Compositions and methods for using purified human RNA editing enzymes

Abstract

In an alternative embodiment, a method for eradicating or reducing the in vivo number of cancer stem cells is provided, comprising administering to an individual in need thereof an ADAR1 (adenosine deaminase associated with RNA1) inhibitor, wherein the ADAR1 inhibitor reduces or significantly reduces ADAR1 Nano-luc reporter activity in cell lines and human cancer stem cell assays. In an alternative embodiment, a method for inhibiting an RNA virus or retrovirus, optionally SARs-CoV-2 virus, is provided, comprising lentiviral ADAR1 overexpression, and in vivo administration, optionally intravenous (IV) administration, of lentiviral ADAR1 transduced stem cells, optionally wherein the stem cells are cord blood CD34+ cells or mesenchymal stromal cells.

Description

[Technical field]

[0001] Related Applications This PCT international application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. (USSN) 63 / 224,818, filed July 22, 2021. The foregoing application is expressly incorporated by reference herein in its entirety for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated by reference herein for all purposes.

[0002] Technical Field The present invention generally relates to molecular biology and medicine. In an alternative embodiment, a method for eradicating or reducing the in vivo number of cancer stem cells is provided, comprising administering an ADAR1 (adenosine deaminase associated with RNA1) inhibitor to an individual in need thereof, wherein the ADAR1 inhibitor reduces or significantly reduces ADAR1 Nano-luc reporter activity in cell lines and human cancer stem cell assays. In an alternative embodiment, a method for inhibiting RNA viruses or retroviruses, optionally SARs-CoV-2 viruses, is provided, comprising lentiviral ADAR1 overexpression and in vivo administration, optionally intravenous (IV) administration, of lentiviral ADAR1 transduced stem cells, optionally wherein the stem cells are cord blood CD34+ cells or mesenchymal stromal cells. [Background technology]

[0003] background Antiviral deamination by ADAR1 induces adenosine-to-inosine (A-to-I) editing that limits the replication of RNA viruses such as coronaviruses and influenza, as well as retroviruses such as HIV. Targeted base editing by ADAR1 has also emerged as a powerful means to introduce single-base changes into RNA to alter transcript susceptibility to splice acceptor sites and microRNA targeting, ultimately altering translation. Furthermore, Z alpha DNA binding by ADAR1 can alter the epigenome within selected Alu-containing regions, whereas Z alpha RNA binding can induce changes in transcript stability. Hyperediting by ADAR1 induces RNA modifications in survival and stem cell transcripts, lncRNAs and primary microRNAs, primarily in association with double-stranded RNA loops formed by Alu sequences, promoting therapy resistance in cancer stem cells as well as self-renewal of normal human hematopoietic stem cells.

[0004] As an innate immune antiviral deaminase, ADAR1 is transcriptionally activated following proinflammatory cytokine signaling via JAK2 / STAT and interferon α, β, and γ signaling. Thus, selective JAK2 and STAT3 inhibition prevents ADAR1 activation. Summary of the Invention [Means for solving the problem]

[0005] overview In an alternative embodiment, there is provided a method for inhibiting an RNA virus or retrovirus, optionally SARs-CoV-2 virus, optionally inhibiting an RNA virus or retrovirus in vivo, in an individual in need thereof, comprising lentiviral ADAR1 expression or overexpression, and in vivo administration, optionally intravenous (IV) administration, of lentiviral ADAR1 transduced stem cells, optionally wherein the stem cells are cord blood CD34+ cells or mesenchymal stromal cells.

[0006] In an alternative embodiment, there is provided a method for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need thereof, comprising delivering or administering an ADAR1 catalytic domain nanoprotein in vivo, optionally contained or formulated in a liposome, lipid nanoparticle (LNP) or nanoliposome, optionally comprising delivering the ADAR1 catalytic domain nanoprotein by intravenous administration or inhalation.

[0007] In an alternative embodiment, there is provided a method for inhibiting the replication of an RNA virus or retrovirus in vivo, optionally SARs-CoV-2 virus, optionally in an individual in need of inhibition of an RNA virus or retrovirus, optionally an RNA virus or retrovirus, comprising delivering or administering an ADAR1 full length nanoprotein in vivo, optionally wherein the ADAR1 full length nanoprotein is comprised in or formulated with a liposome, lipid nanoparticle (LNP) or nanoliposome, optionally wherein the ADAR1 full length nanoprotein is delivered or administered intravenously or by inhalation.

[0008] In an alternative embodiment, there is provided a method for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus in vivo, optionally in an individual in need thereof, comprising in vivo delivery or administration of an ADAR1 catalytic domain nanoprotein delivery, optionally an ADAR1 Z alpha domain deletion nanoprotein delivery, optionally wherein the ADAR1 Z alpha domain deletion nanoprotein is comprised in or formulated with a liposome, lipid nanoparticle (LNP) or nanoliposome, and optionally wherein the ADAR1 Z alpha domain deletion nanoprotein is delivered or administered intravenously or by inhalation.

[0009] In an alternative embodiment there is provided the use of lentiviral ADAR1 transduced stem cells, optionally umbilical cord blood CD34+ cells or mesenchymal stromal cells, for inhibiting an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need of inhibition of an RNA virus or retrovirus, comprising lentiviral ADAR1 expression or overexpression in vivo and in vivo administration, optionally intravenous (IV) administration.

[0010] In an alternative embodiment, there is provided a use of an ADAR1 catalytic domain nanoprotein contained in or formulated in a liposome, lipid nanoparticle (LNP) or nanoliposome, for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need of inhibition of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, comprising in vivo delivery or administration of the ADAR1 catalytic domain nanoprotein.

[0011] In an alternative embodiment, there is provided use of an ADAR1 full length nanoprotein contained in or formulated with a liposome, lipid nanoparticle (LNP) or nanoliposome for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need of inhibition of the RNA virus or retrovirus, comprising in vivo delivery or administration of the ADAR1 full length nanoprotein, optionally where the ADAR1 full length nanoprotein is delivered or administered intravenously or by inhalation.

[0012] In an alternative embodiment, there is provided use of an ADAR 1 Z alpha domain deleted nanoprotein contained in or formulated with a liposome, lipid nanoparticle (LNP) or nanoliposome for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need of inhibition of the RNA virus or retrovirus, comprising in vivo delivery or administration of the ADAR1 Z alpha domain deleted nanoprotein delivery, optionally where the ADAR1 Z alpha domain deleted nanoprotein is delivered or administered intravenously or by inhalation.

[0013] In an alternative embodiment, a method is provided for eradicating or reducing the in vivo number of cancer stem cells, comprising administering to an individual in need thereof an ADAR1 (adenosine deaminase associated with RNA1) inhibitor, wherein the ADAR1 inhibitor reduces or significantly reduces ADAR1 Nano-luc reporter activity in cell lines and human cancer stem cell assays.

[0014] In an alternative embodiment of the methods provided herein, - the ADAR1 inhibitor comprises a JAK2 inhibitor, optionally wherein the JAK2 inhibitor comprises fedratinib, or INREBIC™, or ruxolitinib, or JAKAFI™; -ADAR1 inhibitors include STAT3 inhibitors; -ADAR1 inhibitors include 8-aza-adenosine, nucleoside analogs or integrase inhibitors; -ADAR1 inhibitors include raltegravir (or ISENTRESS™) or dolutegravir (or TIVICAY™); - the ADAR1 inhibitor comprises a lentiviral shRNA ADAR1 knockdown vector; - the ADAR1 inhibitor comprises a lentiviral ADAR1 mutant vector; - the ADAR1 inhibitor comprises a lentiviral ADAR1 Z alpha domain deletion vector; -ADAR1 inhibitors include interferon inhibitor compounds; - the ADAR1 inhibitor comprises a lentiviral ADAR1 or a lentiviral ADAR1 shRNA; - the ADAR1 inhibitor comprises recombinant human full-length ADAR1 protein; - the ADAR1 inhibitor comprises a recombinant human ADAR1 catalytic domain protein; - the ADAR1 inhibitor comprises a recombinant human Z alpha domain deleted ADAR1 protein; and / or - the ADAR1 inhibitor comprises a JAK2 expression vector, optionally a retroviral or lentiviral JAK2 expression vector, optionally a retroviral or lentiviral JAK2 overexpression vector.

[0015] In an alternative embodiment, a method for identifying an ADAR1 agonist is provided, comprising contacting an ADAR1 Nano-luc reporter interferon-responsive cell line and an interferon cell line with a candidate ADAR1 agonist, optionally wherein the candidate ADAR1 agonist comprises recombinant human full-length ADAR1, optionally wherein the candidate ADAR1 agonist comprises recombinant human ADAR1 catalytic domain, optionally wherein the candidate ADAR1 agonist comprises recombinant human Z alpha domain deleted ADAR1, optionally wherein the candidate ADAR1 agonist comprises a lentiviral JAK2 overexpression vector.

[0016] In an alternative embodiment, a stably transduced human non-interferon responsive cell line is provided, comprising a lentiviral ADAR1 overexpression vector and a Nano-luc reporter within the cell line for the purpose of detecting RNA virus inhibition, where optionally the RNA virus is SARS-CoV-2 or influenza A or B.

[0017] In an alternative embodiment, a stably transduced human interferon responsive cell line is provided, comprising a lentiviral ADAR1 overexpression vector and a Nano-luc reporter within the cell line for the purpose of detecting RNA virus inhibition following infection with an RNA virus or retrovirus, optionally wherein the virus is SARS-CoV-2, or influenza A or B, or HIV.

