Analgesic compositions and methods thereof

Abstract

Certain embodiments of the invention provide a composition (e.g., a pharmaceutical composition for analgesia) described herein. Certain embodiments of the invention provide a method of activating a mu opioid receptor(s), comprising administering to a subject in need of, a mu opioid receptor (MOR) agonist, and an inhibitor of exon 7-associated mu opioid receptor (MOR) variant(s) (e.g., antibody or fragment, ASO, siRNA, or shRNA). Certain embodiments provide an isolated exon 7 or homolog specific antibody or fragment thereof, or ASO, siRNA, or shRNA, and the use thereof.

Description

[0001] ANALGESIC COMPOSITIONS AND METHODS THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number

[0004] 63 / 522,965 that was filed on June 23, 2023. The entire content of the application referenced above is hereby incorporated by reference herein.

[0005] GOVERNMENT FUNDING

[0006] This invention was made with government support under DA029244, DA040888, DA006421, and DA007242 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Mu opioids, including those derived from opium, such as morphine, and synthetic ones such as fentanyl and methadone, remain in the mainstream for severe pain management in the clinic despite their side-effects such as tolerance, dependence, addiction, and respiratory depression. However, misusing these mu opioids can magnify their side-effects and promote the development of opioid use disorder (OUD), a main cause of the opioid epidemic and opioid overdose deaths. New methods and compositions for effective and safer pain management are needed.

[0009] SUMMARY OF THE INVENTION

[0010] Certain embodiments of the invention provide a composition described herein. In certain embodiments, the composition comprises a mu opioid receptor (MOR) agonist, and an inhibitor of an exon 7-associated mu opioid receptor (MOR) variant(s).

[0011] Certain embodiments of the invention provide a method of activating a mu opioid receptor(s), comprising administering to a subject in need of, a mu opioid receptor (MOR) agonist, and an inhibitor of exon 7-associated mu opioid receptor (MOR) variant(s).

[0012] Certain embodiments of the invention provide an inhibitor of exon 7-associated mu opioid receptor (MOR) variant(s) as described herein (e.g., antibody or fragment thereof, or ASO, siRNA or shRNA).

[0013] Certain embodiments of the invention provide an isolated exon 7-specific antibody or fragment thereof, or ASO, siRNA or shRNA, and the use thereof.

[0014] Certain embodiments of the invention provide a composition comprising an isolated exon 7-specific antibody or fragment thereof.

[0015] Certain embodiments of the invention provide a vector (e.g., Adeno-associated viruses (AAV)) comprising a nucleic acid or an expression cassette as described herein.

[0016] Certain embodiments of the invention provide a cell comprising a nucleic acid, expression cassette or vector as described herein.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] Figures 1A-1D. Characterization of RabmAb (clone 3H4-8) targeting mouse E7 variants. (Figs.lA, IB & 1C) Immunohistachemical (IHC) study with 3H4-8 (1 :200, 6.25pg / ml) using sagittal brain section of WT male B6 mice (Fig.lA) and male E1ZE11-KO mice in which all Oprml variants are disrupted (Fig.lB). The PAG from A & B. PAG, periaqueductal gray; VTA, ventral tegmental area; HYP, Hypothalamus. The stained sections were co-stained with DAPI (Fig.lC), imaged and scanned with a fluorescence microscope. (Fig.lD) Western blot. E7 variants of mouse (mMOR-lC), rat (rMOR-lCl) and human (hM0R-10) or a mouse E4 variant (mMOR- 1) constructs were transiently transfected into HEK293 cells. Western blot was performed using whole cell lysate 36 hrs after transfection and antibody 3H4-8 (1 :2000, 1.25pg / ml).

[0019] Figure 2. Efficient penetration of Qdot605-labeled 3H4-8 into Neuro-2a cells. 3H4-8 was labeled at the Fc region of heavy chains with Qdot605 by SiteClick antibody labeling kit (Molecular Probe). Neuro-2a cells, a mouse neuroblastoma cell line, were cultured in 8-well slide chamber. The labeled 3H4-8 (100 ug / ml) was added into culture medium for 16 hrs. Neuro-2a cells were then washed, fixed, co-stained with DAPI and imaged using a confocal microscope.

[0020] Figures 3A-3B. Monoclonal antibodies attenuated morphine tolerance in CD-I mice (Radiant-Heat tail-flick assay). 12.5 ug in 5 ul of 3H4-8 or 3G12-12 was i.c.v. administered 2 hrs before the first morphine dosing every day (lOmg / kg, s.c. twice / day) for 5 days. Analgesia was measured by radiant heat tail-flick assay. Fig.3 A: time course. Fig.3B: Dose-response. ED50 of mice without i.c.v. injection on Day 0 is 2.9 mg / kg; ED50 of Control (Ctr) IgG on Day 5 is 11.5 mg / kg; ED50 of 3H4-8 on Day 5 is 3.9 mg / kg; ED50 of 3G12-12 on Day 5 is 3.5 mg / kg. Statistical analysis: 2-way ANOVA with Bonferroni’s post hoc test. ****■. p < 0.0001.

[0021] Figures 4A-4B. Monoclonal antibody attenuated morphine tolerance in CD-I mice (Hot-plate assay). 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 2 hrs before the first morphine dosing every day (lOmgk / kg, s.c. twice / day) for 5 days. Analgesia was measured by Hot-plate assay. Fig.4A: time course; Fig.4B: Dose-response. ED50 of mice without i.c.v. injection on Day 0 is 2.3 mg / kg; ED50 of Control (Ctr) IgG on Day 5 is 10.7 mg / kg; ED50 of 3H4-8 on Day 5 is 5.9 mg / kg. Statistical analysis: 2-way ANOVA with Bonferroni’s post hoc test. **: p < 0.01; ***: p < 0.001. Figure 5. 3H4-8 reduced morphine conditioned place preference (CPP) in B6 mice. 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 2 hrs before every morphine conditioning. Morphine CPP was conducted in a 6-day protocol (lOmg / kg). *: <0.05, two-tailed Z-test.

[0022] Figure 6. 3H4-8 reduced fentanyl analgesia in mMOR-lO-KI (mM!OAct / Act) mice. 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 3 hrs before fentanyl (0.08mg / kg, s.c.). Analgesia was measured by radiant heat tail-flick assay 15 min after fentanyl. **: p < 0.01, two-tailed Z-test.

[0023] Figure 7. Mu opioid dose-response study before and after chronic opioids. ED50 values with 95% Confidence Intervals (Cis) (parentheses) were determined by nonlinear regression analysis (Prism). ED50 values with nonoverlapping 95% Cis were considered significantly different (1). * : p < 0.05, compared with WT day 5.

[0024] Figures 8A-8B. Exon 7-associated variants facilitate fentanyl and methadone tolerance. For fentanyl tolerance (Fig.8A), mice were subcutaneously (s.c.) administered with fentanyl via a subcutaneously implanted osmotic pump (1 mg / kg / day for 7 days). For methadone tolerance (Fig.8B), mice were s.c. administered with methadone (10 mg / kg, twice a day for 5 days. Analgesia was measured by using radiant heat tail-flick assay on indicated days using a UGO Basile radiant heat tail-flick machine. The results were analyzed as percent maximal possible effect (% MPE). Statistical analysis: 2-way ANOVA with Bonferroni’s post hoc test. . ***: p < 0.001; ****: / ? < 0.0001.

[0025] Figure 9. Exon 7-associated variants facilitate fentanyl and methadone reward.

[0026] Fentanyl and methadone CPP were performed in a three-chamber apparatus (Med Associates) with a 6-day paradigm (Day 1, pre-test; Days 2-5, conditioning with i.p. opioid in morning and saline in afternoon; Day 6, post-test). Fentanyl (0.2 mg / kg, s.c.) and methadone (10 mg / kg, s.c.) were used during conditioning sessions. Results were calculated by different scores (seconds) in drug-paired chamber between the test day and preconditioning day. Statistical analysis: two-tailed t-test; *: p < 0.05.

[0027] Figures 10A-10D. Fentanyl-induced respiratory depression in mE7M-B6 Mut mice.

[0028] Fig.10A & Fig.10B). Fentanyl-induced changes in Breath frequency (f) measured by whole body plethysmography (WBP, DSI) in male (Fig. lOA) and female (Fig. lOB) B6 or mE7M-B6 mice following the Hypercapnia WBP protocol in which mice were monitored for respiratory functions by WBP under 5% CO2, 21% O2 and 74% N2 condition. The respiratory parameters, including breath frequency (f), tidal volume (TVb), minute volume (MVb), inspiratory time (Ti), peak inspiratory flow (PIFb) and apnea, were automatically recorded by FinePointe software. Advantages of WBP are: 1) animals are under awake, unanesthetized and unrestricted conditions during the measurement. 2) multiple respiratory functions or parameters can be collected and analyzed. 3) various gas challenges can be applied such as hypercapnia conditions (5% CO2), which can increase sensitivity of the measurement and is particularly useful in opioid-induced respiratory depression studies. (Fig. IOC) Summary of partial WBP data of the 4thdose (0.24 mg / kg) in WT B6 and mE7M-B6 Mut mice. J, & $: significantly reduced or increased; <- no significant change. (Fig.lOD) Area under the curve (AUC) of Fig. lOA & Fig.lOB. 2-Way ANOVA. *. p < 0.05; **. p < 0.01.

[0029] Figures 11A-11E. Morphine analgesia, reward, and tolerance in mMOR-lOAct / Actmice. (Fig.11 A) Gene targeting strategy to generate mM0R-10Act / Actmice. A mouse model (mMlO-KI, mM10Act / Act) was generated in which the coding exon 1 (El) was replaced by mM0R-10. Knocking out El eliminates all 7TM variants, while undisrupted El promoter can drive mM0R-10 expression. Thus, only a single E7 variant, mM0R-10, no other 7TM variants, is expressed in mM10Act / Actmice. (Fig.1 IB) Opioid receptor binding studies with [3H]DAMGO using brain of mM0R-10Act / Actmice. [3H]DAMG0 is a specific mu opioid receptor agonist. Saturation study determined the KD value (receptor binding affinity) and Bmaxvalue (receptor number). Compared to WT B6 mice, the KD value in mM0R-10Act / Actmice was not changed, but with slight lower level of receptor numbers. (Fig.11 C) Morphine analgesia in mM0R-10Act / Actmice. Indicated mice were s.c. administered with morphine (10 mg / kg). Analgesia was measured by using radiant heat tail-flick assay. Morphine analgesia was lost in mM0R-10Ina / Inamice in which mM0R-10 cassette is in reverse orientation and cannot be expressed. Morphine analgesia was normal in mM0R-10Act / Actmice. (Fig.1 ID) Morphine CPP mM0R-10Act / Actmice. CPP was performed in a three-chamber apparatus (Med Associates) with a 6-day paradigm (Day 1, pre-test; Days 2-5, conditioning with i.p. opioid in morning and saline in afternoon; Day 6, post-test), indicated mice were s.c. administered with morphine (3 mg / kg) during conditioning sessions. Results were calculated by different scores (seconds) in drug-paired chamber between the test day and preconditioning day. Statistical analysis: two-tailed t-test; **: p < 0.01. Compared to WT B6 mice, morphine CPP was enhanced in mM0R-10Act / Actmice. (Fig. HE) Morphine tolerance in mM0R-10Act / Actmice. Indicated mice were s.c. administered with morphine (10 mg / kg) for 3 days. Analgesia was measured by using radiant heat tail-flick assay. Left: Time course; Right: Dose-response curves before (Day 1) and after (Day 3) chronic morphine. Statistical analysis: 2- way ANOVA with Bonferroni’s post hoc test. ***: p<0.001; ****: p<0.0001. ED50 values with 95% Confidence Intervals (Cis) (parentheses) were determined by nonlinear regression analysis (Prism). ED50 values with nonoverlapping 95% Cis were considered significantly different. *: p < 0.05, compared with day 1. Morphine analgesia was not changed in mM0R-10Act / Actmice, compared to WT mice, as indicated comparable ED50 values on Day 1. However, morphine tolerance was developed faster and enhanced in mM0R-10Act / Actmice.

