. G. Figure showing the effects of CIPN and AIBP (compound 7) treatment on microglia gene expression and lipid droplet accumulation. Black dots in the plasma membrane and ER represent cholesterol.

[0272] Figure 11 hypothesizes a model of exposure of the TLR4 binding site of AIBP in a modified AIBP sequence: Figure 11. A model of unfolding or exposing the latent N-terminal domain in the AIBP molecule. This figure summarizes the results of the experiments shown in Figures 12–14, demonstrating that in natural AIBP, the N-terminal domain (green) is either hidden, latent, or not sufficiently exposed to mediate AIBP binding to TLR4 (upper panel). Extending the N-terminus with additional amino acids (orange) alters the AIBP conformation, allowing the N-terminal domain of AIBP (green) to access TLR4 binding (lower panel). Figure 12. An example of an amino acid sequence of an exemplary manipulated AIBP provided herein. The amino acid sequence of the extended AIBP molecule is shown in the lower panel of Figure 11. Blue text, amino acids of the natural AIBP sequence; green box, TLR4 binding sequence (amino acids 25–51 of the human AIBP sequence); black text and red box, additional amino acids. Figure 13 reveals the TLR4 binding of a specific modified AIBP sequence derived from a baculovirus expression system.

[0273] Figure 13. TLR4 binding of various manipulated forms of AIBP. All proteins were expressed and purified from baculovirus / insect cell lines. The top figure, representing His-d24AIBP, corresponds to the amino acid sequence shown in Figure 12. The amino acid sequence below the top figure shows the sequence of the orange-bordered "Cleavable His tag". All other figures show various modifications of the amino acid sequence introduced into the AIBP molecule and the corresponding changes. The green "N-terminal domain" box represents amino acid sequences 25-51 of natural AIBP. The right-hand column shows the results of co-immunoprecipitation experiments of AIBP variants with recombinant TLR4 external domains. Figure 14 demonstrates TLR4 binding of specific modified AIBP sequences from mammalian expression systems.

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

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

[0276] Figure 16 confirms that N-terminal modification of AIBP is required for TLR4 binding in the E. coli expression system: Figure 16. TLR4 binding of various manipulated forms of AIBP, continuation 2. All proteins were expressed and purified from E. coli. The right-hand column shows the results of co-immunoprecipitation experiments of AIBP variants using the recombinant external domain of TLR4. No TLR4 binding was observed with AIBP variants d24AIBP-his or d51AIBP-his.

[0277] Figures 17A–D (or Figure S1 or Supplementary Figure 1) provide validation of the specificity of the TLR4 antibody used for flow cytometry and microscopy, and also show TLR4 dimerization and lipid rafts measured in dorsal root ganglion macrophages:

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

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

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

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

[0282] Figure 18A shows the sorting strategies 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, as well as CD11b and TMEM119;

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

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

[0285] Figures 18D-E show heatmaps of CIPN repressor genes upregulated by AIBP (group 4) (Figure 18D) and CIPN-inducing genes downregulated by AIBP (group 3) (Figure 18E) in wild-type mice; the Log2 normalized gene counts scaled by rows and columns represent all technical replicas from the three biological samples.