[0018] In an alternative embodiment, there is provided a use of an ADAR1 inhibitor to eradicate or reduce the in vivo number of cancer stem cells, wherein the ADAR1 inhibitor is administered to an individual in need thereof, and optionally the ADAR1 inhibitor comprises a JAK2 inhibitor, and optionally the JAK2 inhibitor is selected from the group consisting of fedratinib, or INREBIC™, or ruxolitinib, or JAKAFI™; a STAT3 inhibitor; an 8-aza-adenosine, nucleoside analog, or an integrase inhibitor; raltegravir (or ISENTRESS™) or dolutegravir (or TIVICAY™); a retroviral or lentiviral shRNA ADAR1 knockdown vector; a retroviral or lentiviral ADAR1 mutant expression vector; a lentiviral ADAR1 Z alpha domain deletion vector; an interferon inhibitor compound; a lentiviral ADAR1 or lentiviral ADAR1 Uses are provided which include an shRNA; a recombinant human full-length ADAR1 protein; a recombinant human ADAR1 catalytic domain protein; a recombinant human Z alpha domain deleted ADAR1 protein; and / or a JAK2 expression vector, optionally a retroviral or lentiviral JAK2 expression vector, optionally a retroviral or lentiviral JAK2 overexpression vector.

[0019] In an alternative embodiment, an ADAR1 inhibitor for use in eradicating or reducing the in vivo number of cancer stem cells, wherein the ADAR1 inhibitor is administered to an individual in need thereof, and optionally the ADAR1 inhibitor comprises a JAK2 inhibitor, and optionally the JAK2 inhibitor is selected from the group consisting of fedratinib, or INREBIC™, or ruxolitinib, or JAKAFI™; a STAT3 inhibitor; an 8-aza-adenosine, nucleoside analog, or an integrase inhibitor; raltegravir (or ISENTRESS™) or dolutegravir (or TIVICAY™); a retroviral or lentiviral shRNA ADAR1 knockdown vector; a retroviral or lentiviral ADAR1 mutant expression vector; a lentiviral ADAR1 Z alpha domain deletion vector; an interferon inhibitor compound; a lentiviral ADAR1 or a lentiviral ADAR1 ADAR1 inhibitors for use in eradicating or reducing the in vivo numbers of cancer stem cells are provided, comprising shRNA; recombinant human full-length ADAR1 protein; recombinant human ADAR1 catalytic domain protein; recombinant human Z alpha domain deleted ADAR1 protein; and / or a JAK2 expression vector, optionally a retroviral or lentiviral JAK2 expression vector, optionally a retroviral or lentiviral JAK2 overexpression vector.

[0020] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0021] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] The drawings described herein are intended to illustrate exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims. [Brief description of the drawings]

[0024] [Figure 1-1]As discussed in more detail in Example 1 below, Figures 1A-1K show an exemplary process for expression and purification of recombinant human ADAR1 catalytic domain (hADAR1 CD) in a BJ2168 yeast expression system: Figure 1A graphically illustrates the hADAR1 catalytic domain (CD) codon optimization for expression in yeast. Figure 1B shows the hADAR1 catalytic domain (CD) amino acid sequence (SEQ ID NO: 1) with the colored (or light colored) amino acids deleted in the delta-loop construct. Figure 1C shows a schematic of an exemplary pEG(KT)GST-TEV-hADAR1 catalytic domain (CD) and pEG(KT)GST-TEV-hADAR1CD delta-loop vector map. Figure 1D shows a schematic of an exemplary galactose-inducible expression system. Figure 1E shows an image of a Coomassie blue stained α-ADAR1 Western blot confirming galactose-inducible expression of GST-tagged hADAR1 catalytic domain (CD). FIG. 1F shows an example workflow overview illustrating the steps involved in protein purification from yeast cell extract. FIG. 1G shows an image of a Coomassie blue stained gel showing successful cleavage of the GST tag by TEV enzyme. FIG. 1H shows an image of a silver stained gel demonstrating the purity of the hADAR1 catalytic domain (CD) protein product after the final purification step. FIG. 1I graphically shows data from size-exclusion chromatography of purified hADAR1 catalytic domain (CD) using a SUPERDEX 200 10 / 300 GL™ gel filtration column. FIG. 1J graphically shows data from protein mass determination of purified hADAR1 catalytic domain (CD) protein product by mass spectrometry. FIG. 1K graphically shows data from analytical ultracentrifugation of purified hADAR1 catalytic domain (CD) demonstrating the purity of the final protein product. [Figure 1-2] Same as above. [Diagram 2]As discussed in more detail in Example 1 below, Figures 2A-2C show an exemplary process for expression and purification of recombinant human full-length ADAR1 catalytic domain (hADAR1 CD) in a BJ2168 yeast expression system: Figure 2A shows a schematic of an exemplary p424 10xHis-tagged full-length ADAR1 vector map; Figure 2B shows a schematic of an exemplary galactose-inducible expression system; and Figure 1C graphically depicts data from a Coomassie blue stained gel confirming galactose-inducible expression of 10xHis-tagged full-length ADAR1. [Diagram 3] As discussed in more detail in Example 1 below, Figures 3A-3C show an exemplary nanoluciferase-based RNA editorase activity reporter assay in vitro: Figure 3A shows a schematic of an exemplary nanoluciferase reporter design. Figure 3B shows a schematic of an exemplary lentiviral nanoluciferase RNA editorase reporter expression vector. The top panel of Figure 3C graphically depicts a nanoluciferase activity assay showing concentration-dependence and specificity for ADAR1 editorase activity in HEK293T cells after co-transfection with FLAG-tagged ADAR1 constructs and NanoLuciferase reporter. The bottom panel of Figure 3C shows an image of an α-FLAG Western blot analysis demonstrating increased FLAG-ADAR protein levels. The top panel of Figure 3D shows a schematic of an exemplary nanoluciferase activity assay comparing ADAR1 RNA editorase activity in K562 cells after co-transduction of pCDH / ADAR1 with nanoluciferase reporter. Figure 3D bottom panel shows images of α-ADAR1 Western blot analysis demonstrating equal ADAR1 protein levels for all conditions (left), and RT-PCR showing equal expression of the NanoLuciferase reporter for all conditions, as well as in parental untransduced K562 cells as a control (right). [Figure 4]As discussed in more detail in Example 1 below, Figures 4A-B show exemplary in vitro nanoluciferase-based RNA editorase activity reporter assays: Figure 4A top panel graphically depicts a NanoLuciferase activity assay comparing ADAR1 RNA editorase activity in K562 cells following co-transduction of pCDH vector / ADAR1 with the NanoLuciferase reporter. Figure 4A bottom panel shows an image of an α-ADAR1 Western blot analysis demonstrating equal ADAR1 protein levels for all conditions (left), and RT-PCR showing equal expression of the NanoLuciferase reporter for all conditions, as well as in parental untransduced K562 cells as a control (right). FIG. 4B shows images from IVIS™ imaging of 6.5-week-old mice following neonatal intrahepatic transplantation with K562 cells co-transduced with pCDH / wild-type ADAR1 / editase-deficient ADAR1 E912A and a nanoluciferase reporter, demonstrating in vivo visualization of RNA editorase activity. [Diagram 5]As discussed in more detail in Example 1 below, Figures 5A-5D show exemplary assays for stable lentiviral overexpression of ADAR1 wild type (WT) and ADAR1 mutants following shADAR1 knockdown: Figure 5A graphically depicts expression levels of total ADAR1 (left) and ADAR1 p150 isoform (right) in TF1a cells following transduction with shSchramble and shADAR1, as shown by qPCR (normalized to HPRT). Figure 5B shows images of Western blot analysis showing protein levels of ADAR1 in TF1a cells following transduction with shSchramble and shADAR1. Figure 5C shows schematic diagrams of exemplary lentiviral expression vectors for HA-tagged shADAR1-resistant (shR) ADAR1 wild type, ADAR1 editase-deficient mutant E921A, ADAR1 DNA-binding domain-deficient mutant dZa, and ADAR1 mutant E912A dZa constructs. Figure 5D (left) graphically depicts a NanoLuciferase activity assay comparing ADAR1 RNA editorase activity in TF1a cells following co-transduction of pCDH / ADAR1 shR vector and NanoLuciferase reporter into the background of shRNA-mediated ADAR1 knockdown, and Figure 5D (right) graphically depicts an α-HA Western blot analysis demonstrating similar ADAR1 protein levels for all conditions. [Figure 6]As discussed in more detail in Example 1 below, Figures 6A-6D show data demonstrating the involvement of ADAR1 in the JAK / STAT pathway and demonstrating that JAK inhibitors such as ruxolitinib and fedratinib can be used as ADAR-1 inhibitors: Figure 6A graphically depicts ADAR1 p150 isoform expression levels in TF1a cells as shown by qPCR 16 hours after treatment with PBS (control) or interferon alpha (normalized to HPRT). Figure 6B depicts images of Western blot analysis of TF1a cells showing protein levels of ADAR1 and various members of the JAK / STAT pathway 16 hours after treatment with PBS (control) or interferon alpha. Figure 6C depicts images of Western blot analysis of secondary AML (patient 672) CD34+ cells showing protein levels of ADAR1, STAT3 and phospho-STAT3 Y705 16 hours after treatment with PBS (control), interferon alpha, beta or gamma. Figure 6D shows images of Western blot analysis of secondary AML (patient 255) CD34+ cells treated with FDA approved JAK2 inhibitors (ruxolitinib and fedratinib) compared to JAK3 inhibitor (FM-381) at concentrations of 1 nM, 10 nM and 100 nM. Like reference numbers in the various figures indicate like elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description In alternative embodiments, methods are provided for the purification and production of human functional antiviral RNA editing enzyme, ADAR1 (adenosine deaminase associated with RNA1) and related lentiviral vectors, editing reporters and compounds, as well as methods of use for antiviral compound discovery, stem cell expansion, inhibition of cancer stem cells and selective RNA base editing, and inhibition of RNA viruses, including SARS CoV-2 and retroviruses. In alternative embodiments, methods are provided for producing large amounts of recombinant human full-length ADAR1, Z alpha binding domain deleted ADAR1, and the catalytic domain of ADAR1.