[0030] Figure 12. Alignment of C-terminal sequences from E4- and E7-associated variants among mouse, rat, and human.

[0031] DETAILED DESCRIPTION

[0032] Most opioid analgesics used clinically, including morphine and fentanyl, act primarily through the mu opioid receptor (MOR), a class A Rhodopsin-like G protein-coupled receptor (GPCR). All mu opioid receptors (MORs) are encoded by the mu opioid receptor gene, OPRM1, which undergoes extensive alternative splicing, creating multiple splice variants via a variety of alternative splicing events. These 0PRM1 splice variants are conserved from rodent to primate such as human and can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (7TM) Carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. 0PPM1 splice variants are known in the arts and described herein, for example, in Pasternak, et al., Pharmacological Reviews October 2013, 65 (4) 1257-1317; Kang et al., Int J Mol Sci. 2022 Mar 10;23(6):3010; Liu et al., Biomolecules. 2021 Oct 15; 11(10): 1525; and Xu et al., J Clin Invest. 2017 Apr 3; 127(4): 1561 -1573, which are incorporated by reference herein.

[0033] For mu opioid receptor (MOR) variants that comprise exon 7, exon 7 (E7) is at or near the MOR C-terminus and is located intracellularly. It was discovered herein that exon 7- associated variants of the mu opioid receptor gene, OPRM1, are responsible for several sideeffects associated with traditional mu opioids and can be targeted or inhibited to reduce the sideeffects without altering the analgesia in pain treatment. For example, it was unexpectedly found herein that although exon 7 is not an accessible, extracellular domain target of a membrane associated receptor, administration of exon 7 specific antibodies to a subject can surprisingly reduce the side-effects (e.g., tolerance, reward) of MOR agonistic analgesic drugs without altering the analgesia efficacy in pain treatment.

[0034] Accordingly, certain embodiments of the invention provide a method of activating a mu opioid receptor(s), comprising administering to a subject in need of, a mu opioid receptor (MOR) agonist, and an inhibitor of exon 7-associated mu opioid receptor (MOR) variant(s). In certain embodiments, the MOR agonist activates non E7-associated MOR variant(s), for example, to induce analgesia, while the inhibitor inhibits E7-associated MOR variant(s) related activities.

[0035] In certain embodiments, the MOR agonist is a morphine or analog thereof. In certain embodiments, the morphine or analog is selected from the group consisting of morphine, codeine, oxycodone, Morphine-6P-glucuronide (M6G), buprenorphine, hydrocodone, hydromorphone, oxymorphone, levorphanol, and butorphanol, as well as drug of abuse heroin. In certain embodiments, the morphine or analog is selected from the group consisting of morphine, codeine, oxycodone, hydrocodone, hydromorphone, oxymorphone, levorphanol, and butorphanol. In certain embodiments, the morphine or analog is selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, and oxymorphone. In certain embodiments, the morphine or analog is morphine, or oxycodone. In certain embodiments, the morphine or analog is Morphine-6P-glucuronide (M6G). In certain embodiments, the morphine or analog is buprenorphine. In certain embodiments, the morphine or analog is heroin.

[0036] In certain embodiments, the MOR agonist is a synthetic opioid compound. In certain embodiments, the synthetic opioid compound is a 4-anilidopiperidine compound, such as fentanyl or analog thereof, including but not limited to sufentanil, remifentanil, alfentanil, carfentanil, lofentanil, and ohmefentanil. In certain embodiments, the MOR agonist is methadone.

[0037] In certain embodiments, the MOR agonist has a molecular weight that is no greater than lOOOg / mol. In certain embodiments, the MOR agonist is administered orally, intravenously, transdermally, intradermally, subcutaneously, intramuscularly, or intrathecally.

[0038] In certain embodiments, the method provides analgesia for a subject. For example, the subject is in need of treatment of pain, or pain management. In certain embodiments, the subject has a disease induced pain. In certain embodiments, the subject has a surgery related pain. In certain embodiments, the subject has a trauma induced pain. In certain embodiments, the subject has chronic pain. In certain embodiments, the subject has acute pain.

[0039] In certain embodiments, the subject has opioid use disorder (OUD). In certain embodiments, the subject is in need of treatment for OUD. In certain embodiments, the subject is in need of substance dependence treatment, such as heroin dependence treatment.

[0040] In certain embodiments, the method is for treatment of pain.

[0041] In certain embodiments, the method is for treatment of OUD.

[0042] In certain embodiments, the method is for treatment of opioid dependence and addiction.

[0043] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is mouse, rat, dog, cat, horse, cow, sheep, goat, pig, or rabbit. In certain embodiments, the subject is a primate (e.g., human, or non -human primate such as rhesus or cynomolgus macaques). In certain embodiments, the subject is human. In certain embodiments, the subject is a male. In certain embodiments, the subject is a female.

[0044] The term "exon-7 associated mu opioid receptor (MOR) variant" as used herein refers to MOR variant that comprises exon 7, or homolog thereof (e.g., exon O). For example, in human, the exon 7-associated MOR variant is hM0R-10 (NCBI accession number NP OO 1008503.2, NM_00 1008503.3), which comprises a 30 amino acid sequence (SEQ ID NO:2) encoded by exon O of human 0PRM1 gene. It is to be understood that among the population of a species (e.g., human), there might be single nucleotide polymorphism (SNP) (e.g., rs75433176) that may lead to, e.g., an amino acid substitution within the 30 amino acids sequence of exon 7 or homolog thereof (e.g., P / S substitution, see SEQ ID NO: 1). In certain embodiments, the sequence encoded by human 0PRM1 exon O is PPLAVSMAQIFTRYSPPTHREKTCNDYMKR (SEQ ID NO:1). In certain embodiments, the sequence encoded by human 0PRM1 exon O is PPLAVSMAQIFTRYPPPTHREKTCNDYMKR (SEQ ID NO:2). In mice, the exon 7- associated MOR variants are mMOR-lC, mM0R-10, mMOR-lU, and mMOR-lM, each of which comprises a 30 aa sequence PTLAVSVAQIFTGYPSPTHVEKPCKSCMDR (SEQ ID NO:3) encoded by exon 7 of mouse 0PPM1 gene. In rat, the MOR variants are rMOR-lCl and rMOR-lC2, each of which comprises a 30 aa sequence PALAVSVAQIFTGYPSPTHGEKPCKSYRDR (SEQ ID NO:4) encoded by exon 7 of rat 0PRM1 gene.

[0045] In certain embodiments, the mouse exon 7-associated MOR variants are mMOR-lC, mM0R-10, and mMOR-lU. In certain embodiments, the mouse exon 7-associated MOR variant is mMOR-lC (NCBI accession number NP_001034741.1, NM_001039652.2). In certain embodiments, the mouse exon 7-associated MOR variant is mM0R-10 (NCBI accession number AAO18365.1, AY160190.1) In certain embodiments, the mouse exon 7-associated MOR variant is mMOR-lU (NCBI accession number NP_001291884, NM_001304955.1). In certain embodiments, the mouse exon 7-associated MOR variant is mMOR-lM (NCBI accession number NP_001289724, NM_001302795.1).

[0046] In certain embodiments, the rat exon 7-associated MOR variant is rMOR-lCl (NCBI accession number NP_001033689.1, NM_001038600.2). In certain embodiments, the rat exon 7-associated MOR variant is rMOR-lC2 (NCBI accession number NP 001033690, NM_001038601.2).

[0047] In certain embodiments, the 30 aa sequences of exon 7 and homolog have a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In certain embodiments, the 30 aa sequences of exon 7 and homolog have a sequence identity of at least 60%, 61%, 62%, 63%, 64%, 65%, or 66%. In certain embodiments, the 30 aa sequence of exon 7 of a non-human primate has a sequence identity of at least 96%, 97%, 98%, 99%, or 100% to that of human exon O.

[0048] The term "inhibitor of exon 7-associated MOR variant(s)" as used herein refers to an agent that inhibits the activity of one or more exon 7-associated mu opioid receptor (MOR) variant(s). For example, the inhibitory agent may downregulate expression level of exon 7- associated MOR variant(s) or may interfere with or reduce activity (e.g., signaling activities) of exon 7-associated MOR variant(s). Without wanting to be bound by theory, while a MOR agonist may still engage exon 7-associated MOR variant(s), the exon 7-associated MOR variant(s) mediated activities could be reduced or abrogated by the inhibitor. Nonetheless, a MOR agonist could still activate other non E7-associated MOR variants that lack exon 7, for example, so that analgesia induced by other non E7-associated MOR variants could be preserved.

[0049] In certain embodiments, the inhibitor reduces the expression (e.g., mRNA or protein level) of exon 7-associated MOR variant(s) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to a control (e.g., with vehicle control, or in the absence of the inhibitor).

[0050] In certain embodiments, the inhibitor inhibits the activity of exon 7-associated MOR variant(s) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to a control (e.g., with vehicle control, or in the absence of the inhibitor).

[0051] After continued use of a MOR agonist, a subject may develop MOR agonist tolerance. Thus, to maintain the same level of analgesia, dose-escalation of the MOR agonist may be needed. Alternatively, rotation into a different MOR agonist may be needed if cross-tolerance for the different MOR agonist has not developed in the subject.

[0052] In certain embodiments, the method (or inhibitor) as described herein reduces MOR agonist tolerance in the subject by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more as compared to a control (e.g., MOR agonist administered with a vehicle control or in the absence of the inhibitor (e.g., antibody) as described herein).

[0053] In certain embodiments, the method (or inhibitor) as described herein reduces the amount of MOR agonist needed to maintain same level of analgesia in the subject by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more as compared to a control (e.g., MOR agonist administered with a vehicle control or in the absence of the inhibitor (e.g., antibody) as described herein).

[0054] In certain embodiments, the amount (e.g., dose or ED50) of MOR agonist needed to maintain same level of analgesia in the subject using the method (or inhibitor) described herein is less than about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amount of MOR agonist needed to maintain same level of analgesia in a subject under a control regimen (e.g., MOR agonist administered with a vehicle control or in the absence of the inhibitor (e.g., antibody) as described herein). Namely, a subject under a control regimen may need a higher amount of MOR agonist to achieve the same level of analgesia efficacy as compared to a subject using the method (or inhibitor) described herein. Hence, the method (or inhibitor) described herein may effectively achieve a dose-sparing effect for maintaining same or comparable level of analgesia as compared to a control regimen that may require a doseescalation. Likewise, the method (or inhibitor) described herein may also reduce the need or frequency for MOR agonist rotation into a different MOR agonist.

[0055] In certain embodiments, the method (or inhibitor) described herein reduces the risk of MOR agonist addition / reward in the subject by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more as compared to a control (e.g., MOR agonist administered with a vehicle control or in the absence of the inhibitor (e.g., antibody) as described herein).

[0056] In certain embodiments, the method (or inhibitor) described herein reduces the MOR agonist induced respiratory depression (e.g., breathing frequency drop) in the subject by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more as compared to a control (e.g., MOR agonist administered with a vehicle control or in the absence of the inhibitor (e.g., antibody) as described herein).

