Endonuclease v-assisted fluorescence-based assays for human adenosine deaminase acting on RNA 1 (hadar1) and methods thereof

Abstract

Assays and methods of use thereof are provided. Exemplary embodiments include endonuclease V-assisted fluorescence-based assays for hADAR1 and methods of use thereof, including but not limited to screening hADAR1 activity and screening small molecule hADAR1 inhibitors. A method to convert, detect, and / or quantify the adenosine-to-inosine (A-to-I) editing by hADAR1 into a readable signal is provided, the method including mixing a fluorophore-labeled short RNA substrate with hADAR1 under conditions such that the hADAR1 deaminates adenosine at the target site to inosine on the RNA; denaturing the hADAR1; adding an endonuclease V and at least one of magnesium and manganese; and denaturing the endonuclease V under conditions such that inosine-containing RNA is cleaved to produce a readable fluorescence signal.

Description

[0001] Docket No.: 021413 / WO

[0002] ENDONUCLEASE V-ASSISTED FLUORESCENCE-BASED ASSAYS FOR HUMAN ADENOSINE DEAMINASE ACTING ON RNA 1 (hADARI) AND METHODS THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority from U.S. Provisional Application Serial No. 63 / 778,806 filed on 27 March 2025, which is incorporated herein by reference in its entirety.

[0005] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under GM144075 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] MATERIAL INCORPORATED BY REFERENCE

[0008] The Sequence Listing, which is a part of the present disclosure, includes a computer-readable form comprising nucleotide and / or amino acid sequences of the present invention (file name “021413-WO_2026-03027_Sequence-Listing” created on 27 March 2026; 17,374 bytes). The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

[0009] FIELD

[0010] The present disclosure generally relates to hADARI activity assays and their methods of use.

[0011] BACKGROUND

[0012] Human Adenosine Deaminase Acting on RNA 1 (hADARI) is proven as a promising biological target for cancer therapy. However, the lack of a suitable and convenient assay to determine the adenosine-to-inosine (A-to-l) editing in vitro is a major limitation in identifying small molecule inhibitors / drugs for hADARI by high-throughput screening. Costly HEK celldependent editing assays are difficult to handle, require careful observation, are prone to growth variation in cells, and are time-consuming. Conventional and commercially available ADAR activity assay kits are solely dependent on binding activity.Docket No.: 021413 / WO

[0013] BRIEF DESCRIPTION OF THE DISCLOSURE

[0014] Among the various aspects of the present disclosure is the provision of endonuclease V-assisted cleavage-based and / or fluorescence-based assays for hADARI and methods of use thereof, including but not limited to screening small molecule hADARI inhibitors.

[0015] In an aspect of the present disclosure, a method to detect the A-to-l editing or deamination activity of ADAR is provided. The method comprising: providing an RNA sample, wherein the RNA sample comprises a fluorescent probe; adding a DNA guide to the RNA sample; adding an endonuclease V (EndoV) and at least one of Mg and Mn to the RNA sample to form a reaction mixture; adding salt to the reaction mixture; and detecting and quantifying a fluorescence-based signal from the fluorescent probe, wherein a strength of the fluorescence-based signal is based on a concentration of inosine residues in the RNA sample.

[0016] In some embodiments, the fluorescent probe comprises an RNA hairpin probe. In certain embodiments, the RNA hairpin probe comprises a fluorophore.

[0017] In some embodiments, providing the RNA sample is selected from: providing an RNA oligomer mixture comprising RNA oligomers containing one or more inosine residues; and providing an RNA oligomer mixture comprising RNA oligomers and hADARI, wherein the hADARI incorporates inosine into each RNA oligomer via hADARI -mediated adenosine-to-inosine editing.

[0018] In some embodiments, the DNA guide is selected from an unlabeled DNA guide and a labeled DNA guide. In certain embodiments, the labeled DNA guide comprises at least one of a fluorophore, a quencher, and a large functional group.

[0019] In some embodiments, the EndoV is a histidine-tagged, maltose-binding proteincontaining fusion protein.

[0020] In some embodiments, the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV). In certain embodiments ,the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

[0021] In some embodiments, adding the EndoV cleaves the RNA oligomer into RNA fragments, and wherein the RNA fragments subsequently dissociates from the RNA oligomer, resulting in a detectable change to the fluorescence signal of the fluorescent probe.Docket No.: 021413 / WO

[0022] In some embodiments, the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

[0023] In another aspect of the present disclosure, a method of detecting A-to-l editing or deamination activity of ADAR in a high-throughput inhibitor screen is provided. The method comprising: adding hADARI to each candidate molecule of a small molecule library to form a respective candidate mixture; adding an RNA hairpin probe to each respective candidate mixture; adding a DNA guide to each respective candidate mixture; adding EndoV and at least one of Mg and Mn to each candidate mixture; adding a stop agent to each candidate mixture; and reading an RNA fluorescence-based signal of each candidate mixture such that a fluorescence signal of each candidate mixture indicates the hADARI activity of each respective candidate molecule of the small molecule library.