[0026] In an alternative embodiment, methods are provided for purifying human full-length ADAR1, Z alpha domain deleted ADAR1 and the catalytic domain of human ADAR1.

[0027] In an alternative embodiment, a method is provided for producing lentiviral ADAR1 Nano-luc reporter transduced interferon responsive and non-responsive cell lines with ADAR1 overexpression and shRNA knockdown for the purpose of screening for antiviral compounds capable of inhibiting the replication of RNA viruses and retroviruses.

[0028] In an alternative embodiment, methods are provided for identifying ADAR1 antagonists, including lentiviral ADAR1 shRNA knockdown, mutant and Z alpha domain deleted ADAR1 vectors that can inhibit cancer stem cells.

[0029] In an alternative embodiment, methods are provided for detecting ADAR1 agonists, including lentiviral ADAR1 overexpression vectors that can enhance stem cell survival and self-renewal, and vectors with antiviral activity.

[0030] ADAR1 in vivo delivery and gene delivery vehicles In an alternative embodiment, the method provided herein includes inhibiting a virus, e.g., an RNA virus or retrovirus, including lentivirus or adeno-associated virus (AAV)-mediated ADAR1 overexpression, optionally SARs-CoV-2 virus, and optionally in vivo administration of a virus, e.g., lentivirus or AAV-ADAR1-transduced stem cell, optionally by intravenous (IV) administration, where the stem cell is optionally an umbilical cord blood CD34+ cell or a mesenchymal stromal cell. In an alternative embodiment, the viral vector is delivered to a cell or cells in vitro, ex vivo, or in vivo, e.g., as an ADAR1 delivery vehicle.

[0031] In alternative embodiments, the expression vehicle, vector, recombinant virus, or equivalent used to carry out the methods provided herein is or comprises an adeno-associated virus (AAV), a lentiviral vector, or an adenoviral vector; AAV serotypes AAV5, AAV6, AAV8, or AAV9; AAV derived from rhesus monkeys, or AAV derived from rhesus monkeys AAVrh.10hCLN2; organotropic AAV; and / or AAV capsid mutants or AAV hybrid serotypes. In alternative embodiments, AAV is engineered to increase efficiency in targeting specific cell types that are non-permissive to wild-type (wt) AAV, and / or to improve efficacy in infecting only the cell type of interest. In alternative embodiments, hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications including 1) transcapsid formation, 2) adsorption of bispecific antibodies to the capsid surface, 3) engineering of mosaic capsids, and / or 4) engineering of chimeric capsids. Methods for engineering the adeno-associated virus (AAV) capsid to increase the efficiency of targeting specific cell types that are non-permissive to wild-type (wt) virus and to improve the effectiveness of infecting only cell types of interest are well known in the art, see, e.g., Wu et al., Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006 Jul 7; Choi et al., Curr. Gene Ther. 2005 Jun;5(3):299-310.

[0032] For example, rhesus monkey-derived AAV AAVrh.10hCLN2 or its equivalent can be used, and rhesus monkey-derived AAV cannot be inhibited by pre-existing immunity in humans.See, for example, Sondhi et al., Hum Gene Ther.Methods.2012 Oct;23(5):324-35,Epub 2012 Nov 6;Sondhi et al., Hum Gene Ther.Methods.2012 Oct 17, which teaches that direct administration of AAVrh.10hCLN2 to the CNS of rats and non-human primates at doses scalable to humans has an acceptable safety profile and mediates significant payload expression in the CNS.

[0033] Since adeno-associated virus (AAV) is a common infectious agent in primates, and thus healthy primates harbor a large pool of AAV-specific neutralizing antibodies (NAb) that inhibit AAV-mediated gene transfer therapeutic strategies, the methods provided herein also include screening patient candidates for AAV-specific NAb prior to treatment, particularly with the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy.See, for example, Sun et al., J.Immunol.Methods.2013 Jan 31;387(1-2):114-20,Epub 2012 Oct11.

[0034] Any lentiviral vector can be used to practice the methods provided herein, e.g., to deliver cells, such as stem cells, expressing ADAR1 or ADAR1 in vitro, ex vivo, or in vivo, as described, e.g., in U.S. Pat. Nos. 11,299,752, 11,208,669, 11,078,495, 11,007,209, and 10,954,530.

[0035] Pharmaceutical Compositions and Formulations In alternative embodiments, the ADAR1 inhibitors, including drugs, vectors, liposomes, lipid nanoparticles (LNPs), nanoliposomes or nanoparticles used to carry out the methods provided herein, are formulated for administration by any or various means, including oral, parenteral, by inhalation spray, nasal, topical, intrathecal, intrathecal, intracerebral, epidural, intracranial or rectal. The ADAR1 inhibitors, including drugs, vectors, liposomes, lipid nanoparticles (LNPs), nanoliposomes or nanoparticles used to carry out the methods provided herein can further include pharma- ceutical acceptable carriers, adjuvants and vehicles. In alternative embodiments, the drugs provided herein and the therapeutic combinations of drugs used to carry out the methods provided herein are formulated for parenteral administration, including intrathecal, intracerebral or epidural (into the intrathecal, intracerebral, epidural space), subcutaneous, intravenous, intramuscular and / or intraarterial administration, including, for example, by injection route, but also various infusion techniques. Intra-arterial, intrathecal, intracranial, epidural, intravenous and other injections used in some embodiments may include administration via a catheter or pump, such as an intrathecal pump, or an implantable medical device, which may be an intrathecal pump or catheter.

[0036] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, are formulated according to routine procedure(s) compatible with the desired route of administration. In alternative embodiments, the drugs provided herein and the therapeutic combinations of drugs used to practice the methods provided herein are formulated or manufactured as lyophilisates, powders, lozenges, liposomes, lipid nanoparticles (LNPs), nanoliposomes, suspensions, solutions or emulsions in oily or aqueous vehicles and may include formulation agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0037] In alternative embodiments, ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, can be formulated as preparations for implantation or injection.Thus, for example, the compound can be formulated with suitable polymers or hydrophobic materials (for example, as emulsions in acceptable oils) or ion exchange resins, or as poorly soluble derivatives (for example, as poorly soluble salts).Alternatively, the active ingredient can be in powder form for constitution with suitable vehicle, for example, sterile pyrogen-free water, before use.For alternative and exemplary formulations suitable for each of these administration methods, see, for example, Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams&Wilkins, Philadelphia, Pa.

[0038] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, are formulated with sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of synthetic or vegetable origin, hydrogenated naphthalenes, etc. In alternative embodiments, the drugs provided herein and the therapeutic combinations of drugs used to practice the methods provided herein can be formulated in or with biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers, which can be useful excipients for controlling the release of the active compounds.

[0039] In alternative embodiments, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, is administered using parenteral delivery systems such as ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, intrathecal catheters, pumps and implants, and / or by using liposomes, lipid nanoparticles (LNP), nanoliposomes. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration. Formulations for inhalation administration may contain, for example, lactose as an excipient, or may be an aqueous solution containing, for example, polyoxyethylene-9-auryl ether, glycocholate and deoxycholate, or an oily solution for administration in the form of nasal drops, or a gel applied intranasally.

[0040] In an alternative embodiment, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, is administered intranasally.When administered by this route, the suitable dosage form is nasal spray or dry powder, as known to those skilled in the art.For example, nasal formulations can include conventional surfactants, generally non-ionic surfactants.When surfactants are used in nasal formulations, the amount present varies according to the specific surfactant selected, the specific mode of administration (e.g., drops or spray) and the desired effect.

[0041] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, are in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to known techniques using these suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol, or can be prepared as a lyophilized powder. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In alternative embodiments, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. In alternative embodiments, fatty acids such as oleic acid can also be used to prepare injectables. Formulations for intravenous administration can include solutions in sterile isotonic aqueous buffer solutions. If necessary, the formulation may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. When the compound is administered by infusion, it can be dispensed in a formulation that includes an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. When the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0042] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, further comprise aqueous and non-aqueous sterile injection solutions which may contain (or include) antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the body fluids of the intended recipient, and / or aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0043] In an alternative embodiment, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, is formulated for local administration, for example in the form of liquid, lotion, cream or gel.Local administration can be achieved by directly applying to the treatment area.For example, such application can be achieved by rubbing the formulation (such as lotion or gel) on the skin of the treatment area, or by applying or spraying the liquid formulation on the treatment area.

[0044] In alternative embodiments, ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, including bioimplants or bioimplant materials, can also be coated with a compound of the present invention or other compounds to improve interactions between cells and the implant.

[0045] In alternative embodiments, the ADAR1 inhibitor containing drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle contains minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0046] In an alternative embodiment, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, is formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0047] In alternative embodiments, ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, are included in oral formulations such as tablets, pills, troches, lozenges (see, for example, those described in U.S. Pat. No. 5,780,055), aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules or gel tabs, gels, jellies, syrups and / or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents, including sweeteners, flavorings, flavorings, colorings and preservatives, to provide a palatable preparation. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc. Tablets containing the active ingredient mixed with non-toxic pharma- ceutically acceptable excipients suitable for tablet manufacture may be acceptable. These excipients may be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used alone or with a wax.