[0057] In certain embodiments, the inhibitor is a nucleic acid (e.g., ASO, siRNA, or shRNA, etc.).

[0058] In certain embodiments, the inhibitor is a single stranded nucleic acid.

[0059] In certain embodiments, the inhibitor is an antisense oligonucleotide (ASO). In certain embodiments, the ASO may hybridize with and span an intron-exon 7 or intron-exon O junction to achieve exon-skipping during pre-mRNA processing. In certain embodiments, the ASO may block binding site for splicing protein(s), and thereby modulate splicing to reduce the mRNA level of exon 7-associated MOR variant(s). In certain embodiments, the mRNA level of exon 7- associated MOR variant(s) (e.g., exon O) is reduced.

[0060] In certain embodiments, the ASO blocks splicing from exon 3 to exon 7 and specifically reduced expression of E7-associated variants, but does not affect other non E7- associated variants that lack exon 7 or homolog (e.g., exon O).

[0061] In certain embodiments, the ASO is a vivo morpholino oligonucleotide.

[0062] In certain embodiments, the ASO comprises a sequence of 5’- AGGGTTGGCTGTACAAGAAAAAGGA-3’ (SEQ ID NO:30).

[0063] In certain embodiments, nucleic acid (e.g., ASO, siRNA, or shRNA, etc.) is conjugated with a cell-penetrating peptide (CPP), such as TAT.

[0064] In certain embodiments, the inhibitor is not a nucleic acid. In certain embodiments, the inhibitor is not a single stranded nucleic acid. In certain embodiments, the inhibitor is not an antisense oligonucleotide (ASO).

[0065] In certain embodiments, the inhibitor is a double stranded nucleic acid. In certain embodiments, the inhibitor is a siRNA. In certain embodiments, the siRNA hybridizes with a segment within the exon 7 or homolog RNA sequence and may reduce the mRNA level of exon 7-associated MOR variant(s).

[0066] In certain embodiments, the expression of the exon 7-associated MOR variant(s) is reduced on the mRNA and / or protein level.

[0067] In certain embodiments, the expression of the exon 7-associated MOR variant(s) is not reduced on the mRNA and / or protein level. In certain embodiments, the mRNA level of exon 7- associated MOR variant(s) is not reduced. In certain embodiments, the protein level of exon 7- associated MOR variant(s) is not reduced.

[0068] Certain embodiments of invention provide an exon 7 or homolog specific antibody, or fragment thereof.

[0069] In certain embodiments, the inhibitor is an exon 7 or homolog specific antibody, or fragment thereof, that specifically binds exon 7-associated MOR variant(s) at exon 7 domain (i.e., the 30 amino acids such as any one of SEQ ID NOs: l-4). Accordingly, an exon 7 or homolog specific antibody, or fragment thereof, would not bind other MOR variants that lack exon 7.

[0070] In certain embodiments, the inhibitor is a single domain antibody.

[0071] In certain embodiments, the inhibitor is a monoclonal antibody or fragment thereof.

[0072] In certain embodiments, the antibody or fragment binds human hM0R-10.

[0073] In certain embodiments, the antibody or fragment binds rat rMOR-lCl and rMOR-lC2. In certain embodiments, the antibody or fragment binds rat rMOR-lCl. In certain embodiments, the antibody or fragment binds rat rMOR-lC2.

[0074] In certain embodiments, the antibody or fragment binds mouse mMOR-lC, mM0R-10, mMOR-lU, and mMOR-lM. In certain embodiments, the antibody or fragment binds mouse mMOR-lC, mM0R-10, and mMOR-lU. In certain embodiments, the antibody or fragment binds mouse mMOR-lC. In certain embodiments, the antibody or fragment binds mouse mM0R-10. In certain embodiments, the antibody or fragment binds mouse mMOR-lU. In certain embodiments, the antibody or fragment binds mouse mMOR-lM.

[0075] In certain embodiments, the antibody or fragment has cross-reactivity for exon 7 and homolog thereof. For example, in certain embodiments, the antibody or fragment has crossreactivity for mouse exon 7 and rat exon 7. In certain embodiments, the antibody or fragment has cross-reactivity for mouse exon 7, rat exon 7, non-human primate exon 7, and / or human exon O. In certain embodiments, the antibody or fragment has cross-reactivity for mouse exon 7, rat exon 7, and / or human exon O. In certain embodiments, the antibody or fragment has crossreactivity for mouse exon 7 and human exon O.

[0076] In certain embodiments, the antibody or fragment specifically binds an epitope of exon 7 or homolog (e.g., exon O). Exon 7 or homolog protein domain has 30 aa in length (e.g., also see Figure 12). In certain embodiments, the epitope is the 30 amino acid sequences of exon 7 or homolog. In certain embodiments, the epitope is a fragment (e.g., an N-terminal fragment, a middle segment fragment, or C-terminal fragment) of the 30 amino acid sequences of exon 7 or homolog. For example, in certain embodiments, the epitope has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids. In certain embodiments, the epitope has a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In certain embodiments, the epitope has a length of 22, or 23 amino acids. In certain embodiments, the epitope has a length of 23 amino acids. In certain embodiments, the epitope has a length of 22 amino acids. In certain embodiments, the epitope has a length of 5-25, 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-20 amino acids.

[0077] There is a predicated phosphorylation site (having 6 or 7 aa in length, PxPxxE / D or PxxPxxE / D) for high-affinity beta arrestin binding (see Figure 12, underlined) within exon 7 or homolog. In certain embodiments, the epitope comprises the predicated phosphorylation site for beta arrestin. For example, in certain embodiments, the epitope (e.g., having 10-30aa, or 15-25aa in length) comprises SPPTHRE (SEQ ID NO:5), SPTHVE (SEQ ID NO:6), or SPTHGE (SEQ ID NO:7). For example, in certain embodiments, the antibody or fragment specifically binds an exon O epitope comprising or consisting of PPLAVSMAQIFTRYPPPTHREK (SEQ ID NO:8). In certain embodiments, the antibody or fragment specifically binds an exon O epitope comprising or consisting of PPLAVSMAQIFTRYSPPTHREK (SEQ ID NO:29). In certain embodiments, the antibody or fragment specifically binds an exon O epitope comprising or consisting of PPLAVSMAQIFTRYPPPTHREKT (SEQ ID NO:31). In certain embodiments, the antibody or fragment specifically binds an exon O epitope comprising or consisting of PPLAVSMAQIFTRYSPPTHREKT (SEQ ID NO:32). In certain embodiments, the antibody or fragment specifically binds a mouse exon 7 epitope comprising or consisting of PTLAVSVAQIFTGYPSPTHVEK (SEQ ID NO:9). In certain embodiments, the antibody or fragment specifically binds a mouse exon 7 epitope comprising or consisting of PTLAVSVAQIFTGYPSPTHVEKP (SEQ ID NO:33). In certain embodiments, the antibody or fragment specifically binds a rat exon 7 epitope comprising or consisting of PALAVSVAQIFTGYPSPTHGEK (SEQ ID NO: 10). In certain embodiments, the antibody or fragment specifically binds a rat exon 7 epitope comprising or consisting of PALAVSVAQIFTGYPSPTHGEKP (SEQ ID NO:34). In certain embodiments, the antibody or fragment reduces exon 7 or homolog mediated signaling transduction. In certain embodiments, the antibody or fragment reduces exon 7 or homolog domain mediated binding with an endogenous binding partner. In certain embodiments, the antibody or fragment reduces beta arrestin binding with exon 7-associated MOR variant(s) and / or beta arrestin mediated signaling. Without wanting to be bound by theory, in certain embodiments, the antibody or fragment may block interaction of an endogenous binding partner or signaling molecule (e.g., beta arrestin) with exon 7 sequence without reducing the exon 7-associated MOR variant(s) protein expression level. However, in certain embodiments, the antibody or fragment may also promote degradation of exon 7-associated MOR variant(s) protein.

[0078] In certain embodiments, the antibody is a mouse, rat, or rabbit antibody. In certain embodiments, the antibody is a chimeric antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.

[0079] In certain embodiments, the antibody is a single domain antibody.

[0080] In some embodiments, the exon 7 specific antibody (e.g., chimeric or humanized Ab), or fragment thereof, comprises: (1) one or more (e.g., 1, 2, 3, 4, 5, or 6) complementarity determining region (CDR) sequences; (2) a heavy chain variable region sequence; and / or (3) a light chain variable region sequence, as described herein (e.g., as described in Table A in Example 1 below). In some embodiments, the exon 7 specific antibody is a chimeric antibody comprising rabbit variable region and human constant region. In some embodiments, the exon 7 specific antibody is a humanized antibody, for example, wherein one or more (e.g., 1, 2, 3, 4, 5, or 6) rabbit CDRs are grafted onto human framework regions. In some embodiments, the human framework regions of a humanized antibody may optionally contain one or more non-CDR residues (i.e., FR residue) derived from the rabbit antibody if such residue may improve binding.

[0081] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO: 11 and further comprises three CDRs of SEQ ID NO: 12. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (TYGVN, SEQ ID NO:35), H-CDR2 (IIHANGGTYYASWAKG, SEQ ID NO:36), H-CDR3 (GVFDSAGAHYGDDL, SEQ ID NO:37) and L-CDR1 (QSSQSVYSNNLS, SEQ ID NO:38), L-CDR2 (RASTLAS, SEQ ID NO:39), L- CDR3 (LGTYVSSGWYSNV, SEQ ID NO:40).

[0082] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO: 13 and further comprises three CDRs of SEQ ID NO: 14. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (NYDIF, SEQ ID NO:41), H-CDR2 (YIGHGGDIWYASWAKG, SEQ ID NO:42), H-CDR3 (WDL, SEQ ID NO:43) and L-CDR1 (QASQSVYSRNFLS, SEQ ID NO:44), L-CDR2 (GASTLAS, SEQ ID NO:45), L-CDR3 (LGGYADGDNA, SEQ ID NO:46).

[0083] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO: 15 and further comprises three CDRs of SEQ ID NO: 16. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (TFGVN, SEQ ID NO:47), H-CDR2 (IIHANGGTYYANWAKG, SEQ ID NO:48), H-CDR3 (GVFDNTGAHYGDDL, SEQ ID NO:49) and L-CDR1 (QSSQVVYSNNLS, SEQ ID NO:50), L-CDR2 (RASTLAS, SEQ ID NO:39), L-CDR3 (LGTYVSSGWYSNV, SEQ ID NO:40).

[0084] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO: 17 and further comprises three CDRs of SEQ ID NO: 18. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (TYGVS, SEQ ID NO:51), H-CDR2 (IIHANGGTYYASWVNG, SEQ ID NO:52), H-CDR3 (GVFDSAGAHYGDDL, SEQ ID NO:37) and L-CDR1 (QSSQSVYSNNLS, SEQ ID NO:38), L-CDR2 (RASTLAS, SEQ ID NO:39), L-CDR3 (LGTYVSSGWYSNV, SEQ ID NO:40).

[0085] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO: 19 and further comprises three CDRs of SEQ ID NO:20. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (SYAMG, SEQ ID NO:53), H- CDR2 (YISYGGTTFYASWAKG, SEQ ID NO: 54), H-CDR3 (QGI, SEQ ID NO: 55) and L- CDR1 (RSSQSVYSNNYLA, SEQ ID NO:56), L-CDR2 (DASTLAS, SEQ ID NO:57), L-CDR3 (LGGYDCSRADCSA, SEQ ID NO:58).