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

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

[0288] Figures 20A–E (or Figure S4 or Supplementary Figure 4) show tactile allodynia data in tamoxifen-treated WT mice in itLPS and CIPN experiments. Additional RNA-seq data for ABC-imKO-dependent genes and the cisplatin effect of ABC-imKO on WT mice are also provided. As controls for ABC-imKO mice, wild-caught littermates bred in-house were administered 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), and 2 hours later, itLPS (0.1 μg / 5 μL) (n=4 for itSaline, n=5 for itAIBP); (Figure 20B) cisplatin (2.3 mg / Kg) was administered via ip injection 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-series analysis. C, ABC-imKO mice were injected with TAM, then cisplatin, as described above, followed by injection of it saline (5 μL), AIBP (0.5 μg / 5 μL), or hp-β-CD (0.25 mg / 5 μL) on day 7. Tactile threshold 24 hours after it injection is shown. Mean ± SEM (n=3~4 / group). **P<0.01. One-way ANOVA using Dunnett's multiple comparison test. Figure 20D, Heatmap of differentially regulated genes across all conditions (naive, cisplatin / saline or cisplatin / AIBP-induced) regulated in the ABC-imKO method. All significant genes (condition: genotype) obtained from likelihood ratio tests 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 cutoff P<0.05, enrichment >1.5 and minimum overlap of 3 genes in the pathway. Heatmaps represent 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 cord microglia of tamoxifen-induced AIBP-imKO mice. This also demonstrates that the BE-1 monoclonal antibody has similar affinity to wtAIBP and mutAIBP. Figure 21A: IHC of frozen spinal cord sections from vehicle and tamoxifen-induced AIBP-imKO mice showing AIBP staining and colocalization with IBA1 (microglia), NeuN (neurons), and GFAP (astrocytes). PrologGold containing DAPI was mounted on the slide. Confocal images were acquired with a 63x objective lens and analyzed for colocalization in ImageJ software. Colocalization masks and Pearson's R-values, Munder's colocalization coefficient, and randomized Costes' P-values ​​were calculated for each animal in the experiment as described in at least one slide of the method. Representative images and values ​​shown correspond to one animal per condition. Scale bar, 50 μm. Figure 21B: Sandwich ELISA using BE-1 as the capture antibody in a microtiter plate. Dose-response curves for wtAIBP and mutAIBP were detected using rabbit polyclonal anti-AIBP antibody. Two-way ANOVA with Bonferroni post-hoc test for multiple comparisons was used, and no statistically significant differences were found in BE-1 affinity for wtAIBP and mutAIBP. 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 external domain of TLR4. The results of this example are unexpected in that the activity descriptions in Figures 13 and 14 demonstrate that not all N-terminal modifications expose the TLR4-binding domain. For example, putative cleavage products of N-terminal His tags containing cleavage sites do not demonstrate TLR4 binding. These unexpected data suggest that N-terminal modifications to AIBP polypeptides have specific amino acid composition requirements.

[0291] A pull-down assay was performed using the compounds provided herein. Compounds 3, 7, 8, or 9, as well as other constructs shown in Figures 13 and 14, were subjected to baculovirus (BD). TLR4 was purified from either a Bioscience or CHO (ExpiCHO, expression system, ThermoFisher) cell expression system and incubated with TLR4 protein (Sino Biological). Pull-down was performed using the described anti-AIBP antibody. AIBP-bound TLR4 was detected by Western blotting using an anti-his antibody (both modified AIBP and TLR4 have the his tag). Detailed experimental information regarding the pull-down method is provided in Example 1.

[0292] Alternative assays involved transfection of HEK293 cells with modified AIBP and TLR4. For this study, transfected Flag-AIBP (exemplified for compounds 5 and 6) and Flag-TLR4-his constructs were expressed, 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 regarding the pull-down method is provided in Example 1. Example 3: Efficacy demonstrated in an exemplary model: Asthma Decreased AIBP expression in bronchial epithelial cells of asthma 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). The inventors 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 the blood and tissues but also in the lung air spaces. AIBP binds to surfactant protein B and enhances cholesterol efflux from alveolar macrophages to surfactant (4). This normalizes the lipid raft content in the plasma membrane, reduces inflammatory signaling in alveolar macrophages, and decreases the expression of inflammatory cytokines. AIBP is secreted into BALF in response to inhaled LPS lung injury (4). Furthermore, 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 elevated AIBP levels in the lungs may have therapeutic effects.

[0294] Due to the broad anti-inflammatory protection provided by AIBP in the lungs (4) and other tissues (3, 7), this study investigated whether endogenous AIBP pulmonary expression affects asthma patients and whether inhaled AIBP can reduce pneumonia and alleviate airway hyperresponsiveness in a mouse model of asthma.

[0295] Immunohistochemistry of postmortem human lung tissue obtained from non-asthmatic subjects revealed the major AIBP protein expression patterns in bronchial epithelial cells. Interestingly, AIBP expression was significantly reduced in bronchial epithelial cells of postmortem lungs from asthmatic subjects (see Figure 22A). Furthermore, primary bronchial epithelial cells isolated from postmortem lungs of asthmatic subjects had significantly lower APOA1BP mRNA expression compared to non-asthmatic subjects (see Figure 21D). Similar to human asthma, endogenous AIBP expression in bronchial epithelium was significantly reduced in dust mite (HDM)-loaded mice compared to control mice administered intranasal PBS (see Figure 22C). This pattern of reduced AIBP expression in bronchial epithelium after acute HDM loading in mice was not observed in a mouse acute lung injury model (4). Furthermore, the lung cell types expressing the highest levels of AIBP differed between 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 primary recruited inflammatory cells after acute HDM loading (i.e., eosinophils) did not express high levels of AIBP.