[0048] In an alternative embodiment, formulations for oral use are hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium phosphate or kaolin, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0049] In an alternative embodiment, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, comprise aqueous suspensions that contain active materials mixed with excipients suitable for producing aqueous suspensions.Exemplary excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic, and dispersing or wetting agents such as natural phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0050] In alternative embodiments, ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, include oil suspensions, which can be formulated by suspending the active ingredient (e.g., the compound of the present invention) in vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil, or mineral oil, such as liquid paraffin. Oral suspensions may contain thickening agents, such as beeswax, hard paraffin or cetyl alcohol. Sweeteners and flavoring agents, such as those mentioned above, may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant, such as ascorbic acid.

[0051] In an alternative embodiment, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, includes an agent that controls the release of the compound, thereby providing a timed or sustained release compound.

[0052] In alternative embodiments, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle, is formulated or made as a multiparticulate and / or solid dispersion formulation comprising a hydrophobic matrix forming agent, e.g., a water-insoluble, non-swelling amphiphilic lipid, and a hydrophilic matrix forming agent, e.g., a meltable, water-soluble excipient, e.g., as described in U.S. Patent Application Publication No. 20080118560. In one embodiment, the ADAR1 inhibitors containing drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles are contained in tablets, pills, capsules, lozenges, etc., which contain any combination of the following: binders, such as starch, polyvinylpyrrolidone, gum tragacanth or gelatin; fillers, such as microcrystalline cellulose or lactose; disintegrants, such as crospovidone, sodium starch glycolate, cornstarch; lubricants, such as magnesium stearate, stearic acid, glyceryl behenate; lubricants, such as colloidal silicon dioxide and talc; sweeteners, such as sucrose or saccharin, aspartame, acesulfame-K; and / or flavors, such as peppermint, methyl salicylate or orange flavor.When the unit dosage form is a capsule, it can also contain a liquid carrier, such as fatty oil.

[0053] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles used to practice the methods provided herein, include (or are contained in or packaged in) a unit dosage formulation with a coating, e.g., a coating including sugar, shellac, sustained release and / or other enteric coating agent, or any pharma- ceutically pure and / or non-toxic agent.

[0054] In an alternative embodiment, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein, comprise (or are contained in or packaged in) unit dosage formulations, and each different compound of the composition or article of manufacture is contained in different layers of a pill, tablet or capsule, for example, with an outer base-soluble layer and an inner acid-soluble layer, as described, for example, in U.S. Patent No. 7,384,653. In an alternative embodiment, the therapeutic combination of drugs provided herein and drugs used to carry out the methods provided herein comprise (or are contained in or packaged in) unit dosage formulations, and each different compound of the composition or article of manufacture is contained in liquids or gels of different viscosities, as described, for example, in U.S. Patent Application Publication No. 20050214223. In alternative embodiments, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle used to practice the methods provided herein, comprises (or is contained in or packaged in) a unit dosage formulation with reduced abuse potential, including, for example, a bittering agent, a bright deterrent / indicator dye, or fine insoluble particulate matter, e.g., as described in U.S. Patent Application Publication No. 20040228802.

[0055] Carrier In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, used to carry out the methods provided herein, include or are formulated with or as aqueous or non-aqueous solutions, suspensions, emulsions and solids. Examples of non-aqueous solvents suitable for use as disclosed herein include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In alternative embodiments, aqueous carriers can include water, ethanol, alcohol / aqueous solutions, glycerol, emulsions and / or suspensions, including saline and buffered media. Oral carriers can be elixirs, syrups, capsules, tablets.

[0056] In an alternative embodiment, liquid carriers, including carriers for preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compounds, are used to manufacture or formulate ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, used to carry out the methods provided herein. The active ingredient can be dissolved or suspended in a pharma- ceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of both, or a pharma-ceutically acceptable oil or fat. The liquid carrier can contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, coloring agents, viscosity regulators, stabilizers or osmotic pressure regulators.

[0057] In alternative embodiments, the liquid carriers used to manufacture or formulate the compounds of the present invention include water (partially containing additives such as cellulose derivatives, or sodium carboxymethylcellulose solution as described above), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the carriers can also include oily esters such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid forms containing compounds for parenteral administration. Liquid carriers for pressurized compounds disclosed herein can be halogenated hydrocarbons or other pharmaceutically acceptable propellants.

[0058] In an alternative embodiment, the solid carrier is used to manufacture or formulate the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein, and includes solid carriers including substances such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicalcium phosphate, mannitol, etc. The solid carrier may further include one or more substances that act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet disintegrants, and may be encapsulating materials. In powders, the carrier may be a finely divided solid mixed with the finely divided active compound. In tablets, the active compound is mixed with a carrier having the necessary compression properties in suitable proportions and compressed into the desired shape and size. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form, such as powder or granules, mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactant or dispersant, as required, in a suitable machine. Molded tablets can be made by molding a mixture of powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets can be coated or scored as required, and can be formulated to provide slow or controlled release of the active ingredient therein, for example, using various percentages of hydroxypropylmethylcellulose to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0059] In alternative embodiments, parenteral carriers are used to manufacture or formulate ADAR1 inhibitors (including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods disclosed herein), and parenteral carriers suitable for use as disclosed herein include, but are not limited to, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also include, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

[0060] In alternative embodiments, carriers used to manufacture or formulate ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein, can be mixed as necessary with disintegrants, diluents, granulating agents, lubricants, binders, etc., using conventional techniques known in the art. The carriers can also be sterilized using methods that do not deleteriously react with the compounds, as is generally known in the art.

[0061] The present invention also provides articles of manufacture and kits that contain (include) ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles, used to carry out the methods provided herein, including pharmaceutical compositions and formulations. By way of example, the kit or article of manufacture can only include a container (such as a bottle) that contains a desired amount of a compound (or a pharmaceutical composition of a compound) as described herein. Such kits or articles of manufacture can further include instructions for using the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles as described herein. The instructions can be attached to the container or can be included in the package (such as a box or a plastic or foil bag) that holds the container.

[0062] The ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles used to practice the methods provided herein, can be delivered to the body by any method or protocol, including, for example, ex vivo "cell loading" with a therapeutic combination of drugs provided herein and drugs used to practice the methods provided herein, or can be targeted to a specific tissue or organ (e.g., muscle or brain), where the "loaded cells" are cells administered intramuscularly, or intrathecally, intracerebrally, or epidurally into the central nervous system (CNS), for example, as described in U.S. Patent No. 20050048002.

[0063] In an alternative embodiment, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle used to practice the methods provided herein, is first lyophilized and then suspended in a hydrophobic medium, including, for example, aliphatic, cyclic or aromatic molecules, as described, for example, in US 20080159984.

[0064] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles, used to carry out the methods provided herein, comprise pharmaceutically acceptable salts or are formulated as pharmaceutically acceptable salts.Pharmaceutically acceptable salts can comprise their suitable acid addition salts or base salts.In alternative embodiments, the compounds can be formulated as described in Berge et al, J Pharm Sci, 66, 1-19 (1977).

[0065] In alternative embodiments, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector or nanoparticle used to practice the methods provided herein, is selected from the group consisting of strong inorganic acids, such as mineral acids, e.g., hydrochloride, hydrobromide and hydroiodide, sulfuric acid, phosphoric acid, and the like. They are formulated as salts formed with hydrohalic acids such as sulfates, bisulfates, hemisulfates, thiocyanates, persulfates and sulfonic acids; with strong organic carboxylic acids such as alkane carboxylic acids of 1-4 carbon atoms, such as acetic acid, which is unsubstituted or substituted (e.g., by halogen); with saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; with hydroxy carboxylic acids, such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids, such as aspartic acid or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids, such as methane- or p-toluenesulfonic acid, which is unsubstituted or substituted (e.g., by halogen). The compounds of the present invention also include salts that are not pharma- ceutically acceptable, for example, salts where the salts may still be of value as intermediates in synthetic or analytical processes or protocols.

[0066] In alternative embodiments, the ADAR1 inhibitor, including drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector, or nanoparticle used to practice the methods provided herein, may be any acceptable salt, such as acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, or the like. , pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivorate, camphorate, undecanoate and succinate, organic sulfonic acids such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, hemisulfate, thiocyanate, persulfate, phosphoric acid and sulfonic acid. The pharmaceutical compositions disclosed herein can be prepared according to routine methods well known in the art. For example, see Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 2006. th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0067] In some embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles provided herein are provided in the form of pharma- ceutically acceptable salts that contain amines that are basic in nature and can react with inorganic or organic acids to form pharma- ceutically acceptable acid addition salts, e.g., such salts can be obtained with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as with para-toluenesulfonic acid, methanesulfonic acid, oxalic acid, paclitaxel, tetrahydrofuran ... or, where appropriate, such pharma- ceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionate, phosphate salts, and organic acids such as la-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, as well as related inorganic and organic acids. Salt, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate These include salts such as phosphate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, methylene-bis-b-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate laurylsulfonate.

[0068] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles used to practice the methods provided herein include compositions manufactured under "Good Manufacturing Practice" or GMP, or "current Good Manufacturing Practice" (cGMP) conditions.

[0069] Method of administration In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein, are administered by any or various means, including oral, parenteral, inhalation spray, nasal, topical, intrathecal, intrathecal, intracerebral, epidural, intracranial or rectal. The ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein can be administered with pharma- ceutically acceptable carriers, adjuvants and vehicles. In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to carry out the methods provided herein, are administered by injection routes, including various infusion techniques. Intra-arterial, intrathecal, intracranial, epidural, intravenous and other injections can include administration via a catheter or pump, such as an intrathecal pump, or an implantable medical device, which can be an intrathecal pump or catheter.