[0086] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO:21 and further comprises three CDRs of SEQ ID NO:22. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (NYGMS, SEQ ID NO:59), H- CDR2 (IIHATSGAYYASWAKG, SEQ ID NO:60), H-CDR3 (GVFDSDGGHYGDDL, SEQ ID NO:61) and L-CDR1 (QSSQNVGNNRWLS, SEQ ID NO:62), L-CDR2 (DASDLAS, SEQ ID NO:63), L-CDR3 (LGTYVGSGWYANA, SEQ ID NO:64).

[0087] In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises three CDRs of SEQ ID NO:23 and further comprises three

[0088] CDRs of SEQ ID NO:24. In some embodiments, the exon 7 specific antibody (e.g., rabbit Ab, or humanized Ab), or a fragment thereof, comprises H-CDR1 (TYAVN, SEQ ID NO:65), H-CDR2 (IIHATSGAYYASWAKG, SEQ ID NO:60), H-CDR3 (GVFDSDGGHYGDDL, SEQ ID NO:61) and L-CDR1 (QSSQSVYHNNLS, SEQ ID NO:66), L-CDR2 (RASTLAS, SEQ ID NO:39), L-CDR3 (LGTYVGSGWYANA, SEQ ID NO:64).

[0089] In some embodiments, the exon 7 specific antibody, or a fragment thereof, comprises a light chain sequence, or a fragment thereof, and / or a heavy chain sequence, or a fragment thereof, derived from any of the following antibody clones described herein:3H4-8, 3G12-12, 2D4-3, 2G7-1, 22G9-11, 23D9-4, and 23F11-1. The amino acid sequences of the light chain variable domain (VL) and heavy chain variable domain (VH) of these antibody clones are set forth in Table A below.

[0090] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 11; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 12.

[0091] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 13; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 14.

[0092] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 15; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 16.

[0093] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 17; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 18.

[0094] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:20.

[0095] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:21; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:22.

[0096] In some embodiments, the exon 7 specific antibody, or fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:23; and further comprises a light chain variable region comprising an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:24.

[0097] In certain embodiments, the inhibitor is an antibody or fragment thereof that competes with an antibody clone described herein (e.g., 3H4-8, 3G12-12, or a clone in Table A) with respect to binding to the exon 7 or homolog associated MOR variant(s). Competitive binding assays are known in the art and described herein. In certain embodiments, the binding competition assay is ELISA, or biosensor-based assay, such as surface plasmon resonance (SPR). In certain embodiments, the target (e.g., used in binding assay) is exon 7 or exon O protein domain as described herein (e.g., any one of SEQ ID NOs: l-4). In certain embodiments, the target is an exon 7 or homolog epitope (e.g., 5-29 aa in length) as described herein. In certain embodiments, the target is an epitope of any one of SEQ ID NOs:8-10, 29, and 31-34. In certain embodiments, the inhibitor is an antibody or fragment thereof that competitively reduces the binding of an antibody clone (e.g., 3H4-8, 3G12-12) described herein with the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, for example, as determined by a binding competition assay (e.g., ELISA or SPR).

[0098] In certain embodiments, the inhibitor comprises an exon 7 or homolog specific antibody, or fragment thereof, and further comprises a cell penetrating peptide (CPP) amino acid sequence, such as TAT fused to the antibody or fragment. For example, a cell penetrating peptide (CPP) amino acid sequence may be fused to the N terminus or C terminus of the exon 7 or homolog specific antibody, or fragment thereof. Cell penetrating peptides (CPPs) and fusion with antibody or fragment thereof are known in the art and described herein, for example, Slastnikova et al., Front Pharmacol. 2018; 9: 1208 is incorporated for reference herein. In certain embodiments, the cell penetrating peptide (CPP) is TAT peptide, Nona-arginine (R9), cyclic R10 peptide, Protein transduction domain mimics MePhl3-b-dG5 (P13D5), or Cell-penetrating poly(disulfide)s.

[0099] In certain embodiments, administration of an inhibitor of an exon 7-associated mu opioid receptor (MOR) variant (e.g., antibody or fragment) in the methods described herein comprises administering, to the subject in need of, a vector comprising an expression cassette or nucleic acid sequence encoding the inhibitor (e.g., antibody or fragment, such as a single domain antibody). In certain embodiments, the vector is a viral vector. Suitable viral vectors include, for example, retroviral vectors, herpes virus-based vectors and parvovirus-based vectors (e.g., adeno-associated virus (AAV) based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors). In certain embodiments, the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), self-complementary AAV (scAAV), poliovirus, HSV, or murine Maloney -based viral vector. In certain embodiments, the vector is an Adeno-associated virus (AAV) vector. In certain embodiments, the inhibitor is an antibody or fragment, including but not limited to, a single domain antibody. In certain embodiments, the inhibitor is an ASO, siRNA or shRNA.

[0100] After the vector described herein has been created, the vector may be purified. Vector purification to enhance the concentration of the vector in a composition can be accomplished by any suitable method, such as by density gradient purification, by chromatography techniques, or limiting dilution purification. The specific purification techniques are known to those versed in the art and will vary depending on the vector type (e.g., type of virus, such as type of AAV).

[0101] The present invention also provides a stock, preferably a homogeneous stock, comprising the viral vector (e.g., AAV vector). The preparation and analysis of viral stocks (e.g., AAV stocks) is well known in the art. Viral stocks vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. In certain embodiments, such a stock has a viral titer of at least about 105plaque-forming units (pfu), such as at least about 106pfu or even more specifically at least about 107pfu. In still more specific embodiments, the titer can be at least about 108pfu, or at least about 109pfu. In certain embodiments, the stock is a high titer stock of at least about IO10pfu or at least about 1011pfu. The invention additionally provides a composition comprising a vector described herein (e.g., an AAV vector) and a carrier.

[0102] In certain embodiments, the inhibitor of exon 7-associated MOR variant(s) (e.g., nucleic acid, antibody or fragment, vector) is administered orally, intravenously, subcutaneously, intradermally, intramuscularly, intrathecally, or intracerebroventricularly.

[0103] In certain embodiments, the MOR agonist is administered orally, intravenously, transdermally, intradermally, subcutaneously, intramuscularly, intrathecally, or intracerebroventricularly.

[0104] In certain embodiments, the MOR agonist and the inhibitor of exon 7-associated MOR variant(s) are co-administered (e.g., intravenously, subcutaneously, intradermally, intramuscularly, intrathecally, or intracerebroventricularly).

[0105] As used herein, the term “co-administration” refers to simultaneous administration of both a MOR agonist and an inhibitor of exon 7-associated MOR variant(s), or sequential administration of both a MOR agonist and an inhibitor of exon 7-associated MOR variant(s) within about 1, 5, 10, 15, 20, 30, 45, 60 minutes, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, or 72 hours.

[0106] In certain embodiments, the inhibitor of exon 7-associated MOR variant(s) (e.g., nucleic acid, antibody or fragment) is administered prior to the administration of the MOR agonist, for example, the inhibitor of exon 7-associated MOR variant(s) (e.g., nucleic acid, antibody or fragment) is administered 4, 5, 6, 7 days or more prior to administration of the MOR agonist.

[0107] In certain embodiments, the MOR agonist is administered, for example, 4, 5, 6, 7 days or more prior to the administration of the inhibitor of exon 7-associated MOR variant(s) (e.g., nucleic acid, antibody or fragment).

[0108] In certain embodiments, the MOR agonist and the inhibitor of exon 7-associated MOR variant(s) are administered using two separate formulations (i.e., one MOR agonist formulation such as a tablet, transdermal patch; and one inhibitor formulation such as a liquid formulation suitable for injection).

[0109] In certain embodiments, the MOR agonist and the inhibitor of exon 7-associated MOR variant(s) are administered using a single formulation that comprises both MOR agonist and the inhibitor of exon 7-associated MOR variant(s) (e.g., as described herein, or also see below).

[0110] Compositions Accordingly, certain embodiments of the invention provide a pharmaceutical composition, comprising an inhibitor (e.g., antibody or fragment) of an exon 7-associated mu opioid receptor (MOR) variant(s) as described herein, and a pharmaceutically acceptable carrier.

[0111] Certain embodiments of the invention also provide a pharmaceutical composition, comprising: a mu opioid receptor (MOR) agonist as described herein, and an inhibitor of an exon 7-associated mu opioid receptor (MOR) variant(s) as described herein, and a pharmaceutically acceptable carrier.

[0112] In certain embodiments, the MOR agonist is a morphine or analog thereof. In certain embodiments, the morphine or analog is selected from the group consisting of morphine, codeine, oxycodone, hydrocodone, hydromorphone, oxymorphone, levorphanol, and butorphanol. In certain embodiments, the morphine or analog is selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, and oxymorphone. In certain embodiments, the morphine or analog is morphine, or oxycodone.

[0113] In certain embodiments, the MOR agonist is a synthetic opioid compound. In certain embodiments, the synthetic opioid compound is a 4-anilidopiperidine compound, such as fentanyl or analog thereof, including but not limited to sufentanil, remifentanil, alfentanil, carfentanil, lofentanil, and ohmefentanil. In certain embodiments, the MOR agonist is methadone.

[0114] In certain embodiments, the MOR agonist has a molecular weight that is no greater than lOOOg / mol.

[0115] In certain embodiments, the inhibitor of an exon 7-associated MOR variant(s) is a nucleic acid as described herein (e.g., ASO, siRNA, or shRNA, etc.).

[0116] In certain embodiments, the inhibitor is an exon 7 or homolog specific antibody, or fragment thereof, that specifically binds exon 7-associated MOR variant(s) as described herein. In certain embodiments, the antibody or fragment thereof specifically binds an epitope comprising or consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO: 33, and / or SEQ ID NO:34.

[0117] In certain embodiments, the antibody is a mouse, rat, or rabbit antibody. In certain embodiments, the antibody is a chimeric antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a human antibody.

[0118] In certain embodiments, the antibody is a single domain antibody.

[0119] In some embodiments, the exon 7 specific antibody (e.g., chimeric or humanized Ab), or fragment thereof, comprises: (1) one or more (e.g., 1, 2, 3, 4, 5, or 6) complementarity determining region (CDR) sequences; (2) a heavy chain variable region sequence; and / or (3) a light chain variable region sequence, as described herein (e.g., as described in Table A in Example 1 below). In some embodiments, the exon 7 specific antibody is a chimeric antibody comprising rabbit variable region and human constant region. In some embodiments, the exon 7 specific antibody is a humanized antibody, for example, wherein one or more (e.g., 1, 2, 3, 4, 5, or 6) rabbit CDRs are grafted onto human framework regions.

[0120] In some embodiments, the exon 7 specific antibody, or a fragment thereof, comprises a light chain sequence, or a fragment thereof, and / or a heavy chain sequence, or a fragment thereof, derived from any of the following antibodies described herein:3H4-8, 3G12-12, 2D4-3, 2G7-1, 22G9-11, 23D9-4, and 23F11-1. The amino acid sequences of the light chain variable domain (VL) and heavy chain variable domain (VH) of these antibody clones are set forth in Table A below. For in vivo use, active ingredient(s) of the invention, such as MOR agonist and / or an inhibitor (e.g., antibody) of the invention, is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, an inhibitor of the invention may be present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease or side effects of MOR agonist). A pharmaceutical composition comprises active ingredient(s) of the invention in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition.

[0121] In certain embodiments, the composition is in liquid dosage form.

[0122] In certain embodiments, the composition is in solid dosage form (e.g., lyophilized formulation).