[0296] Since endogenous AIBP expression is reduced in asthma (Figure 21), and administration of AIBP as a recombinant protein or via adeno-associated virus delivery has produced anti-inflammatory and protective effects in neuroinflammation and neuropathic pain (7), vasculitis and atherosclerosis (3), and acute lung injury (4), we investigated whether intranasal delivery of recombinant AIBP (compound 7) has a therapeutic effect in a mouse model of asthma.

[0297] Compound 7 was administered 2 hours before the administration of HDM.

[0298] Intranasal administration of HDM four times weekly to female mice induces eosinophilic inflammation of the lung and airway hyperresponsiveness (AHR) to metacholine overload (8). Two doses of compound 7, 2.5 and 25 μg, or a vehicle (PBS) were administered weekly to 8-week-old C57BL / 6J female and male mice by intranasal drop infusion two hours prior to intranasal HDM. Intranasal compound 7 did not produce any apparent adverse effects. As expected, HDMI-loaded female mice pre-treated with PBS developed AHR. In contrast, pre-treatment with compound 7 dose-dependently reduced HDM-induced AHR, resulting in near-complete inhibition of AHR at a dose of 25 μg (Figure 23A), and also resulted in dose-dependent reduction of HDM-induced pulmonary and BAL eosinophilia (Figures 23B-C). In this model, HDM did not result in any significant change in the number of alveolar macrophages or neutrophils (8). Similar results were obtained for compound 7 in male mice (Figure 23D-F).

[0299] In summary, our research demonstrates that AIBP expression in human bronchial epithelial cells of asthma patients is significantly reduced compared to non-asthma patients, and that it is also significantly reduced in bronchial epithelial cells after HDM exposure in a mouse model of asthma. This result is consistent with the finding of decreased ApoA-I levels in BALF in asthma patients compared to non-asthma patients (5). Since airway epithelium and bronchial inflammation are major components of asthma that lead to airway smooth muscle contraction, airway obstruction, and asthma exacerbation (9), restoring the level of AIBP, which has anti-inflammatory properties, may present a novel therapeutic strategy for asthma. Our results regarding 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 basis for treating moderate / severe asthma, further preclinical and subsequent human trials in asthma-positive subjects are needed to determine whether compound 7 has an additive anti-inflammatory effect on asthma control when used in combination with ICS, and / or whether it is an alternative anti-inflammatory agent to ICS in asthma patients who do not respond well to ICS or experience side effects from ICS. Materials and Method of Example 3 Human lung specimens

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

[0301] Primary bronchial epithelial cells were isolated from the bronchi of postmortem lungs. In short, the bronchi were dissected, and the inside of each bronchus was scraped with a Cell Lifter (Corning, Inc.) to obtain bronchial epithelial cells. The bronchial epithelial cells were collected and cultured in CnT-17 medium (Cellntec, Bern, Switzerland). Evaluated by E-cadherin expression using flow cytometry, these primary bronchial epithelial cells were over 95% pure. Immunohistochemistry of human and mouse lungs

[0302] Paraffin-embedded lung sections were stained using a cocktail (6, 7) of mouse anti-human and anti-mouse AIBP monoclonal antibodies A7 and BE-1 developed in the inventors' laboratory, and mixed in a 1:2 ratio. Due to the high homology between mouse and human AIBP, 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 the results were expressed as the AIBP-positive area of ​​bronchial epithelium per 1 μm of bronchial basement membrane length in human specimens. AIBP expression in mouse lungs was measured using the mean gray value tool in Image J (NIH), and the cytosolic values ​​of bronchial epithelium in bronchioles with an inner diameter of 150-200 μm were normalized to the values ​​in adjacent alveoli. The operators were not informed of the nature of the samples. Quantification of APOA1BP mRNA

[0303] To quantify APOA1BP mRNA in human bronchial epithelial cells from asthma and non-asthma 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 using the Oligo-dT and SuperScript II kits (Life Technologies). qPCR was performed using the TaqMan PCR Master Mix and TaqMan primers for human APOA1BP (Hs.PT.58.22278956, Integrated DNA Technologies, Coralville, IA). The relative amounts of APOA1BP mRNA were normalized against the relative amounts of the housekeeping gene hypoxanthine phosphoribosyltransferase-1 (HPRT1). Manufacturing 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 substitution. The product was over 90% pure, with no detectable aggregates (HPLC-SEC), and residual endotoxin was less than 0.2 EU / mg. Storage stability tests of compound 7 at -80°C for up to 6 months or at 4°C for 1 week showed no loss of titer or purity. Acute HDM mouse model of asthma