[0070] In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors, or nanoparticles used to practice the methods provided herein are administered by any known method or route, including intranasally, intramuscularly, intravenously, topically, or orally, or a combination thereof.

[0071] One embodiment includes an article of manufacture including a pharmaceutical composition or formulation, blister package, blister with lid or blister card or packet, clamshell, tray or shrink wrap, or kit comprising an ADAR1 inhibitor, including a drug, vector, or nanoparticle preparation provided herein for oral administration.

[0072] In alternative embodiments, all components may be in one blister package, blister with lid or blister card or packet, clamshell, tray or shrink wrap, or kit, but may be formulated as separate components, e.g., for topical, oral or local application. Each component may be packaged separately or may be formulated as one unit dose, e.g., in one tube (e.g., with a gel, lotion, etc.), ampoule, blister packet, etc.

[0073] Dosage In alternative embodiments, the ADAR1 inhibitors, including drugs, liposomes, lipid nanoparticles (LNPs), nanoliposomes, vectors or nanoparticles used to practice the methods provided herein, are formulated and administered in a variety of different dosages and treatment regimens depending on the disease or condition to be ameliorated, the condition of the individual being treated, the goals of treatment, etc., as routinely determined by a clinician, see, e.g., the latest edition of Remington: The Science and Practice of Pharmacy, Mack Publishing Co., supra.

[0074] In alternative embodiments, an effective amount of an ADAR1 inhibitor, including a drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector, or nanoparticle, including a stereoisomer, salt, hydrate, or solvate, used to practice the methods provided herein is about 0.1 mg to about 20.0 mg per kg of body weight of an individual or subject (e.g., a patient). In another variation, the effective amount is about 0.1 mg to about 10.0 mg per kg of body weight of an individual or subject (e.g., a patient), or about 0.1 mg to about 5.0 mg per kg of body weight of a patient. Alternatively, the effective amount is about 0.2 mg to about 2 mg per kg of body weight of an individual or subject (e.g., a patient).

[0075] In alternative embodiments, an effective amount of an ADAR1 inhibitor, including a drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector, or nanoparticle, used to practice the methods provided herein (e.g., as a solid dosage form, such as a pill, tablet, or lozenge) is about 0.1 mg to about 10.0 mg per kg of body weight, or about 0.1 mg to about 2.0 mg per kg of body weight, or about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, or about 0.5 mg per kg of body weight of the individual, subject, or patient. g, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, or about 1.0 mg, or an effective amount of a drug or compound provided herein, or a composition used to practice a method provided herein, is about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, or about 0.3 mg per kg of body weight.

[0076] In alternative embodiments, an effective amount of an ADAR1 inhibitor, including a drug, liposome, lipid nanoparticle (LNP), nanoliposome, vector, or nanoparticle, used to practice the methods provided herein (e.g., as a solid dosage form, such as a pill, tablet, or lozenge) is about 0.25 mg to about 100 mg, about 0.5 mg to about 200 mg, or about 1 mg to about 400 mg, or about 0.25 mg to about 100 mg, about 0.5 mg to about 200 mg, or about 1 mg to about 400 mg. A solid dosage form containing about 200 mg, or about 1 mg to about 250 mg, or a solid dosage form containing about 5 mg to about 150, or a solid dosage form (e.g., as a pill, tablet or lozenge) containing about 1 mg to about 75, or a solid dosage form containing about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg or about 75 mg.

[0077] Nanoparticles, Nanolipoparticles and Liposomes Nanoparticles, nanolipoparticles, vesicles and liposomal membranes are provided that contain the compounds and compositions used to implement the methods and embodiments provided herein, including, for example, ADAR1 inhibitors.Multilayer liposomes, lipid nanoparticles (LNPs), nanoliposomes that contain the compounds used to implement the embodiments provided herein are as described, for example, in US Patent Publication No. 20070082042 to Park et al.Multilayer liposomes, lipid nanoparticles (LNPs), nanoliposomes can be prepared with a particle size of about 200 to 5000 nm using a mixture of oily components that include squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithin to entrap the compositions used to implement the embodiments provided herein.

[0078] Liposomes, lipid nanoparticles (LNPs), nanoliposomes can be made using any method, including a method of producing liposomes encapsulating an active agent (e.g., an ADAR1 inhibitor, or any compound used to practice the methods provided herein), as described, for example, in U.S. Patent Application Publication No. 20070042031 to Park et al., which includes providing an aqueous solution in a first reservoir, providing an organic lipid solution in a second container, and then mixing the aqueous solution with the organic lipid solution in a first mixing area to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce liposomes encapsulating the active agent, and immediately thereafter mixing the liposome solution with a buffer to produce a dilute liposome solution.

[0079] In one embodiment, as described, e.g., in U.S. Patent Application Publication No. 20070110798, liposome compositions used to practice the embodiments provided herein include substituted ammonium and / or polyanions to target delivery of a compound provided herein or a compound used to practice a method provided herein to a desired cell type or organ, e.g., the brain.

[0080] Nanoparticles comprising the compounds provided herein are provided and used to practice the methods provided herein, e.g., in the form of active agent-containing nanoparticles (e.g., secondary nanoparticles), e.g., as described in U.S. Patent Application Publication No. 20070077286. In one embodiment, nanoparticles are provided that include a fat-soluble active agent, or a fat-solubilized water-soluble active agent for interaction with a divalent or trivalent metal salt, used to practice the embodiments provided herein.

[0081] In one embodiment, the compositions used to practice the embodiments provided herein can be formulated and delivered to mammalian cells in vivo, in vitro or ex vivo using solid lipid suspensions, for example, as described in U.S. Patent Application Publication No. 20050136121.

[0082] In alternative embodiments, the ADAR1-encoding nucleic acid or vector used to practice the methods provided herein is an ADAR1-encoding nucleic acid or vector used to perform the methods provided herein. The nucleic acid is delivered in vivo and may be in the form of or may include RNA, which may be formulated in lipid formulations or liposomes, and may be injected, for example, intramuscularly (IM) using formulations and methods such as those described in U.S. Patent Application No. US20210046173A1, which describes delivering to a subject (e.g., via intramuscular administration) a nucleic acid encoding ADAR1 that includes (or consists of, or consists essentially of) a nucleic acid encoding ADAR1, or an RNA (e.g., mRNA) that includes an open reading frame (ORF) encoding ADAR1, and optionally the RNA (or DNA-bearing expression vehicle) is coupled to a non-cationic lipid, including a mixture of cholesterol and DSPC, or a PEG-lipid, or a PEG-modified lipid, or an LNP, or an ionizable cationic lipid; or (13Z,16Z)-N,N-dimethyl-2-nonylhenicos-12,15-dien-1-amine, cholesterol, DSPC and PEG-2000. The PEG-lipid is formulated into a liposome, or lipid nanoparticle (LNP), or nanoliposome, containing a mixture of DMG. In an alternative embodiment, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA), or the PEG-lipid is PEG conjugated to dimyristoylglycerol (PEG-DMG). In an alternative embodiment, the LNP comprises 20-99.8 mol % of ionizable cationic lipid, 0.1-65 mol % of non-cationic lipid, and 0.1-20 mol % of PEG-lipid.In an alternative embodiment, the LNP comprises an ionizable cationic lipid selected from the group consisting of (2S)-1-({6-[(3)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16dien-1-amine; and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine; or a pharma-ceutically acceptable salt thereof, or a stereoisomer of any of the foregoing. In an alternative embodiment, the PEG-modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In alternative embodiments, the ionizable cationic lipids include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine. In one embodiment, the lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16dien-1-amine or N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, each of which is described in PCT / US2011 / 052328, the entire contents of which are incorporated herein by reference.In some embodiments, the non-cationic lipids of the present disclosure are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DO ... 1-Oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or a mixture thereof.

[0083] Delivery Vehicle In alternative embodiments, any delivery vehicle can be used to carry out the methods provided herein, for example, the compounds and compositions provided herein, or compounds used to carry out the methods provided herein, such as ADAR1 inhibitors, can be delivered to mammalian cells, for example, in vivo, in vitro or ex vivo. For example, delivery vehicles including polycations, cationic polymers and / or cationic peptides, such as polyethyleneimine derivatives, can be used, for example, as described in US Patent Publication No. 20060083737.

[0084] In one embodiment, a dried polypeptide-surfactant complex is used to formulate the compounds and compositions provided herein, or the compounds used to practice the embodiments provided herein, for example, as described in U.S. Patent Application Publication No. 20040151766.

[0085] In one embodiment, the ADAR1 inhibitor used to carry out the method provided herein can be applied to cells using a vehicle with cell membrane penetrating peptide conjugate, for example, as described in U.S. Patent No. 7,306,783, U.S. Patent No. 6,589,503.In one aspect, the composition to be delivered is conjugated to cell membrane penetrating peptide.In one embodiment, the composition to be delivered and / or delivery vehicle is conjugated to transport mediating peptide, for example, as described in U.S. Patent No. 5,846,743, which describes a transport mediating peptide that is highly basic and binds to polyphosphoinositides.

[0086] In one embodiment, electropermeabilization is used as a primary or secondary means to deliver compositions to cells, e.g., using any of the electroporation systems described in U.S. Pat. Nos. 7,109,034, 6,261,815, and 5,874,268.