[0123] The composition or active ingredient(s) of the invention (e.g., inhibitor of E7-associated MOR variant) may be administered, e.g., intravenously, subcutaneously, intradermally, or intrathecally, by infusion or injection. Solutions of the composition or active ingredient(s) of the invention may be prepared in water, saline buffer, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0124] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising active ingredient(s) of the invention that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes or nanoparticles such as a lipid nanoparticle. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0125] Lyophilized formulation may also contain bulking agent (e.g., mannitol or glycine) and cryoprotectant / lyoprotectant (e.g., trehalose or sucrose). Lyophilized formulation can be reconstituted into a liquid dosage form using saline, 5% dextrose solution or water before administration.

[0126] The composition or active ingredient(s) of the invention such as the inhibitor (e.g., antibody) can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A), and may comprise pharmaceutically acceptable carriers and / or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising active ingredient(s) of the invention such as the inhibitor (e.g., antibody) of the invention, and pharmaceutically acceptable carriers and / or additives. Exemplary carriers include surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, and corrigents. However, the carriers that may be employed in the present invention are not limited to this list. In fact, other commonly used carriers can be appropriately employed: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, and so on. The composition may also comprise other low-molecular- weight polypeptides, proteins such as serum albumin, gelatin, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it can comprise an isotonic solution comprising, for example, physiological saline, dextrose, and other adjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which can also contain an appropriate solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol and PEG), and non-ionic detergent (polysorbate 80 and HCO-50).

[0127] In certain embodiments, the composition or active ingredient(s) of the invention such as MOR agonist and / or the inhibitor (e.g., antibody or a fragment) may be systemically administered, e.g., intravenously, subcutaneously, intradermally, orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be freeze-dried into lyophilized formulation (e.g., lyophilized cake), may be formulated or reconstituted as a liquid dosage form, may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, active ingredient(s) of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active ingredient(s) of the present invention. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the active ingredient(s) of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0128] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the composition or active ingredient(s) of present invention may be incorporated into sustained-release preparations and devices. In certain embodiments, the composition or active ingredient(s) of the invention is incorporated into a prefilled syringe.

[0129] For topical administration, the present composition or active ingredient(s) of the invention may be applied in pure form, z.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0130] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present inhibitor can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0131] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0132] Examples of useful dermatological compositions that can be used to deliver the present invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0133] Useful dosages of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0134] The amount of the present invention required for use in treatment will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician or pharmacist.

[0135] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0136] Composition or active ingredient(s) of the invention can also be administered in combination with other therapeutic agents and / or treatments, such as other agents or treatments that are useful for the treatment of pain, OUD, or substance dependence.

[0137] As used herein, the term “therapeutic agent” refers to any agent or material that has a beneficial effect on the mammalian recipient.

[0138] Certain embodiments of the invention provide a method, a composition, or an inhibitor of E7-associated MOR variant (e.g., an antibody or fragment thereof) as described herein for providing analgesia or for the prophylactic or therapeutic treatment of pain, OUD, or substance dependence. In certain embodiments, the invention provides an inhibitor of E7-associated MOR variant (e.g., an antibody or fragment thereof) in combination with a MOR agonist for providing analgesia or for the prophylactic or therapeutic treatment of pain, OUD, or substance dependence.

[0139] Certain embodiments of the invention provide the use of a composition, or an inhibitor of E7-associated MOR variant (e.g., an antibody or fragment thereof) as described herein to prepare a medicament for providing analgesia or the treatment of pain, OUD, or substance dependence in a subject.

[0140] Certain embodiments of the invention provide a kit comprising a composition, or an inhibitor of E7-associated MOR variant (e.g., an antibody or fragment thereof) as described herein, packaging material, and instructions for administering the composition, or inhibitor to a subject for providing analgesia or the treatment of pain, OUD, or substance dependence. In certain embodiments, the kit comprises a composition comprising a MOR agonist and an inhibitor of E7-associated MOR variant. In certain embodiments, the kit comprises two separate formulations (i.e., one MOR agonist formulation such as a tablet, transdermal patch; and one inhibitor formulation such as a liquid formulation suitable for injection), packaging material, and instructions for administering to the subject.

[0141] Certain embodiments of the invention provide a method, a composition, or an inhibitor of E7-associated MOR variant (e.g., an antibody or fragment thereof) as described herein for use in medical therapy.

[0142] Certain embodiments of the invention provide an isolated exon 7 or homolog specific antibody, or fragment thereof.

[0143] Certain embodiments of the invention provide a composition comprising an exon 7 or homolog specific antibody, or fragment thereof, as described herein and a carrier.

[0144] Certain embodiments of the invention provide an isolated polynucleotide comprising a nucleotide sequence encoding the isolated exon 7 or homolog specific antibody, or fragment thereof, as described herein.

[0145] Certain embodiments of the invention provide a vector comprising the polynucleotide as described herein. Certain embodiments of the invention provide a cell comprising a polynucleotide as described herein or a vector as described herein.

[0146] Certain Definition

[0147] As used herein, the term “antibody” includes a single-chain variable fragment (scFv), single domain antibody (sdAb, also referred to as nanobody), humanized, fully human or chimeric antibodies, single-chain antibodies, diabodies, and antigen-binding fragments of antibodies that do not contain the Fc region (e.g., Fab fragments). In certain embodiments, the antibody is a human antibody or a humanized antibody. A “humanized” antibody contains only the three CDRs (complementarity determining regions) and sometimes a few carefully selected “framework” residues (the non-CDR portions of the variable regions) from each donor antibody variable region recombinantly linked onto the corresponding frameworks and constant regions of a human antibody sequence. A “fully human antibody” is created in a hybridoma from mice genetically engineered to have only human-derived antibody genes or by selection from a phagedisplay library of human-derived antibody genes.

[0148] A scFv is a fusion protein of the variable region of the heavy (VH) and light chains (VL) of an immunoglobulin that is connected by means of a linker peptide. The linker is usually short, about 10-25 amino acids in length. If flexibility is important, the linker will contain a significant number of glycines. If solubility is important, serines or theonines will be utilized in the linker. The linker may link the amino-terminus of the VH to the carboxy -terminus of the VL, or the linker may link the carboxy -terminus of the VH to the amino-terminus of the VL. Divalent (also called bivalent) scFvs can be generated by linking two scFvs. For example, a divalent scFv can be made by generating a single peptide containing two VH and two VL regions. Alternatively, two peptides, each containing a single VH and a single VL region can be dimerized (also called “diabodies”). Holliger et al., “Diabodies: small bivalent and bispecific antibody fragments,” PNAS, July 1993, 90:6444-6448. Bivalency allows antibodies to bind to multimeric antigens with high avidity, and bispecificity allows the cross-linking of two antigens.

[0149] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, an antibody group wherein the antibodies constituting the group are homogeneous except for naturally occurring mutants that exist in a small amount. Monoclonal antibodies are highly specific and interact with a single antigenic site. Furthermore, each monoclonal antibody targets a single antigenic determinant (epitope) on an antigen, as compared to common polyclonal antibody preparations that typically contain various antibodies against diverse antigenic determinants. In addition to their specificity, monoclonal antibodies are advantageous in that they are typically produced from hybridoma cultures not contaminated with other immunoglobulins.

[0150] The adjective "monoclonal" indicates a characteristic of antibodies obtained from a substantially homogeneous group of antibodies, and does not specify antibodies produced by a particular method. For example, a monoclonal antibody to be used in the present invention can be produced by, for example, hybridoma methods (Kohler and Milstein, Nature 256:495, 1975) or recombination methods (U.S. Pat. No. 4,816,567). The monoclonal antibodies used in the present invention can be also isolated from a phage antibody library (Clackson et al., Nature 352:624-628, 1991; Marks et al., J. Mol. Biol. 222:581-597, 1991). The monoclonal antibodies of the present invention may comprise "chimeric" antibodies (immunoglobulins), wherein a part of a heavy (H) chain and / or light (L) chain is derived from a specific species or a specific antibody class or subclass, and the remaining portion of the chain is derived from another species, or another antibody class or subclass. Furthermore, mutant antibodies and antibody fragments thereof are also comprised in the present invention (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855, 1984).

[0151] As used herein, the term "mutant antibody" refers to an antibody comprising a variant amino acid sequence in which one or more amino acid residues have been altered. For example, the variable region of an antibody can be modified to improve its biological properties, such as antigen binding. Such modifications can be achieved by site-directed mutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)), PCR-based mutagenesis, cassette mutagenesis, and the like. Such mutants comprise an amino acid sequence which is at least 70% identical to the amino acid sequence of a heavy or light chain variable region of the antibody, more specifically at least 75%, even more specifically at least 80%, still more specifically at least 85%, yet more specifically at least 90%, and most specifically at least 95% identical. As used herein, the term “sequence identity” is defined as the percentage of residues identical to those in the antibody's original amino acid sequence, determined after the sequences are aligned and gaps are appropriately introduced to maximize the sequence identity as necessary.

[0152] Specifically, the identity of one nucleotide sequence or amino acid sequence to another can be determined using the algorithm BLAST, by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Programs such as BLASTN and BLASTX were developed based on this algorithm (Altschul et al., J. Mol. Biol. 215: 403-410, 1990). To analyze nucleotide sequences according to BLASTN based on BLAST, the parameters are set, for example, as score=100 and wordlength=12. On the other hand, parameters used for the analysis of amino acid sequences by BLASTX based on BLAST include, for example, score=50 and wordlength=3. Default parameters for each program are used when using the BLAST and

[0153] Gapped BLAST programs. Specific techniques for such analyses are known in the art (see the website of the National Center for Biotechnology Information (NCBI), Basic Local Alignment Search Tool (BLAST); http: / / www.ncbi.nlm.nih.gov).

[0154] Polyclonal and monoclonal antibodies can be prepared by methods known to those skilled in the art.

[0155] In another embodiment, antibodies or antibody fragments can be isolated from an antibody phage library, produced by using the technique reported by McCafferty et al. (Nature 348:552-554 (1990)). Clackson et al. (Nature 352:624-628 (1991)) and Marks et al. (J. Mol. Biol. 222:581-597 (1991)) reported on the respective isolation of mouse and human antibodies from phage libraries. There are also reports that describe the production of high affinity (nM range) human antibodies based on chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), and combinatorial infection and in vivo recombination, which are methods for constructing large-scale phage libraries (Waterhouse et al., Nucleic Acids Res. 21 :2265-2266 (1993)). These technologies can also be used to isolate monoclonal antibodies, instead of using conventional hybridoma technology for monoclonal antibody production.

[0156] Antibodies to be used in the present invention can be purified by a method appropriately selected from known methods, such as the protein A-Sepharose method, hydroxyapatite chromatography, salting-out method with sulfate, ion exchange chromatography, and affinity chromatography, or by the combined use of the same.

[0157] The present invention may use recombinant antibodies, produced by gene engineering. The genes encoding the antibodies obtained by a method described above are isolated from the hybridomas. The genes are inserted into an appropriate vector, and then introduced into a host (see, e.g., Carl, A. K. Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies, Published in the United Kingdom by Macmillan Publishers Ltd, 1990). The present invention provides the nucleic acids encoding the antibodies of the present invention, and vectors comprising these nucleic acids. Specifically, using a reverse transcriptase, cDNAs encoding the variable regions (V regions) of the antibodies are synthesized from the mRNAs of hybridomas. After obtaining the DNAs encoding the variable regions of antibodies of interest, they are ligated with DNAs encoding desired constant regions (C regions) of the antibodies, and the resulting DNA constructs are inserted into expression vectors. Alternatively, the DNAs encoding the variable regions of the antibodies may be inserted into expression vectors comprising the DNAs of the antibody C regions. These are inserted into expression vectors so that the genes are expressed under the regulation of an expression regulatory region, for example, an enhancer and promoter. Then, host cells are transformed with the expression vectors to express the antibodies. The present invention provides cells expressing antibodies of the present invention. The cells expressing antibodies of the present invention include cells and hybridomas transformed with a gene of such an antibody.