[0305] All experiments were conducted 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 prior to each HDM administration, 50 μl of PBS control or either 2.5 μg or 25 μg of Compound 7 solution was administered intranasally. The control group received intranasal PBS instead of HDM. On day 24, airway hyperresponsiveness to metacholine was assessed as described (8), and mice were sacrificed and BAL and lungs were collected. BAL was collected by washing with 1 mL of PBS via tracheal catheter, centrifuged, and the pellet was resuspended in 1 mL of PBS. After measuring the total number of cells in the BAL, the number of differentiated cells was quantified using Wright-Giemsa stained slides (8). Lung eosinophil count was quantified in the peribronchial space of lung paraffin-embedded sections stained with anti-mouse major basic protein (MBP) rabbit polyclonal antibody (courtesy of the Mayo Foundation for Medical Education and Research). The results are expressed as the number of positively stained peribronchial cells per bronchiole with an inner diameter of 150–200 μm. At least five bronchioles were counted in each slide. The operators were not informed of the nature of the samples.

[0306] statistics: All results are presented as mean ± SEM. Statistical software package (GraphPad Prism) was used for analysis. The Mann-Whitney test was used for two-group analysis. For comparisons of more than two groups, two-way or one-way ANOVA and post-hoc Tukey multiple comparison tests were used. P-values ​​less than 0.05 were considered statistically significant. Legend for the figure in Example 3

[0307] Figure 22. Decreased AIBP expression in bronchial epithelium. A. Postmortem lung specimens from non-asthmatic and asthmatic individuals, stained with anti-AIBP antibody. Quantification of AIBP-positive bronchial epithelium (n=6). B. APOA1BP mRNA from bronchial epithelial cells isolated from non-asthmatic (n=9) and asthmatic (n=11) individuals, normalized to HPRT1. C. Lungs of mice treated with anti-AIBP antibody-stained vehicle or 100 μg of HDM intranasally four times weekly. 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 pneumonia in acute HDM models of asthma in female and male mice. Female (A-C) and male (D-F) C57BL / 6J mice were given intracavitary infusions of 2.5 μg or 25.0 μg of RFT1081 or PBS four times weekly. Two hours later, the mice were given intranasal infusions of 100 μg of HDM or vehicle. Three days after the last loading, the mice were tested for airway resistance to methacholine (A, D) and collected for analysis as shown in the lungs (B, E) and BAL (C, F). Mean ± SEM; n=8 mice per group. * P<0.05; ** P<0.01; **** P<0.001; *** P<0.0001 Example 4: Efficacy demonstrated by exemplary method: Glaucoma

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

[0310] A mouse model of a glaucoma DBA / 2J (D2) patient. The advantage of using the genetic D2 model with age-matched non-glaucomatous control D2-Gpnmb+ mice is that it can recapitulate the elevation of chronic IOP in human glaucoma, and the retinal pathology develops at about 9-10 months of age (1, 2). The inventors recognize that D2 has limitations, similar to other animal models. This is because in these mice, glaucomatous-like pathology develops secondary to anterior segment abnormalities with adhesions and pigment dispersion (1, 2). In preliminary studies, the inventors observed a significant increase in cholesterol content in the retina of Apoa1bp - / - compared to WT mice (see Figure 24A), suggesting that AIBP deficiency induces excessive cholesterol accumulation in the retina. Notably, in 10-month-old glaucomatous D2 mice, the inventors found that the cholesterol content in the retina was also significantly increased compared to D2-Gpnmb + mice (Figure 24B). Therefore, the inventors tested whether overexpression of AIBP by in vivo delivery of AAV-AIBP could reverse excessive cholesterol accumulation, protect RGCs and their axons, and maintain the central visual pathway in glaucomatous D2 mice. The AAV serotype used by the inventors in the mouse model was AAV-DJ / 8, which expressed mouse AIBP with a fibronectin signal peptide to ensure robust protein secretion. The inventors 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. The inventors evaluated RGC and axon survival by staining for RNA-binding proteins using 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 shows the entire retinal projection via active uptake and transport. 3、4AIBP protein expression was detected in the retina at 10 months of age after AAV-AIBP injection (Figure 24E). AAV-AIBP, rather than AAV-Null, significantly reduced cholesterol content (Figures 24C and 24D) and protected RGCs in the central and peripheral regions of glaucoma D2 retina (Figures 24G and 24H). Furthermore, we observed a significant improvement in CTB transport to SCs (Figures 24J-M), suggesting that AAV-AIBP helped maintain the structural and functional integrity of the optic nerve.