[0087] Manufactured articles, preparations and kits For example, articles of manufacture, formulations, pharmaceutical compositions or preparations, and kits for practicing the methods provided herein are provided that include components and compositions for practicing the methods provided herein, including, for example, ADAR1 (adenosine deaminase associated with RNA1) inhibitors, such as, for example, fedratinib, or INREBIC™, or ruxolitinib, or JAK2 inhibitors such as JAKAFI™, or STAT3 inhibitors, or 8-aza-adenosine, nucleoside analogs or integrase inhibitors, or raltegravir (or ISENTRESS™) or dolutegravir (or TIVICAY™), or lentiviral shRNA ADAR1 knockdown vectors, or lentiviral ADAR1 mutant vectors, or lentiviral ADAR1 Z alpha domain deletion vectors, or interferon inhibitory compounds, or lentiviral ADAR1 or lentiviral ADAR1 shRNA, and the like; and, optionally, the articles of manufacture and kits can further include instructions for practicing the methods provided herein.

[0088] Any of the above aspects and embodiments may be combined with any other aspect or embodiment as disclosed herein in the Summary, Figures and / or Detailed Description sections.

[0089] As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0090] As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0091] As used herein, unless otherwise specified or clear from the context, the term "about" is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean.About (use of the term "about") can be understood to be within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0092] Unless otherwise specified or clear from the context, as used herein, the terms "substantially all," "substantially most of," "substantially all of" or "the majority of" encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more of a reference amount of a composition.

[0093] Each patent, patent application, publication, and document referenced herein is incorporated herein by reference in its entirety. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the above is pertinent prior art, nor does it constitute any admission as to the contents or dates of these publications or documents. Incorporation by reference of these documents alone should not be construed as a claim or admission that any portion of the contents of any document is believed to be essential to satisfy any national or local legal disclosure requirements for patent applications. The right is nevertheless reserved for reliance on any of such documents, where appropriate, to provide material believed essential to the subject matter claimed by an examining authority or court.

[0094] Modifications may be made to the above without departing from the basic aspects of the invention. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements are within the scope and spirit of the invention. The present invention illustratively described herein may suitably be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms. Thus, the terms and expressions used are used as terms of description rather than limitation, and equivalents of the features shown and described or portions thereof are not excluded, recognizing that various modifications are possible within the scope of the invention. Embodiments of the invention are described in the following claims.

[0095] The present invention will be further described with reference to the examples described herein, but it will be understood that the invention is not limited to such examples. EXAMPLES

[0096] Working Example Unless otherwise stated in the examples, all recombinant DNA techniques are carried out according to standard protocols, for example as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY, and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0097] Example 1: Purification and production of human functional antiviral RNA editing enzyme, ADAR1 and related lentiviral vectors, editing reporters and compounds This example demonstrates that methods as provided herein using exemplary methods are effective and can be used to eradicate or reduce the number of cancer stem cells in vivo.

[0098] method Human ADAR1 catalytic domain (CD) purification protocol Yeast strain: Saccharomyces cerevisiae BJ2168 (From Goode lab / Expression vector: pEG(KT) (URA / LEU minus)(from Zakarian lab / Princeton) Gene: Codon-optimized ADAR1-CD (GenScript)

[0099] Yeast Growth Medium (1 liter) Minimal selective medium (URA / LEU minus growth medium): 100 mM potassium phosphate pH 6.0, 6.7 g yeast nitrogen base, 1.92 g synthetic amino acid dropout mix (minus URA / LEU), 5.0 g ammonium sulfate, 10.0 g Succinic acid, 2% glycerol, 3% lactic acid, 2% raffinose. pH media to pH 6.0 using NaOH pellets and sterilize using a 0.22 um filter. 5x Induction Medium: 50g / L Select Yeast Extract, 100g / L Bacto-Tryptone, 10% D(+)-Galactose. Filter the medium using a 0.22um sterile filter.

[0100] purification buffer Yeast "popcorn" buffer: 20mM Hepes pH8.0, 150mM NaCl -Add fresh 1 mM PMSF and 1 Roche protease inhibitor cocktail pill YeastBuster lysis buffer (Novagen) -Add 1x THP, 1 mM 1,4-dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 Roche protease cocktail pill;

[0101] GST pull-down buffer GST binding buffer: 20 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton® X100, 5% glycerol, 1 mM DTT -Add fresh 1 mM PMSF and 1 Roche protease inhibitor cocktail pill High Salt Wash Buffer: 20mM Hepes pH 8.0, 500mM NaCl, 0.1% Triton X100, 5% Glycerol, 1mM DTT Low Salt Wash Buffer: 20mM Hepes pH 8.0, 75mM NaCl, 0.1% Triton X100, 5% Glycerol, 1mM DTT

[0102] HiTrap Heparin Column (GE) Buffer Buffer A: 20mM Hepes pH8.0, 75mM NaCl, 5% Glycerol, 1mM DTT, 0.22um Filtration Buffer B: 20mM Hepes pH8.0, 1M NaCl, 5% Glycerol, 1mM DTT, 0.22um filtration

[0103] Dialysis buffer / protein storage buffer: 20mM Hepes pH8.0, 150mM NaCl, 5% glycerol, 1mM DTT

[0104] Superdex 200 10 / 300 GL(GE) buffer Buffer A: 20mM Hepes pH8.0, 150mM NaCl, 5% glycerol, 1mM DTT, 0.22um filtration

[0105] Yeast growth procedure 1.) In a sterile 150 mL baffled flask, inoculate 15 mL of minimal selective medium (URA / LEU minus medium) with a large single yeast colony taken from a freshly streaked URA / LEU minus plate. Grow the culture overnight at 30 °C in a shaker set at 250 RPM. NOTE: This protocol is designed for preparing a 3 liter yeast culture. 2.) The next day, inoculate 800 mL of minimal selective medium in a 2 liter baffled flask with 1.5 mL of each starter culture. Grow the cultures overnight at 30° C. in a shaker set at 250 RPM. 3.) The next morning, measure the optical density (OD) of the cultures using a wavelength of 600 nm using a Spectronic 200 spectrophotometer. For ideal gene expression, OD 600 nm should be between 1.0 and 2.0. To induce protein expression, add 200 mL of 5x induction medium to each flask. Grow the yeast 24 hours at 30°C in a shaker set at 250 RPM. 4.) Harvest the yeast by spinning down the culture using a 500mL spin bottle at 5K RPM for 10 minutes. Repeat this step until all of the culture has been pelleted. 5.) Discard the supernatant and combine the pellets by resuspending them in yeast "popcorn" buffer. First, vortex and resuspend the pellets in 20 mL of buffer. Next, transfer the yeast to a 50 mL conical tube and re-pellet the yeast using a tabletop centrifuge at 5K RPM for 10 minutes. Discard the wash and save the pellet. 6.) Resuspend the pellet in yeast "popcorn" buffer containing 1 mM PMSF and one Roche protease inhibitor cocktail pill. For resuspension, use half of the pellet volume and thoroughly resuspend the yeast by vortexing (e.g., use 5 mL of popcorn buffer for 10 mL of wet yeast pellet). NOTE: The Roche protease inhibitor cocktail pill is pre-solubilized in popcorn buffer at this point prior to use throughout this protocol. 7.) Finally, "popcorn" the yeast by dropping the yeast drop-by-drop into liquid nitrogen in a 50 mL conical tube. Store the popcorn at -80°C for long term storage.

[0106] Yeast lysis protocol 1.) Begin the dissolution protocol by pre-cooling the ceramic mortar and pestle. To do so, place the mortar in a bucket with dry ice. Be sure to completely cover the sides of the mortar with dry ice. Additionally, fill the mortar with liquid nitrogen and allow the liquid nitrogen to dissipate completely before use. The pestle and spatula should also be pre-cooled by immersing them in liquid nitrogen prior to step 2. NOTE: Always use blue cryogenic gloves when handling liquid nitrogen. 2.) Next, place a small amount of yeast popcorn into the mortar and begin grinding by carefully manipulating the pestle in a circular motion until a very fine powder is formed, which may take up to 10 minutes per round of grinding. 3.) Transfer the ground yeast material, or "powder", to a new 50 mL conical tube. The yeast powder can be stored at -80°C before beginning the next step, if desired. 4.) Thaw yeast powder at room temperature. While the powder is thawing, activate 100mL of YeastBuster Lysis Buffer. To activate the buffer, add 1mL of 100x THP, 1mM DTT, 1mM PMSF, 100uL Cyanase (50U / uL) and 1 Roche protease inhibitor cocktail pill. 5.) Resuspend the powder in 100mL all-activated Yeast Buster Lysis Buffer. Do not shake or vortex the sample (this will damage the protein and create foam)! 6.) Place the sample in a 500mL beaker equipped with a stir bar and stir at 100 RPM for 1 hour at room temperature. 7.) Complete the lysis protocol by dounceing the sample 30 times in a pre-chilled Dounce homogenizer. This can be done by piecewise dounceing 15 mL of sample at a time. 8.) Finally, pellet unbroken cells and cell debris by spinning the extract in a refrigerated centrifuge at 15K RPM for 25 minutes. NOTE: Use tubes that can withstand these spin speeds (e.g., Nalgene centrifuge tubes). 9.) Discard the pellet and transfer the supernatant containing the protein to a new 50 mL conical tube. Measure the protein concentration of the extract and determine the total amount of protein in the sample. Quick-freeze the extract using liquid nitrogen for later use. Store the frozen extract at -80 °C.