[0158] The antibodies of the present invention also include antibodies which comprise complementarity-determining regions (CDRs), or regions functionally equivalent to CDRs. The term "functionally equivalent" refers to comprising amino acid sequences similar to the amino acid sequences of CDRs of any of the monoclonal antibodies isolated in the Examples. The term "CDR" refers to a region in an antibody variable region (also called "V region"), and determines the specificity of antigen binding. The H chain and L chain each have three CDRs, designated from the N terminus as CDR1, CDR2, and CDR3. There are four regions flanking these CDRs: these regions are referred to as "framework," and their amino acid sequences are highly conserved. The CDRs can be transplanted into other antibodies, and thus a recombinant antibody can be prepared by combining CDRs with the framework of a desired antibody. One or more amino acids of a CDR can be modified without losing the ability to bind to its antigen. For example, one or more amino acids in a CDR can be substituted, deleted, and / or added.

[0159] In certain embodiments, an amino acid residue is mutated into one that allows the properties of the amino acid side-chain to be conserved. Examples of the properties of amino acid side chains comprise: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W). The letters within parenthesis indicate the one-letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues is deleted, added, and / or substituted can retain the original biological activity (Mark D.

[0160] F. et al., Proc. Natl. Acad. Sci. U.S.A. 81 :5662-5666 (1984); Zoller M. J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland

[0161] G. et al., Proc. Natl. Acad. Sci. U.S.A. 79: 6409-6413 (1982)). The number of mutated amino acids is not limited, but in general, the number falls within 40% of amino acids of each CDR, and specifically within 35%, and still more specifically within 30% (e.g., within 25%). The identity of amino acid sequences can be determined as described herein.

[0162] In the present invention, recombinant antibodies artificially modified to reduce heterologous antigenicity against humans can be used. Examples include chimeric antibodies and humanized antibodies. These modified antibodies can be produced using known methods. A chimeric antibody includes an antibody comprising variable and constant regions of species that are different to each other, for example, an antibody comprising the antibody heavy chain and light chain variable regions of a nonhuman mammal such as a mouse, and the antibody heavy chain and light chain constant regions of a human. Such an antibody can be obtained by (1) ligating a DNA encoding a variable region of a mouse antibody to a DNA encoding a constant region of a human antibody; (2) incorporating this into an expression vector; and (3) introducing the vector into a host for production of the antibody.

[0163] A humanized antibody, which is also called a reshaped human antibody, may be obtained by substituting an H or L chain complementarity determining region (CDR) of an antibody of a nonhuman mammal such as a mouse, with the CDR of a human antibody. Conventional genetic recombination techniques for the preparation of such antibodies are known (see, for example, Jones et al., Nature 321 : 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta Curr. Op. Struct. Biol. 2: 593-596 (1992)). Specifically, a DNA sequence designed to ligate a CDR of a mouse antibody with the framework regions (FRs) of a human antibody is synthesized by PCR, using several oligonucleotides constructed to comprise overlapping portions at their ends. A humanized antibody can be obtained by (1) ligating the resulting DNA to a DNA that encodes a human antibody constant region; (2) incorporating this into an expression vector; and (3) transfecting the vector into a host to produce the antibody (see, European Patent Application No. EP 239,400, and International Patent Application No. WO 96 / 02576). Human antibody FRs that are ligated via the CDR are selected where the CDR forms a favorable antigen-binding site. Similar to mouse antibody humanization, rabbit antibody humanization methods are also known (see, for example, Zhang et al., MAbs. 2017 Apr;9(3):419-429; Goydel et al., J Biol Chem. 2020 May l;295(18):5995-6006; and International Patent Application Nos. WO / 2005 / 016950, and WO / 2009 / 155726). The humanized antibody may comprise additional amino acid residue(s) that are not included in the CDRs introduced into the recipient antibody, nor in the framework sequences. Such amino acid residues are usually introduced to more accurately optimize the antibody's ability to recognize and bind to an antigen. For example, as necessary, amino acids in the framework region of an antibody variable region may be substituted such that the CDR of a reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0164] As described herein, an antibody of the invention or a fragment thereof may comprise a synthetic CDR-H3. Additionally, an antibody of the invention may also be a recombinant antibody (e.g., a humanized or chimeric antibody) or a fragment thereof. Accordingly, such an antibody of the invention or fragment thereof would not be a product of nature. Additionally, an antibody of the invention or a fragment thereof may comprise markedly different characteristics (e.g., structural, functional and / or other properties) as compared to naturally occurring antibody.

[0165] The isotypes of the antibodies of the present invention are not limited. The isotypes include, for example, IgG (IgGl, IgG2, IgG3, and IgG4), IgM, IgA (IgAl and IgA2), IgD, and IgE. The antibodies of the present invention may also be antibody fragments comprising a portion responsible for antigen binding, or a modified fragment thereof. The term "antibody fragment" refers to a portion of a full-length antibody, and generally to a fragment comprising an antigen-binding domain or a variable region. Such antibody fragments include, for example, Fab, F(ab')2, Fv, single-chain Fv (scFv) which comprises a heavy chain Fv and a light chain Fv coupled together with an appropriate linker, diabody (diabodies), linear antibodies, and multispecific antibodies prepared from antibody fragments. Previously, antibody fragments were produced by digesting natural antibodies with a protease; currently, methods for expressing them as recombinant antibodies using genetic engineering techniques are also known (see Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al., Science 229:81 (1985); Co, M. S. et al., J. Immunol., 1994, 152, 2968-2976; Better, M. & Horwitz, A. H., Methods in Enzymology, 1989, 178, 476-496, Academic Press, Inc.;

[0166] Plueckthun, A. & Skerra, A., Methods in Enzymology, 1989, 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology, 1989, 121, 663-669; Bird, R. E. et al., TIBTECH, 1991, 9, 132-137).

[0167] An "Fv" fragment is the smallest antibody fragment, and contains a complete antigen recognition site and a binding site. This region is a dimer (VH-VL dimer) wherein the variable regions of each of the heavy chain and light chain are strongly connected by a noncovalent bond. The three CDRs of each of the variable regions interact with each other to form an antigenbinding site on the surface of the VH-VL dimer. In other words, a total of six CDRs from the heavy and light chains function together as an antibody's antigen-binding site. However, a variable region (or a half Fv, which contains only three antigen-specific CDRs) alone is also known to be able to recognize and bind to an antigen, although its affinity is lower than the affinity of the entire binding site. Thus, a specific antibody fragment of the present invention is an Fv fragment, but is not limited thereto. Such an antibody fragment may be a polypeptide which comprises an antibody fragment of heavy or light chain CDRs which are conserved, and which can recognize and bind its antigen.

[0168] A Fab fragment (also referred to as F(ab)) also contains a light chain constant region and heavy chain constant region (CHI). For example, papain digestion of an antibody produces the two kinds of fragments: an antigen-binding fragment, called a Fab fragment, containing the variable regions of a heavy chain and light chain, which serve as a single antigen-binding domain; and the remaining portion, which is called an "Fc" because it is readily crystallized. A Fab' fragment is different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CHI region, which contains one or more cysteine residues from the hinge region of an antibody. A Fab' fragment is, however, structurally equivalent to Fab in that both are antigen-binding fragments which comprise the variable regions of a heavy chain and light chain, which serve as a single antigen-binding domain. Herein, an antigen-binding fragment comprising the variable regions of a heavy chain and light chain which serve as a single antigen-binding domain, and which is equivalent to that obtained by papain digestion, is referred to as a "Fab-like antibody," even when it is not identical to an antibody fragment produced by protease digestion. Fab'-SH is Fab' with one or more cysteine residues having free thiol groups in its constant region. A F(ab') fragment is produced by cleaving the disulfide bond between the cysteine residues in the hinge region of F(ab')2. Other chemically crosslinked antibody fragments are also known to those skilled in the art. Pepsin digestion of an antibody yields two fragments; one is a F(ab')2 fragment which comprises two antigen-binding domains and can cross-react with antigens, and the other is the remaining fragment (referred to as pFc'). Herein, an antibody fragment equivalent to that obtained by pepsin digestion is referred to as a "F(ab')2-like antibody" when it comprises two antigenbinding domains and can cross-react with antigens. Such antibody fragments can also be produced, for example, by genetic engineering. Such antibody fragments can also be isolated, for example, from the antibody phage library described above. Alternatively, F(ab')2-SH fragments can be recovered directly from hosts, such as E. coli, and then allowed to form F(ab')2 fragments by chemical crosslinking (Carter et al., Bio / Technology 10: 163-167 (1992)). In an alternative method, F(ab')2 fragments can be isolated directly from a culture of recombinant hosts.

[0169] The term "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (for example, P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93 / 11161). In general, a diabody is a dimer of two polypeptide chains. In each of the polypeptide chains, a light chain variable region (VL) and a heavy chain variable region (VH) in an identical chain are connected via a short linker, for example, a linker of about five residues, so that they cannot bind together. Because the linker between the two is too short, the VL and VH in the same polypeptide chain cannot form a single chain V region fragment, but instead form a dimer. Thus, a diabody has two antigen-binding domains. When the VL and VH regions against the two types of antigens (a and b) are combined to form VLa-Vm, and VLb-Vua via a linker of about five residues, and then co-expressed, they are secreted as bispecific Dbs. The antibodies of the present invention may be such Dbs.

[0170] The terms “nanobody” and “single-domain antibody” are used interchangeably herein. As used herein, the term “nanobody” or “single-domain antibody” refers to a single monomeric variable antibody domain comprising three complementarity-determining regions (CDRs including CDR1, CDR2, CDR3) and four framework regions (FRs including FR1, FR2, FR3, FR4), such as a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression), which is capable of binding to a specific antigen. Methods of producing sdAb are known, for example, see Serge Muyldermans, FEBS J. 2021 Apr; 288(7): 2084-2102; and Harmand et al., RSC Chem. Biol., 2021, 2, 685-701.

[0171] A single-chain antibody (also referred to as "scFv") can be prepared by linking a heavy chain V region and a light chain V region of an antibody (for a review of scFv see Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, eds. Rosenburg and Moore, Springer Verlag, N.Y., pp. 269-315 (1994)). Methods for preparing single-chain antibodies are known in the art (see, for example, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,091,513; and 5,455,030). In such scFvs, the heavy chain V region and the light chain V region are linked together via a linker, such as a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A, 1988, 85, 5879-5883). The heavy chain V region and the light chain V region in a scFv may be derived from the same antibody, or from different antibodies. The peptide linker used to ligate the V regions may be any single-chain peptide consisting of 12 to 19 residues. A DNA encoding a scFv can be amplified by PCR using, as a template, either the entire DNA, or a partial DNA encoding a desired amino acid sequence, selected from a DNA encoding the heavy chain or the V region of the heavy chain of the above antibody, and a DNA encoding the light chain or the V region of the light chain of the above antibody; and using a primer pair that defines the two ends. Further amplification can be subsequently conducted using a combination of the DNA encoding the peptide linker portion, and the primer pair that defines both ends of the DNA to be ligated to the heavy and light chain respectively. After constructing DNAs encoding scFvs, conventional methods can be used to obtain expression vectors comprising these DNAs, and hosts transformed by these expression vectors. Furthermore, scFvs can be obtained according to conventional methods using the resulting hosts. These antibody fragments can be produced in hosts by obtaining genes that encode the antibody fragments and expressing these as outlined above. Antibodies bound to various types of molecules, such as polyethylene glycols (PEGs), may be used as modified antibodies. Methods for modifying antibodies are already established in the art. The term "antibody" in the present invention also encompasses the above-described antibodies.