[0311] A microbead-induced intraocular pressure model. Recently, the inventors successfully developed a mouse model of microbead-induced ocular hypertension, which showed significant loss of reductase cells (RGCs) at 6 weeks post-treatment in 4-month-old C57BL / 6J mice (Figure 25). To further investigate the protective effects of AAV-AIBP on RGCs and visual function in vivo, the inventors intravitreal-injected AAV-Null or AAV-AIBP 3 weeks prior to microbead injection. AAV-AIBP significantly reduced RGC death (see Figure 25C) and, importantly, improved visual impairment (see Figure 25D).

[0312] Optic nerve crush (ONC) model. Since ONCs also induce RGC death and degeneration, they serve as a useful model not only for traumatic optic neuropathy but also for glaucoma injury. 5 The inventors intravitrealized injection of AAV-Null or AAV-AIBP three weeks prior to ONC, and then evaluated RGC survival by RBPMS staining one week after ONC. Overexpression of AIBP was found to protect RGCs from ONC damage (Figure 26).

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

[0314] Figure 24. AAV-AIBP reduces retinal neurodegeneration in DBA / 2J (D2) glaucoma mice. A and B, Apoa1bp - / - mice. Filipin staining of cholesterol (A). Quantification of Filipin intensity in the inner retina (B). C–M, Glaucoma D2 mice. Filipin staining of cholesterol (C). Quantification of Filipin intensity in the inner retina (D). Confirmation of AIBP expression in the retina by immunoblotting using anti-His antibody (E). IOP measurement (F). RBPMS (green)-positive RGCs in the peripheral retina (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 bar: 20 μm (A and C) and 50 μm (G and I).