[0107] GST-ADAR1-CD purification protocol 1.) Begin by thawing the protein extract at room temperature, preferably in water. Once thawed, dilute the extract with GST binding buffer to a total volume of 500 mL. Add fresh 1 mM DTT, 1 mM PMSF and 1 Roche protease inhibitor cocktail pill to the final diluted extract (starting material). Ideally, the final protein concentration of the extract is 1-2 mg / mL. 2.) While the extract is thawing, pre-equilibrate the GST resin with 150 mL (3 CV) of ice-cold GST binding buffer. 3.) The extract is then passed over the GST resin. This step takes several hours to complete, so keep the extract and flow-through on ice throughout the entire process. Additionally, save 100 uL of starting material and flow-through for anti-ADAR1 or anti-GST Western blot analysis. 4.) After the extract has passed through, begin washing the GST resin with the following wash buffer. a.) 100 mL GST binding buffer; b.) 100 mL high salt wash buffer; c. 100 mL GST binding buffer; d.) 100 mL low salt buffer. 5.) After the final wash, remove the stopcock and replace it with the small yellow cap and seal thoroughly with Parafilm. 6.) Add 50 mL of low salt buffer and carefully resuspend the GST resin and transfer all the beads evenly into 4 x 50 mL conical tubes (approximately 25 mL each tube). 7.) Fill each tube to 50 mL with low salt buffer containing 1 mM DTT. The final elution volume is 150 mL (approximately 3 CV). 8.) Add 300uL of TEV protease to each tube and incubate overnight at 4°C with gentle rotation. 9.) The next day, transfer all GST resin and solution back into the purification column. First release / open the top screw cap to release the internal pressure of the column. Then carefully open the bottom screw cap and capture the TEV eluted protein into a 250 mL beaker on ice. Save 160 uL of the elution for protein concentration measurement and Coomassie Blue staining analysis. 10.) Proceed with purification by pumping the elution onto 5 mL HiTrap heparin columns (GE) mounted in tandem on an AKTA Pure system (FPLC). Use the method script written for the AKTA Pure system. NOTE: The milligram volume obtained from step 9 will determine the number of heparin columns required to capture all ADAR1-CD protein in the sample. 11.) Determine the separation of ADAR1-CD and other contaminating proteins, including TEV protease, by visualizing the heparin fraction on an SDS-PAGE gel and staining it with Coomassie blue stain. NOTE: Determining protein separation is crucial before proceeding to the next step. 12.) Collect the FPLC fractions containing ADAR1-CD but not other contaminating proteins and transfer them to a 50 mL conical tube on ice. 13.) Because the sample is high salt (approximately 300 mM), the sample needs to be dialyzed in dialysis buffer using dialysis tubing pre-equilibrated with 1 mM EDTA pH 8.0 in dH20. Ensure that the dialysis tubing has a MWCO of 10-14 kDa or less. Dialyze overnight at 4°C in 3 liters of buffer with gentle stirring with a stir bar. 14.) The next morning, carefully transfer the sample to a new 50 mL conical tube on ice. At this point, the protein is ready to be concentrated using a Millipore Amicon-Ultra centrifugal spin column with a MWCO of 10-14 kDa. Spin the sample at 5,000 x g for 20 minutes. After 20 minutes, remove the sample from the centrifuge and carefully pipette the protein solution to prevent unwanted protein aggregation. Continue spinning the sample until a final volume of 500 uL is achieved. 15.) Measure the protein concentration of the concentrated sample, save 20uL for further SDS-PAGE analysis, and flash freeze the remainder of the sample in liquid nitrogen. At this point the sample is ready for downstream application. Note: Alternatively, after the concentration step the sample can be applied to a Superdex 200 10 / 300 GL™ gel filtration column to further purify the protein.

[0108] Design of nanoluciferase reporters In vivo RNA editor reporter constructs The reporter is called NanoLuc and contains the following DNA sequence: [ka] was generated by subcloning into the pCDH-EF1-T2A-copGFP lentiviral expression plasmid (CD521A-1, SBI Systems Biosciences). Forward primer: GP17015XbaI / NanoLuc (5'-ctagtctagactagccaaggtgagcgcgtca-3') (SEQ ID NO: 3) and reverse primer GP17023 NotI / NanoLuc The following was used to generate the amplification of the NanoLuc sequence: 5'-atagtttagcggccgccagaatgcgttc gcacag-3' (SEQ ID NO: 4).

[0109] After amplification, the pCDH-EF1-T2A-copGFP vector was digested with restriction enzymes XbaI and NotI. Ligation of the above sequence in frame into XbaI / NotI digested pCDH-EF1-T2A-copGFP generated a NanoLuc reporter responsive to RNA editase activity. A [TAG] stop codon is upstream of nanoluciferase, and in response to RNA editing of adenosine to inosine, the codon is translated as [TGG], relieving the stop codon block and inducing expression of the reporter nanoluciferase. A housekeeping elongation factor 1 alpha (EF1) promoter drives expression of the reporter. Oligonucleotide primers were synthesized by Eton Bioscience (San Diego, CA). Validation of the NanoLuc reporter was completed using both restriction enzyme analysis and DNA sequencing.

[0110] In vivo visualization of ADAR1 editase activity by nanoluc reporter Transduction ATCC-derived K562 cells were first transduced with control, ADAR1 WT or ADAR1 E912A mutant vectors and stably maintained. These stable lines were then co-transduced with an equal MOI of the ADAR1 NanoLuc reporter lentivirus. The cells were then passaged and stably maintained before transplantation into mice.

[0111] Implantation and imaging Immunocompromised RAG2- / -yc- / - mice were bred and housed at the Sanford Consortium vivarium following IACUC approved protocols. Neonatal pups (P2-P3) were intrahepatically transplanted with 100,000 K562 cells transduced with either pCDH, ADAR1 WT or ADAR1 E912A vectors and ADAR1 NanoLuc reporter (all). Mice were monitored and weighed weekly from P21 onwards. Mice with >20% weight loss (approximately 7 weeks of age) compared to non-transplanted controls were imaged by IVIS Lumina Imaging System. Promega NANOLUC™ substrate was prepared 40x (in sterile PBS) and administered intraperitoneally in a volume (ul) equivalent to 10x mouse body weight (g). Mice were euthanized after imaging.

[0112] Figure legend Figure 1. Expression and purification of recombinant human ADAR1 catalytic domain (hADAR1 CD) in the BJ2168 yeast expression system. (A) hADAR1 CD codon optimization for expression in yeast. (B) hADAR1 CD amino acid sequence. Colored amino acids are deleted in the Δloop construct. (C) pEG(KT)GST-TEV-hADAR1 CD and pEG(KT)GST-TEV-hADAR1 CD Δloop vector maps. (D) Schematic of the galactose-inducible expression system. (E) Coomassie blue staining and α-ADAR1 Western blot confirmed galactose-inducible expression of GST-tagged hADAR1 CD. (F) Workflow showing the steps involved in protein purification from yeast cell extracts. (G) Coomassie blue staining indicating successful cleavage of the GST tag by TEV enzyme. (H) Silver staining demonstrating the purity of the hADAR1 CD protein product after the final purification step. (I) Size-exclusion chromatography of purified hADAR1 CD using a Superdex200 10 / 300 GL gel filtration column.

[0113] (J) Protein mass determination of purified hADAR1 CD protein product by mass spectrometry. (K) Analytical ultracentrifugation of purified hADAR1 CD demonstrating the purity of the final protein product.

[0114] Figure 2. Expression and purification of recombinant human full-length ADAR1 in the BJ2168 yeast expression system. (A) p424 10xHis-tagged full-length ADAR1 vector map. (B) Schematic of the galactose-inducible expression system. (C) Coomassie blue staining confirming galactose-inducible expression of 10xHis-tagged full-length ADAR1.

[0115] Figure 3. Nanoluciferase-based RNA editorase activity reporter assay in vitro. (A) Schematic of the nanoluciferase reporter design. The reporter was designed with a UGA stop codon between the promoter sequence and the nanoluciferase sequence (Herbert sequence). In the absence of A-to-I editing in the cell, the stop codon before the nanoluciferase sequence prevents its transcription. Hence, there is no signal. In the presence of ADAR1, the stop codon is edited via the A-to-I RNA editorase activity of ADAR1, thereby no longer preventing the transcription of the nanoluciferase sequence. Thus, there is a luminescent signal that can be detected and quantified. (B) Lentiviral nanoluciferase RNA editorase reporter expression vector. (C) Top panel NanoLuciferase activity assay showing concentration dependency and specificity for ADAR1 editase activity in HEK293T cells after co-transfection with FLAG-tagged ADAR1 construct and NanoLuciferase reporter. Bottom panel α-FLAG Western blot analysis demonstrates increased FLAG-ADAR protein levels. (D) Top panel NanoLuciferase activity assay comparing ADAR1 RNA editorase activity in K562 cells after co-transduction of pCDH / ADAR1 with the NanoLuciferase reporter. Bottom panel α-ADAR1 Western blot analysis demonstrates equal ADAR1 protein levels for all conditions (left) and RT-PCR shows equal NanoLuciferase reporter expression for all conditions as well as in parental untransduced K562 cells as a control (right).

[0116] Figure 4. Nanoluciferase-based RNA editorase activity reporter assay in vivo. (A) Top panel NanoLuciferase activity assay comparing ADAR1 RNA editorase activity in K562 cells after co-transduction of pCDH vector / ADAR1 with NanoLuciferase reporter. Bottom panel α-ADAR1 Western blot analysis demonstrates equal ADAR1 protein levels for all conditions (left) and RT-PCR shows equal NanoLuciferase reporter expression for all conditions as well as in parental untransduced K562 cells as control (right). (B) IVIS™ imaging of 6.5 week old mice after neonatal intrahepatic transplantation with K562 cells co-transduced with pCDH / wild type ADAR1 / editase-deficient ADAR1 E912A and nanoluciferase reporter demonstrates in vivo visualization of RNA editorase activity.