[0172] The antibodies obtained can be purified to homogeneity. The antibodies can be isolated and purified by a method routinely used to isolate and purify proteins. The antibodies can be isolated and purified by the combined use of one or more methods appropriately selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectro-focusing, for example (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography. These chromatographic methods can be practiced using liquid phase chromatography, such as HPLC and FPLC. Columns to be used in affinity chromatography include protein A columns and protein G columns. For example, protein A columns include Hyper D, POROS, and Sepharose F. F. (Pharmacia). Antibodies can also be purified by utilizing antigen binding, using carriers on which antigens have been immobilized.

[0173] As used herein, the term, "antisense oligonucleotide" or ASO refers to an oligonucleotide, 7-100 nucleotides in length, characterized by having a nucleotide sequence that is complementary to a target nucleotide sequence in a pre-mRNA present in a mammalian cell and renders the oligonucleotide operative, when contacted with the mammalian cell in therapeutically effective amounts, to modulate (e.g., down-regulate) an amount of a mRNA splice variant produced from the pre-mRNA in the cell or to increase a degradation rate of the pre-mRNA in the cell and / or decrease a translation rate of a mRNA. In embodiments in which ASOs modulate splicing of a pre-mRNA, such ASOs may comprise a nucleotide sequence complementary to a target sequence in the pre-mRNA that comprises or overlaps: an intron-exon junction; an intronic or exonic splice silencer; and / or an intronic or exonic splice enhancer. In embodiments in which ASOs increase a pre-mRNA degradation rate or decrease a mRNA translation rate, such ASOs may comprise a nucleotide sequence complementary to one or more target sequence(s) in the pre-mRNA that comprise(s) or overlap(s) a nucleotide sequence from a single exon or two or more nucleotide sequences from two or more exons.

[0174] The nucleotides of ASOs can be modified or unmodified DNA nucleotides, RNA nucleotides, or combinations thereof. In some embodiments, an ASO composition comprises at least one of a DNA oligonucleotide, an RNA oligonucleotide, a morpholino oligonucleotide, or a "vivo morpholino" oligonucleotide. In particular, the oligonucleotide can include modified oligonucleotide backbones such as, but not limited to, phosphorothioates, phosphorodiamidates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkyl phosphotriesters, and boranophosphates having normal 3'-5' linkages, as well as 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to various salts, mixed salts and free acid forms are also included. References that teach the preparation of such modified backbone oligonucleotides are provided, for example, in U.S. Pat. Nos. 4,469,863 and 5,750,666, all incorporated by reference herein. The design and synthesis of antisense oligonucleotides is well known in the art. Computer programs for the design of antisense oligonucleotide sequences are also available.

[0175] In some embodiments, at least seven, eight, 10, 15, 20, 25, 30, 40, or 50 contiguous nucleotides in a ASO are complementary to the target nucleotide sequence in the selected pre- mRNA that comprises, or overlaps, a sequence motif selected from the group consisting of a 5' splice site, a 3' splice site, a branch site, an intronic splice enhancer, an intronic splice silencer, an exonic splice enhancer, and an exonic splice silencer. In some embodiments, at least 8-12, 8- 15, 8-20, 10-25, 15-30, 20-40, or 30-50 contiguous nucleotides in a ASO are complementary to the target nucleotide sequence in the selected pre-mRNA that comprises, or overlaps, a sequence motif selected from the group consisting of a 5' splice site, a 3' splice site, a branch site, an intronic splice enhancer, an intronic splice silencer, an exonic splice enhancer, and an exonic splice silencer.

[0176] In some embodiments, an ASO is about 7-90 nucleotides in length, 7-50 nucleotides in length, 7-25 nucleotides in length, 7-15 nucleotides in length, 8-12 nucleotides in length, 8-17 nucleotides in length, 8-25 nucleotides in length, 8-50 nucleotides in length, 9-12 nucleotides in length, 9-25 nucleotides in length, 10-17 nucleotides in length, 10-25 nucleotides in length, 20- 30 nucleotides in length, or 25 nucleotides in length. In some embodiments, an ASO is about 89- 90 nucleotides in length. In embodiments where an ASO comprises at least 75% RNA nucleotides, the ASO can be less than 21 nucleotides in length. In some embodiments, a ASO is entirely single-stranded. In some embodiments, an ASO is partially single-stranded. In some embodiments, an ASO is capable of reversibly or permanently forming stem loop structures, hairpin structures, and the like.

[0177] As used herein, the term "intron-exon junction" refers to sequences of nucleotides in a pre-mRNA that reside at the junction of an intron and an exon in the pre-mRNA. An intron-exon junction may extend up to 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, and 50 nucleotides in both 5'-ward and 3 '-ward directions from the intron-exon border.

[0178] The invention encompasses isolated or substantially purified protein compositions. In the context of the present invention, an "isolated" or "purified" polypeptide is a polypeptide that exists apart from its native or library environment. A polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell such as a bacterium or a mammalian expression system for the production of the polypeptide. For example, an "isolated" or "purified" protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0179] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0180] As used herein, "comparison window" makes reference to a contiguous and specified segment of an amino acid or polynucleotide sequence, wherein the sequence in the comparison window may comprise additions or deletions (z.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least about 20 contiguous amino acid residues or nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.

[0181] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide or polynucleotide sequence in the comparison window may comprise additions or deletions (ie., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0182] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, and at least about 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least about 70%, at least about 80%, 90%, or at least about 95%.

[0183] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

[0184] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity or complementarity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The term "amino acid" includes the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L form, as well as unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, -tetrahydroi soquinoline- 3 -carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxy carbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (Ci-Ce)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T.W. Greene, Protecting Groups In Organic Synthesis,' Wiley: New York, 1981, and references cited therein) The term also comprises natural and unnatural amino acids bearing a cyclopropyl side chain or an ethyl side chain.

[0185] The term "nucleic acid" and “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

[0186] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. In certain embodiments, the promoter is a tissue-specific promoter. In certain embodiments, the promoter is a constitutive promoter.

[0187] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0188] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0189] "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. In certain embodiments, the promoter is a tissue-specific promoter. In certain embodiments, the promoter is a tissue-specific promoter that could drive expression in central nervous system or peripheral nervous system, for example, a tissue-specific promoter may drive expression in cell type(s) such as neuron, microglia, and / or astrocyte. In certain embodiments, the promoter is a constitutive promoter.

[0190] The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0191] Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and / or an initiator.

[0192] "Operably-linked" may refer to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (z.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

[0193] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0194] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of active ingredient(s) (e.g., MOR agonist and / or the inhibitor of E7 associated MOR variant(s)) either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0195] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0196] Certain embodiments of the invention will be illustrated in the following non-limiting Example.

[0197] Example 1. Generation and Characterization of Rabbit monoclonal antibodies targeting mouse E7 variants.

[0198] Results

[0199] Production of MOR exon 7 specific antibodies

[0200] About 30 Rabbit mAb clones were generated against a mouse MOR exon 7 derived peptide (SEQ ID NO:33). Seven representative clones were sequenced (see Table A below). This example describes the characterization of an exemplary Rabbit mAb clone 3H4-8.

[0201] Binding characterization of the antibody

[0202] Rabbit mAb (clone 3H4-8) labeled E7-associated MOR variants in WT B6 mice (Fig.lA), but failed to label it in El / El l-KO mice (Figs. IB and 1C), confirming its target specificity in immunohistochemical (IHC) assay.

[0203] E7 variants of mouse (mMOR-lC), rat (rMOR-lCl) and human (hM0R-10) or a mouse E4 variant (mMOR-1) constructs were transiently transfected into HEK293 cells. Western blot was performed using whole cell lysate 36 hrs after transfection and 3H4-8. In Western blot assay (Fig. ID), 3H4-8 strongly labeled mouse mMOR-lC and labeled rat mMOR-lCl and human hM0R-10 with lower intensity. mMOR-1 C and mMOR-1 share all the sequences that are encoded by El / 2 / 3, including N-terminus, 7TM, 3 intracellular loops, 3 extracellular loops and part of intracellular C-terminus, except for a different intracellular C-terminal tail sequence that is encoded by E7 / 8 / 9 in mMOR-lC and by E4 in mMOR-1, respectively. The exon composition of mMOR-lC is El / 2 / 3 / 7 / 8 / 9, while the exon composition of mMOR-1 is El / 2 / 3 / 4. The 3H4-8 specificity was confirmed by the negative result from lysates from a transfected E4-associated mMOR-1.

[0204] Cellular distribution of the antibody

[0205] 3H4-8 was labeled at the Fc region with Qdot605. Neuro-2a cells, a mouse neuroblastoma cell line, were cultured in 8-well slide chamber. The labeled 3H4-8 (100 ug / ml) was added into Neuro-2a cells (a mouse neuroblastoma cell line) culture for 16 hrs. It was shown (Fig.2) that 3H4-8 can spontaneously penetrate from culture medium into Neuro-2a cells, although the mechanism remains to be investigated.

[0206] Monoclonal antibodies attenuated morphine tolerance in CD-I mice

[0207] Both RabmAbs, 3H4-8 and 3G12-12 were shown to reduce morphine tolerance (Fig.3A) in Radiant-Heat tail-flick assay. In particular, ED50 of mice without i.c.v. injection on Day 0 is 2.9 mg / kg. In the absence of exon-7 specific antibody, control IgG showed morphine tolerance, wherein ED50 of Ctr IgG group on Day 5 is 11.5 mg / kg. In contrast, ED50 of 3H4-8 group on Day 5 is only 3.9 mg / kg; ED50 of 3G12-12 group on Day 5 is only 3.5 mg / kg, suggesting that MOR exon-7 specific monoclonal antibodies attenuated morphine tolerance (Fig.3B) in this assay.

[0208] While radiant heat tail-flick assay mainly measures spinal analgesia, Hot-plate assay likely measure both supraspinal and spinal analgesia. 3H4-8 was studied and shown to reduce morphine tolerance (Fig.4A) in Hot-plate assay. In particular, ED50 of mice without i.c.v. injection on Day 0 is 2.3 mg / kg. In the absence of exon-7 specific antibody, control IgG showed morphine tolerance, wherein ED50 of Ctr IgG group on Day 5 is 10.7 mg / kg. In contrast, ED50 of 3H4-8 on Day 5 is only 5.9 mg / kg, suggesting that MOR exon-7 specific monoclonal antibody attenuated morphine tolerance (Fig.4B) in this assay.

[0209] Monoclonal antibody reduced morphine reward

[0210] In addition, the effect of MOR exon 7 specific antibody on morphine reward was investigated using a conditioned place preference (CPP) paradigm.12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 2 hrs before every morphine conditioning, it was shown that 3H4-8 reduced morphine CPP in B6 mice (Fig.5).

[0211] Fentanyl analgesia in mMOR-lO-KI (mM!OAct / Act) mice mM0R-10-KI (mM10Act / Act) mice have exon 7 associated MOR variant mM0R-10. Only a single exon 7 variant (mM0R-10), and no other 7TM variants, is expressed in mM10Act / Actmice. Inhibition of mM0R-10 by antibody in this model indicates exon 7 specific binding and inhibition by antibody in vivo. 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 3 hrs before fentanyl (0.08mg / kg, s.c.). Analgesia was measured by radiant heat tail-flick assay 15 min after fentanyl. 3H4-8 reduced fentanyl analgesia in mM0R-10-KI (mM10Act / Act) mice (Fig.6), suggesting exon 7 specific binding and inhibition or degradation of exon 7-associated MOR- 10 variant by antibody in vivo.