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

[0316] Figure 26. AAV-AIBP mitigates retinal neurodegeneration and mouse optic nerve crush models. A, representative image of RBPMS-positive RGCs in the central retinal region after ONC injury; B, quantitative analysis of RGC survival in the central and peripheral retinal regions. 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 present 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. The present invention provides, for example, the following items: (Item 1) An isolated or recombinant polypeptide, wherein the polypeptide is composed of an ApoA-I binding protein (AIBP) amino acid sequence and an N-terminal amino acid sequence of the AIBP amino acid sequence. The N-terminal amino acid sequence of the AIBP amino acid sequence consists of at least 8 amino acids, or the N-terminal amino acid sequence of the AIBP amino acid sequence has an amino acid length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more. The N-terminal amino acid sequence of the AIBP amino acid sequence can, under relevant physiological conditions, induce unfolding of a potential domain in the AIBP amino acid sequence, expose a potential domain, or otherwise make it accessible in order to bind the polypeptide to TLR4. Where necessary, “relevant physiological conditions” refers to the relevant physiological conditions that the polypeptide compound experiences in vivo when it is administered to a subject requiring the polypeptide compound. An isolated or recombinant polypeptide, provided that the N-terminal amino acid sequence of the AIBP amino acid sequence does not consist of a His tag and a protein cleavage site that, when acted upon under the conditions described above, causes the His tag to disappear. (Item 2) The isolated or recombinant polypeptide described in item 1, wherein the amino acid sequence at the N-terminus of the AIBP amino acid sequence consists of 8 to 40 consecutive amino acid residues, and 3 to 12 of these amino acid residues are independently selected from the group consisting of arginine (R), histidine (H), and lysine (K). (Item 3) The isolated or recombinant polypeptide compound according to item 1, wherein the N-terminus of the amino acid sequence on the N-terminal side of the AIBP amino acid sequence is a secretion signal amino acid sequence. (Item 4) The isolated or recombinant polypeptide compound according to item 3, wherein the secretory signal amino acid sequence is a fibronectin secretory signaling domain, an immunoglobulin heavy chain secretory signaling domain, an immunoglobulin κ light chain secretory signaling domain, or an interleukin-2 signal peptide secretory signaling domain. (Item 5) The isolated or recombinant polypeptide described in item 4, wherein the fibronectin secretion signaling domain is MLRGPGPGRLLLLAVLCLGTSVRCTETGKSKR (SEQ ID NO: 24). (Item 6) The isolated or recombinant polypeptide according to item 1, wherein the AIBP sequence is hAIBP (sequence number 6) or d24hAIBP (sequence number 8). (Item 7) The isolated or recombinant polypeptide described in item 1, wherein the amino acid sequence at the N-terminus of the AIBP amino acid sequence consists of six consecutive histidine amino acid residues (HHHHHH; SEQ ID NO: 1) at the N-terminus of the TLR4 binding domain of the AIBP amino acid sequence. (Item 8) The isolated or recombinant polypeptide according to item 7, wherein the polypeptide has a thrombin cleavage domain interposed between the N-terminuses of the TLR4 binding domain of the ApoA-I binding protein sequence, and the thrombin cleavage domain has one or more amino acid deletions and / or mutations within this domain, thereby rendering it functionally inoperable. (Item 9) The amino acid sequence at the N-terminal end of the aforementioned AIBP amino acid sequence is The isolated or recombinant polypeptide described in item 1, which is MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2) or MSPIDPMGHHHHHHGRRRASVAAGILVPRGSDGDDGDDDR (SEQ ID NO: 19), each having an amino acid mutation in its thrombin cleavage domain, thereby rendering it functionally inoperable. (Item 10) The amino acid sequence at the N-terminal end of the aforementioned AIBP amino acid sequence is TETGKSKR (Sequence ID 26), MDYKDHDGDYKDHDIDYKDDDDKLAAANS (Sequence ID 33), and MSPIDPMGHHHHHHGRRRASVAAGILVPAASPGLDGICSR(Sequence ID 7) An isolated or recombinant polypeptide as described in item 1, selected from the group consisting of the following: (Item 11) The isolated or recombinant polypeptide compound described in item 10, wherein the AIBP amino acid sequence is the AIBP amino acid sequence of a mammalian AIBP amino acid sequence. (Item 12) The isolated or recombinant polypeptide compound described in item 11, wherein the mammalian AIBP amino acid sequence is the AIBP amino acid sequence of the human AIBP amino acid sequence. (Item 13) The isolated or recombinant polypeptide compound described in item 12, wherein the human AIBP amino acid sequence is a full-length amino acid sequence of 288 amino acid residues including the NCBI reference sequence: NP_658985.2. (Item 14) The isolated or recombinant polypeptide compound described in item 12, wherein the human AIBP amino acid sequence is the human AIBP amino acid sequence that includes the NCBI reference sequence: NP_658985.2 and has amino acids 1 to 24 deleted from the AIBP amino acid sequence. (Item 15) A pharmaceutical composition comprising a polypeptide compound described in any one of items 1 to 15 and at least one excipient suitable for parenteral administration. (Item 16) A pharmaceutical composition as described in item 16, wherein parenteral administration is by intrathecal injection or intrathecal implant. (Item 17) A nucleic acid compound comprising a nucleic acid sequence encoding a polypeptide compound as described in any one of items 1 to 15. (Item 18) An expression vector comprising a nucleic acid sequence encoding a polypeptide compound as described in any one of items 1 through 15. (Item 19) The expression vector