[0117] Figure 5. Stable lentiviral shRNA-mediated knockdown of ADAR1, and stable lentiviral overexpression of ADAR1 wild-type and ADAR1 mutants after shADAR1 knockdown. (A) Expression levels of total ADAR1 (left) and ADAR1 p150 isoform (right) in TF1a cells after transduction with shSchramble and shADAR1, as shown by qPCR (normalized to HPRT), confirmed efficient (90%) shRNA-mediated knockdown of ADAR1. (B) Protein levels of ADAR1 in TF1a cells after transduction with shSchramble and shADAR1, as shown by Western blot analysis, demonstrate efficient (90%) shRNA-mediated knockdown of ADAR1. (C) Lentiviral expression vectors of HA-tagged shADAR1-resistant (shR) ADAR1 wild-type, ADAR1 editase-deficient mutant E921A, ADAR1 DNA-binding domain-deficient mutant dZa, and ADAR1 mutant E912A dZa constructs. (D) Nanoluciferase activity assay comparing ADAR1 RNA editorase activity in TF1a cells after co-transduction of pCDH / ADAR1 shR vector and NanoLuciferase reporter into a background of shRNA-mediated ADAR1 knockdown (left). α-HA Western blot analysis demonstrates similar ADAR1 protein levels for all conditions.

[0118] Figure 6. Involvement of ADAR1 in the JAK / STAT pathway and JAK inhibitors as potential ADAR1 inhibitors. (A) ADAR1 p150 isoform expression levels in TF1a cells shown by qPCR 16 hours after treatment with PBS (control) or interferon alpha (normalized to HPRT). (B) Western blot analysis of TF1a cells showing protein levels of ADAR1 and various members of the JAK / STAT pathway 16 hours after treatment with PBS (control) or interferon alpha. (C) Western blot analysis of secondary AML (patient 672) CD34+ cells showing protein levels of ADAR1, STAT3 and phospho-STAT3 Y705 16 hours after treatment with PBS (control), interferon alpha, beta or gamma. (D) Western blot analysis of secondary AML (patient 255) CD34+ cells treated with FDA-approved JAK2 inhibitors (ruxolitinib and fedratinib) compared with a JAK3 inhibitor (FM-381) at concentrations of 1 nM, 10 nM, and 100 nM.

[0119] Although several embodiments of the invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A composition for use in a method for inhibiting an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need thereof, the method comprising: A composition comprising lentiviral ADAR1 expression or overexpression and in vivo administration, optionally intravenous (IV) administration, of said composition, optionally wherein said stem cells are cord blood CD34+ cells or mesenchymal stromal cells.

2. 1. A composition for inhibiting the replication of an RNA virus or retrovirus, optionally SARs-CoV-2 virus, in vivo, optionally in an individual in need thereof, wherein the composition comprises an ADAR1 catalytic domain nanoprotein, and wherein the composition is delivered or administered in vivo to the individual in need thereof; Optionally, the ADAR1 catalytic domain nanoprotein is contained in or formulated into a liposome, lipid nanoparticle (LNP), or nanoliposome, and optionally, the composition is delivered by intravenous administration or inhalation.

3. A composition for inhibiting the replication of an RNA virus or retrovirus, optionally the SARS-CoV-2 virus, in vivo, and optionally inhibiting an RNA virus or retrovirus, in an individual in need thereof, which composition comprises an ADAR1 full-length nanoprotein, wherein said composition is delivered or administered in vivo to said individual in need of in vivo delivery or administration, optionally wherein said ADAR1 full-length nanoprotein is contained in liposomes or formulated with liposomes, and optionally wherein said composition is delivered or administered by intravenous administration or inhalation, a composition.

4. A composition for inhibiting the replication of an RNA virus or retrovirus, optionally the SARS-CoV-2 virus, in vivo, and optionally inhibiting an RNA virus or retrovirus, in an individual in need thereof, which composition comprises an ADAR1 Z alpha domain deletion nanoprotein delivery, wherein said composition is delivered or administered in vivo to said individual in need of in vivo delivery or administration, optionally wherein said ADAR1 Z alpha domain deletion nanoprotein is contained in liposomes or formulated with liposomes, and optionally wherein said composition is delivered or administered by intravenous administration or inhalation, a composition.

5. A composition for eradicating or reducing the in vivo number of cancer stem cells, which composition comprises an ADAR1 (adenosine deaminase related to RNA 1) inhibitor, wherein said composition is administered to an individual in need of administration, and wherein said ADAR1 inhibitor decreases or significantly decreases ADAR1 Nano-luc reporter activity in a cell line and / or a human cancer stem cell assay, a composition.

6. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a JAK2 inhibitor.

7. The composition according to claim 6, wherein the JAK2 inhibitor comprises fedratinib, or INREBIC (trademark), or ruxolitinib, or JAKAFI (trademark).

8. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a STAT3 inhibitor.

9. The composition according to claim 5, wherein the ADAR1 inhibitor comprises 8-aza-adenosine, nucleoside analogs or integrase inhibitors.

10. The composition according to claim 5, wherein the ADAR1 inhibitor comprises raltegravir (or ISENTRESS (trademark)) or dolutegravir (or TIVICAY (trademark)).

11. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a lentiviral shRNA ADAR1 knockdown vector.

12. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a lentiviral ADAR1 mutant vector.

13. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a lentiviral ADAR1 Z alpha domain deletion vector.

14. The composition according to claim 5, wherein the ADAR1 inhibitor comprises an interferon inhibitory compound.

15. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a lentiviral ADAR1 or lentiviral ADAR1 shRNA.

16. The composition according to claim 5, wherein the ADAR1 inhibitor comprises recombinant human full-length ADAR1.

17. The composition according to claim 6, wherein the ADAR1 inhibitor comprises a recombinant human ADAR1 catalytic domain.

18. The composition according to claim 5, wherein the ADAR1 inhibitor comprises recombinant human Z alpha domain deletion ADAR1.

19. The composition according to claim 5, wherein the ADAR1 inhibitor comprises a JAK2 expression vector, optionally a retroviral or lentiviral JAK2 expression vector, optionally a retroviral or lentiviral JAK2 overexpression vector.

20. the ADAR1 inhibitor is formulated or manufactured as a parenteral formulation, an aqueous solution, a liposome, an injectable solution, a tablet, a pill, a lozenge, a capsule, a caplet, a spray, a sachet, an inhalant, a powder, a lyophilized powder, an inhalant, a patch, a gel, a geltab, a nanosuspension, a nanoparticle, a nanoliposome, a microgel, a pellet, a suppository, or any combination thereof; 20. The composition of any one of claims 5 to 19, wherein the drug delivery device or article of manufacture is or comprises an implant, if desired.

21. 20. The composition of any of claims 5 to 19, wherein the ADAR1 inhibitors are formulated or manufactured together in one parenteral formulation, one aqueous solution, one liposome, one injectable solution, one lyophilized powder, one feed, one food, one dietary supplement, one pellet, one lozenge, one liquid, one elixir, one aerosol, one inhalant, one adhesive, one spray, one powder, one lyophilized powder, one patch, one tablet, one pill, one capsule, one gel, one geltab, one lozenge, one caplet, one nanosuspension, one nanoparticle, one nanoliposome, one microgel, or one suppository.

22. 20. The composition of any of claims 5 to 19, wherein the ADAR1 inhibitor is formulated in a unit dosage ranging from 0.1 mg to about 1 gram, optionally formulated as an immediate release or controlled release formulation.

23. A composition for eradicating or reducing the in vivo number of cancer stem cells in an individual in need thereof, comprising an ADAR1 inhibitor; Optionally, the ADAR1 inhibitor is: - a JAK2 inhibitor, optionally wherein the JAK2 inhibitor is fedratinib, or INREBIC™, or ruxolitinib, or JAKAFI™; a STAT3 inhibitor; an 8-aza-adenosine, nucleoside analog, or integrase inhibitor; raltegravir (or ISENTRESS™) or dolutegravir (or TIVICAY™); a retroviral or lentiviral shRNA ADAR1 knockdown vector; a retroviral or lentiviral ADAR1 mutant expression vector; a lentiviral ADAR1 Z alpha domain deletion vector; an interferon inhibitory compound; a lentiviral ADAR1 or lentiviral ADAR1 A composition comprising shRNA; recombinant human full-length ADAR1 protein; recombinant human ADAR1 catalytic domain protein; recombinant human Z alpha domain deleted ADAR1 protein; and / or a JAK2 expression vector, optionally a retroviral or lentiviral JAK2 expression vector, optionally a retroviral or lentiviral JAK2 overexpression vector.

24. 1. A composition comprising a stably transduced human non-interferon responsive cell line, comprising a lentiviral ADAR1 overexpression vector and a Nano-luc reporter within the cell line for the purpose of detecting RNA virus inhibition, wherein optionally the RNA virus is SARS-CoV-2 or influenza A or B.

25. 1. A composition comprising a stably transduced human interferon-responsive cell line, comprising a lentiviral ADAR1 overexpression vector and a Nano-luc reporter within the cell line for the purpose of detecting RNA virus inhibition following infection with an RNA virus or retrovirus, wherein optionally the virus is SARS-CoV-2, or influenza A or B, or HIV.

26. A method for identifying an ADAR1 agonist, comprising contacting an ADAR1 Nano-luc reporter interferon-responsive cell line and an interferon cell line with a candidate ADAR1 agonist.

27. The method according to claim 26, wherein the candidate ADAR1 agonist comprises recombinant human full-length ADAR1.

28. The method according to claim 26, wherein the candidate ADAR1 agonist comprises a recombinant human ADAR1 catalytic domain.

29. The method according to claim 26, wherein the candidate ADAR1 agonist comprises recombinant human Z alpha domain-deleted ADAR1.

30. The method according to claim 26, wherein the candidate ADAR1 agonist comprises a retrovirus or lentivirus JAK2 overexpression vector.

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