[0212] Methods

[0213] Generation of Rabbit monoclonal antibodies

[0214] A mouse exon 7 derived peptide (PTLAVSVAQIFTGYPSPTHVEKP, SEQ ID NO:33) was synthesized by GenScript and used as immunogen. The mouse exon 7 derived peptide has an added terminal Cysteine (C) residue to facilitate conjugation with a carrier protein Keyhole limpet hemocyanin (KLH) for immunization. Rabbits were immunized with immunogen and followed up with boosting. A week after each boost, antisera from immunized animals were tested by indirect ELISA for monitoring the induced immune response. PBMCs were isolated from fresh blood harvested from rabbits. Antigen-positive enriched B cells were plated into 96- well plates for one round of B cell cloning. For primary binder screening, the conditional medium was screened by ELISA with the target peptide for identifying positive clones. The primary screening was followed by a confirmatory screening, the conditional medium was screened again to confirm positive clones. About 30 positive clones were generated. Seven clones were selected for V-region sequencing. After V-region sequencing of ELISA positive clones, antibody sequences were cloned into mammalian expression vector (expression plasmids) and mammalian cells were transfected for antibody expression. Post-transfection, supernatant was harvested to perform antibody validation with ELISA. Antibodies were purified by protein A affinity chromatography. The purified Ab were dialyzed into PBS buffer for storage.

[0215] Immunohistochemical (IHC) study

[0216] Immunohistochemical (IHC) study with 3H4-8 (1 :200, 6.25pg / ml) using sagittal brain section of WT male B6 mice (Fig.1 A) and male El / El 1-KO mice in which all Oprml variants are disrupted (Fig. IB). The PAG from A & B. PAG, periaqueductal gray; VTA, ventral tegmental area; HYP, Hypothalamus. The stained sections were co-stained with DAPI (Fig.lC), imaged and scanned with a fluorescence microscope. (Fig. ID) Western blot. E7 variants of mouse (mMOR-lC), rat (rMOR-lCl) and human (hMOR-10) or a mouse E4 variant (mMOR-1) constructs were transiently transfected into HEK293 cells. Western blot was performed using whole cell lysate 36 hrs after transfection and antibody 3H4-8 (1 :2000, 1.25pg / ml).

[0217] Incubation of antibody with Neuro-2a cells

[0218] 3H4-8 was labeled at the Fc region of heavy chains with Qdot605 by SiteClick antibody labeling kit (Molecular Probe). Neuro-2a cells, a mouse neuroblastoma cell line, were cultured in 8-well slide chamber. The labeled 3H4-8 (100 ug / ml) was added into culture medium for 16 hrs. Neuro-2a cells were then washed, fixed, co-stained with DAPI and imaged using a confocal microscope.

[0219] Morphine tolerance study in CD-I mice

[0220] For Radiant-Heat tail-flick assay, 12.5 ug in 5 ul of 3H4-8 or 3G12-12 was i.c.v. administered 2 hrs before the first morphine dosing every day (lOmg / kg, s.c. twice / day) for 5 days. Analgesia was measured by radiant heat tail-flick assay.

[0221] For hot-plate assay, 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 2 hrs before the first morphine dosing every day (lOmg / kg, s.c. twice / day) for 5 days. Analgesia was measured by Hot-plate assay.

[0222] Morphine reward (CPP)

[0223] 12.5 ug in 5 ul of 3H4-8 was i.c.v. administered 2 hrs before every morphine conditioning. CPP was performed using a 3 -chamber apparatus (Med Associates) with a 6-day paradigm. On day 1, mice were placed in the central chamber for 1 minute of habituation with the sliding doors closed, followed by a 30-minute preconditioning period of free exploration in the 3 chambers. Time spent in each chamber was recorded. On days 2 to 5, mice were i.p. injected with saline (morning) or morphine (10 mg / kg, afternoon) and confined to either the black or the white chamber for 30 minutes. On day 6, mice were placed for a 1 -minute habituation in the central chamber, followed by a 30-minute free exploration in the 3 chambers. Time spent in each chamber was recorded. Preference was defined as difference between the times spent in the morphine-paired side of the chamber on the test day (day 6) compared with the preconditioning day (day 1).

[0224] Table A. Sequences of exemplary exon 7 or homolog specific antibodies (CDRs are underlined). Example 2. Exon 7 -associated variants facilitate fentanyl and methadone tolerance and reward in mE7M-B6 mutant mice, and enhance morphine tolerance and reward in a mM0R-10 knockin mice.

[0225] Both fentanyl and methadone induced significant tolerance in wide-type (WT, + / +) mice. Truncating exon 7-associated variants resulted in reduced fentanyl and methadone tolerance in mE7M-B6 (exon 7 truncated Mut, - / -) mice (Figs.8A-8B), suggesting that exon 7-associated variants facilitate or promote fentanyl and methadone tolerance. Fentanyl and methadone doseresponse curve studies before and after chronic opioid confirmed the results (Fig. 7). Furthermore, compared to WT B6 mice, both fentanyl and methadone reward (CPP) were significantly decreased in mE7M-B6 Mut mice (Fig. 9). In addition, fentanyl induced a dosedependent respiratory depression in WT B6 mice, however, the respiratory depression by fentanyl was significantly reduced in mE7M-B6 mice (Figs.lOA-lOD) with some differences between male and female mice. In mM0R-10Act / Actmice, morphine reward (CPP) and tolerance were increased compared to WT mice (Figs.l 1A-1 IE). These results were complemented with those from mE7M-B6 mut mice with reduced morphine CPP and tolerance, further suggesting that E7-associated variant, such as mM0R-10, facilitates morphine tolerance and reward.

[0226] Example 3. Discovering and validating Oprml exon 7-associated variants as novel therapeutic targets for mitigating adverse effects of clinically used mu opioids without altering analgesia in pain management.

[0227] The single-copy gene (OPRMP) encoding the mu opioid receptor (MOR) undergoes extensive alternative splicing, generating multiple splice variants. One set of 0PRM1 variants, exon 7-associated full-length 7TM C-terminal variants (E7 variants), contain a unique intracellular C-terminal tail with 30 amino acids encoded by E7 that are conserved from rodents to humans. E7 variants are abundantly expressed in central nervous system with distinct distributions. Increasing evidence described herein has indicated that E7 variants contribute to several mu opioid-induced adverse side-effects. Truncating E7-enocded C-terminal tails in mice (mE7M-B6) attenuated morphine tolerance and reward without effect on analgesia. An antisense oligonucleotide (ASO) targeting E7 variants also reduced morphine tolerance, and reduced morphine reward measured by conditioned place preference (CPP). These observations suggest that Oprml Wl variants mediate several adverse effects associated with clinically used mu opioids, and targeting E7 variants can diminish mu opioid-induced adverse effects but maintain mu opioid analgesic potency via other Oprml 7TM variants. The current studies further establish the role of E7 variants in mediating mu opioid-induced tolerance, reward and respiratory depression in mE7M-B6 or naive mice by newly developed rabbit monoclonal antibodies (RabmAb) that target E7 variants. Both the ASO and RabmAb attenuated morphine tolerance and reward in mice. Additionally, a new mouse model (mM0R-10-KI) in which only a single E7 variant, mM0R-10, is expressed showed enhanced morphine tolerance and reward, complementing those from mE7M-B6 mice. Together, these studies indicate that E7 variants represent novel therapeutic targets for mitigating adverse effects of clinically used mu opioids without altering analgesia in pain management. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method of activating a mu opioid receptor(s) (MOR), comprising administering to a subject in need of: a mu opioid receptor (MOR) agonist, and an inhibitor of an exon 7-associated mu opioid receptor (MOR) variant(s).

2. The method of claim 1, wherein the MOR agonist is morphine or analog thereof (e.g., oxycodone, heroin).

3. The method of claim 1, wherein the MOR agonist is fentanyl or analog thereof.

4. The method of claim 1, wherein the MOR agonist is methadone or buprenorphine.

5. The method of any one of claims 1-4, wherein the subject is in need of analgesia.

6. The method of any one of claims 1-4, wherein the subject is in need of substance (e.g., heroin) dependence or addiction treatment.

7. The method of any one of claims 1-6, wherein the subject is a human, and the MOR vari ant i s hMOR- 10.

8. The method of any one of claims 1-6, wherein the subject is a mouse, and the MOR variants are mMOR-lC, mM0R-10, mMOR-lU, and mMOR-lM.

9. The method of any one of claims 1-8, wherein the inhibitor is a nucleic acid, and the expression of the exon 7-associated MOR variant is reduced.

10. The method of claim 9, wherein the inhibitor is an antisense oligonucleotide (ASO).

11. The method of claim 9, wherein the inhibitor is siRNA or shRNA.

12. The method of any one of claims 1-8, wherein the expression of the exon 7-associated MOR variant is not reduced.

13. The method of any one of claims 1-8 and 12, wherein the inhibitor is an antibody or fragment thereof.

14. The method of claim 13, wherein the antibody or fragment specifically binds an epitope comprising or consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and / or SEQ ID NO:34.

15. The method of any one of claims 1-14, wherein the MOR agonist is administered orally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, or intrathecally.

16. The method of any one of claims 1-15, wherein the inhibitor is administered orally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, intrathecally, or intracerebroventricularly.

17. The method of any one of claims 1-16, wherein the MOR agonist and the inhibitor are co-administered (e.g., intravenously, subcutaneously, intramuscularly, or intrathecally).

18. A pharmaceutical composition, comprising: a mu opioid receptor (MOR) agonist, and an inhibitor of an exon 7-associated mu opioid receptor (MOR) variant(s), and a pharmaceutically acceptable carrier.

19. The composition of claim 18, wherein the MOR agonist is morphine or analog thereof.

20. The composition of claim 18, wherein the MOR agonist is fentanyl or analog thereof.

21. The composition of claim 18, wherein the MOR agonist is methadone.

22. The composition of any one of claims 18-21, wherein the MOR variant is hM0R-10.

23. The composition of any one of claims 18-21, wherein the MOR variants are mMOR-lC, mM0R-10, mMOR-lU and mMOR-lM.

24. The composition of any one of claims 18-23, wherein the inhibitor is a nucleic acid.

25. The composition of claim 24, wherein the inhibitor is an antisense oligonucleotide (ASO).

26. The composition of claim 24, wherein the inhibitor is siRNA or shRNA.

27. The composition of any one of claims 18-21, wherein the inhibitor is an antibody or fragment thereof.

28. The composition of claim 27, wherein the antibody or fragment thereof specifically binds an epitope comprising or consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and / or SEQ ID NO:34.

29. The composition of any one of claims 18-28, which is in liquid dosage form.

30. An isolated exon 7-specific antibody or fragment thereof, wherein the antibody or fragment binds exon 7 or homolog (e.g., exon O) domain of the exon 7-associated mu opioid receptor (MOR) variant(s).

31. The antibody or fragment of claim 30, wherein the antibody or fragment thereof specifically binds an epitope comprising or consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and / or SEQ ID NO:34.

32. The use of a composition according to any one of claims 18-29, or an isolated exon 7- specific antibody or fragment thereof according to any one of claims 30-31 to prepare a medicament for providing analgesia or the treatment of pain, opioid use disorder, or substance dependence in a subject.

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