described in item 19, wherein the expression vector is a recombinant adenovirus. (Item 20) The following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, the pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, the inflammation of the nerve or CNS includes TLR4-mediated inflammation of the nerve or CNS. - Allodynia, If necessary, the allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary, the virus being influenza or coronavirus (where necessary, the coronavirus being COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary, influenza A, B, or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus 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, (By adding ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) to the subjects, or by increasing the level of ApoA-I binding protein), The method described above is (a) To provide a pharmaceutical preparation or composition comprising: (i) 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 AIBP amino terminus, which are amino acid residues or peptides (also referred to as AIBP variants as provided herein) that are not present in wt AIBP or are non-native (to AIBP), If necessary, the heterologous amino-terminal amino acid sequence includes a peptide tag, if necessary, the peptide tag includes a multi-histidine (multi-his) tag, if necessary, the multi-his tag includes six histidine (HHHHHH (SEQ ID NO: 1)), or three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, four, fifteen, six, seven, eight, nine, fifteen, six, seven, eight, nine, fifteen, fifteen, six, seven, eight, nineteen, twenty or more histidine residues. If necessary, the heterologous amino-terminal amino acid sequence includes an enzyme cleavage site, and if necessary, the enzyme cleavage site includes a thrombin cleavage site. If necessary, the heterologous amino-terminal amino acid sequence includes a secretory signal, and if necessary, the secretory signal includes a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin κ light chain secretory peptide, or an interleukin-2 signal peptide. If necessary, the heterologous amino-terminal amino acid sequence includes the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR (SEQ ID NO: 2), The variant comprises a recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition that can fold, expose, or make accessible a potential domain in the AIBP molecule containing amino acids 25-51 that mediate the binding of AIBP to TLR4; (ii)(i) A recombinant nucleic acid encoding the APOA1BP polypeptide, If necessary, nucleic acids expressing or encoding APOA1BP polypeptide or polypeptides having APOA1BP polypeptide activity may be included in the expression vehicle, vector, recombinant virus, or equivalent. If necessary, the vector or virus may be an adenovirus vector or adeno-associated virus (AAV) vector, retrovirus, lentiviral vector, herpes simplex virus, human immunodeficiency virus (HIV), or synthetic vector, or may include these. If necessary, the AAV vector may include or be: Adeno-associated virus (AAV), or adenovirus vector, AAV serotype or variant AAV5, AAV6, AAV8 or AAV9, AAV-DJ or AAV-DJ / 8 (trademark) (Cell Biolabs, Inc., San Diego, CA) AAV derived from rhesus macaques, or the aforementioned AAV derived from rhesus macaques, AAVrh.10hCLN2. AAV capsid variant or AAV hybrid serotype, Organ-directed AAV, or cardiac-directed AAV, or cardiac-directed AAVM41 variant, Here, if necessary, the AAV is engineered to increase its efficiency in targeting specific cell types that are intolerant to wild-type (wt) AAV and / or to improve its efficiency in infecting only the cell types of interest. If necessary, the hybrid AAV is retargeted or manipulated as a hybrid serotype by one or more modifications including: 1) transcapsidation, 2) adsorption of bispecific antibodies to the capsid surface, 3) manipulation of mosaic capsids, and / or 4) manipulation of chimeric capsids, with recombinant nucleic acids; (iii) The recombinant or synthetic ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein is all or part of a human or mammalian APOA1BP, AIBP1, or AIBP2 sequence, or contains thereof, in any of the formulations or pharmaceutical compositions of (i) to (ii); (iv) A formulation or pharmaceutical composition of any of (i) to (iii) formulated for in vivo administration; or for enteral or parenteral administration, or for oral, intravenous (IV) or intrathecal (IT) administration, If necessary, the formulation or pharmaceutical composition, or the recombinant, peptide mimetic or synthetic APOA1BP, or a bioequivalent of APOA1BP, or the nucleic acid encoding APOA1BP, or a vector containing the nucleic acid encoding APOA1BP, may be supported in nanoparticles, particles, micelles, or liposomes or lipoplexes, polymerosomes, polyplexes, or dendrimers, which may further include or express any of the formulations or pharmaceutical compositions of (i) to (iii), if necessary, a cell or CNS permeable portion or peptide, or a CNS targeting portion or peptide; or (v) To provide a formulation or pharmaceutical composition comprising any formulation or pharmaceutical composition of (i) to (iv) formulated as nanoparticles, liposomes, tablets, pills, capsules, gels, gel tablets, liquids, powders, emulsions, lotions, aerosols, sprays, lozenges, aqueous, sterile, or injectable solutions, or implants (e.g., intrathecal implants); (b) Administering the preparation or pharmaceutical composition of (a) to a subject that requires administration, wherein the subject may be a human or an animal, if necessary. As a result, the following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, the pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, the inflammation of the nerve or CNS includes TLR4-mediated inflammation of the nerve or CNS. - Allodynia, If necessary, the allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary, the virus is influenza or coronavirus (where necessary, coronavirus is COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary, influenza A, B or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus or smallpox virus, Epstein-Barr virus (EBV), adenovirus, hantavirus, Flaviviridae or dengue virus, Zika virus, or chikungunya virus infection, or co-occurring conditions thereof, and / or - Vascular inflammation, atherosclerosis, and cardiovascular diseases A method for treating, improving, preventing, reversing, or reducing the severity or duration of the symptoms, or for reducing the severity of the symptoms. (Item 21) A kit comprising a recombinant or isolated polypeptide as described in any one of items 1 through 14, a formulation or pharmaceutical composition as described in item 15 or 16, or one used in item 20, and optionally including instructions for carrying out the method described in item 20. (Item 22) Use in the manufacture of a pharmaceutical product of a recombinant or isolated polypeptide as described in any one of items 1 to 14, a formulation or pharmaceutical composition as described in item 15 or 16, or a pharmaceutical composition used in item 20. (Item 23) The following symptoms of a recombinant or isolated polypeptide as described in any one of items 1 to 14, a formulation or pharmaceutical composition as described in item 15 or 16, or a formulation or pharmaceutical composition used in item 1: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, the pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, the inflammation of the nerve or CNS includes TLR4-mediated inflammation of the nerve or CNS. - Allodynia, If necessary, the allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary, the virus being influenza or coronavirus (where necessary, the coronavirus being COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary, influenza A, B, or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus 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 diseases Use in the manufacture of a pharmaceutical for treating, improving, preventing, reversing, or reducing the severity or duration of the symptoms, or for reducing the severity of the symptoms. (Item 24) The following symptoms: - Neuropathic pain, - Inflammatory neuropathic pain, If necessary, the pro-inflammatory neuropathic pain includes Toll-like receptor 4 (TLR4) mediated pro-inflammatory neuropathic pain. - Inflammation of nerves or CNS, If necessary, the inflammation of the nerve or CNS includes TLR4-mediated inflammation of the nerve or CNS. - Allodynia, If necessary, the allodynia includes TLR4-mediated allodynia. - Pain following nerve or tissue injury, or neuropathic pain, If necessary, pain following nerve or tissue injury or neuropathic pain may be caused by, or resulting from, trauma, chemotherapy, arthritis, diabetes, or viral infection, or may be a sequela thereof. - Postoperative pain or neuropathic pain, - Chemotherapy-induced peripheral neuropathy (CIPN) (e.g., cisplatin-induced CIPN or allodynia), - Neurodegenerative diseases or conditions, chronic or progressive neurodegenerative diseases or conditions as needed, Alzheimer's disease or chronic traumatic encephalopathy (CTE) or related tauopathy as needed, traumatic brain injury (TBI), post-traumatic stress disorder, traumatic war neurosis, or post-traumatic stress syndrome (PTSS), - Primary headache, migraine or cluster headache as needed. - Hyperalgesia, - Glaucoma or other inflammatory eye disease, - Pneumonia and asthma, -Acute respiratory distress syndrome (ARDS), - sepsis, - Viral infections, where necessary, the virus being influenza or coronavirus (where necessary, the coronavirus being COVID-19) or human immunodeficiency virus (HIV) or viruses that cause HIV infection (where necessary, influenza A, B, or C), or hepatitis viruses, 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 varicella virus, orthopoxvirus 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, A formulation, pharmaceutical composition, or therapeutic combination for use in a method for treating, improving, preventing, reversing, or reducing the severity or duration of the symptoms, The formulation or therapeutic combination comprises a recombinant or isolated polypeptide as described in any of items 1 to 14, a formulation or pharmaceutical composition as described in item 15 or 16, or a formulation or therapeutic combination as described in item 20. The aforementioned formulation or therapeutic combination is a formulation, pharmaceutical composition, or therapeutic combination administered to an individual or patient who requires such administration. (Item 25) A method for exposing the potential (or hidden, unexposed, inaccessible) N-terminal TLR4 binding domain of an ApoA-I binding protein (APOA1BP, AIBP, or AI-BP) polypeptide, comprising adding a heterologous amino-terminal amino acid sequence of at least about 10 amino acids, or between about 5 and 50 amino acids, or between about 10 and 100 amino acids, or adding about 20 and 80 amino acids, or between about 30 and 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), to the amino terminus. If necessary, the heterologous amino-terminal amino acid sequence includes a peptide tag, if necessary, the peptide tag includes a multi-histidine (multi-his) tag, if necessary, the multi-his tag includes at least six histidine (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. If necessary, the heterologous amino-terminal amino acid sequence includes an enzyme cleavage site, and if necessary, the enzyme cleavage site includes a thrombin cleavage site. If necessary, the heterologous amino-terminal amino acid sequence includes a secretory signal, and if necessary, the secretory signal includes a fibronectin secretory signal, an immunoglobulin heavy chain secretory signal, or an immunoglobulin κ light chain secretory peptide, or an interleukin-2 signal peptide. If necessary, the heterologous amino-terminal amino acid sequence is the amino acid sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGICSR(Sequence ID 2) A method that includes this.

Claims

[Claim 1] The invention described herein.

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