[0024] In some embodiments, the RNA hairpin probe comprises a fluorophore.

[0025] In some embodiments, the small molecule library is provided as a homogeneous plate assay.

[0026] In some embodiments, the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV). In certain embodiments, the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

[0027] In some embodiments, the stop agent comprises a salt.

[0028] In some embodiments, the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

[0029] In a further aspect of the present disclosure, a method to detect A-to-l editing or deamination activity of ADAR is provided. The method comprising: providing an RNA sample comprising a fluorescent probe and a quencher probe; adding an endonuclease V (EndoV) and at least one of Mg and Mn to the RNA sample to form a reaction mixture; and detecting and quantifying a fluorescence-based signal from the fluorescent probe, wherein a strength of the fluorescence-based signal is based on a concentration of inosine residues in the RNA sample.

[0030] In some embodiments, providing the RNA sample is selected from: providing an RNA oligomer mixture comprising RNA oligomers containing one or more inosine residues; andDocket No.: 021413 / WO

[0031] providing an RNA oligomer mixture comprising RNA oligomers and hADARI, wherein the hADARI incorporates inosine into each RNA oligomer via hADARI -mediated adenosine-to-inosine editing.

[0032] In some embodiments, the EndoV is a histidine-tagged, maltose-binding proteincontaining fusion protein.

[0033] In some embodiments, the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV). In certain embodiments, the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

[0034] In some embodiments, adding the EndoV cleaves the RNA oligomer into RNA fragments, and wherein the RNA fragments subsequently dissociate from the RNA oligomer, resulting in a detectable change to the fluorescence signal of the fluorescent probe.

[0035] In some embodiments, the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

[0036] In an additional aspect of the present disclosure, a method of detecting A-to-l editing or deamination activity of ADAR in a high-throughput inhibitor screen is provided. The method comprising: adding hADARI to each candidate molecule of the small molecule library to form a respective candidate mixture; adding an RNA hairpin probe to each respective candidate mixture; adding EndoV and at least one of Mg and Mn to each respective candidate mixture; and reading an RNA fluorescence of each candidate mixture such that a fluorescence signal of each candidate mixture indicates the hADARI activity of each respective candidate molecule of the small molecule library.

[0037] In some embodiments, the RNA hairpin probe comprises a fluorophore and a quencher.

[0038] In some embodiments, the small molecule library is provided as a homogeneous plate assay.

[0039] In some embodiments, the EndoV is a histidine-tagged, maltose-binding proteincontaining fusion protein.

[0040] In some embodiments, the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV). In certain embodiments, the EndoV isDocket No.: 021413 / WO

[0041] selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

[0042] In some embodiments, the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

[0043] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Those of skill in the art will understand that the drawings described herein are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0046] FIG. 1 is a schematic showing the cellular process of transcription and translation, which includes post-transcription modification of RNAs by RNA-modifying enzymes that change structure and function of the RNAs.

[0047] FIG. 2 is a schematic showing adenosine deaminases acting on RNAs (ADARs) that catalyze adenosine-to-inosine (A-to-l) editing on the RNAs.

[0048] FIG. 3 is a schematic showing how A-to-l editing demarks self RNA from non-self. FIG. 4 is a schematic showing how hADARI from cancer cells performs A-to-l editing, leading to escape from immunotherapy and the human immune system, making hADARI a promising biological target in cancer.

[0049] FIG. 5 is a schematic showing existing strategies and limitations for hADARI assays, which include cell-based assays, in vitro binding-dependent assays, and in silico screening.

[0050] FIG. 6 is a schematic representing the endonuclease V-assisted fluorescence polarization assay of the present disclosure.

[0051] FIG. 7A is a schematic and associated graph of RNA substrate S1a with successful A-to-l editing. The incorporation of G instead of A in PCR corresponds to Inosines in Sanger Sequencing.

[0052] FIG. 7B is a schematic and associated graph of RNA substrate S2a with successful A-to-l editing. The incorporation of G instead of A in PCR corresponds to Inosines in Sanger Sequencing.Docket No.: 021413 / WO

[0053] FIG. 7C is a schematic and associated graph of RNA substrate S3a with successful A-to-l editing. The incorporation of G instead of A in PCR corresponds to Inosines in Sanger Sequencing.

[0054] FIG. 8A is a table showing hADARI concentration-dependent editing assay with S1 to determine the optimized enzyme-to-substrate ratio. A 1:1 molar ratio of substrate-to-enzyme is found to be good for the assay at 20 nM substrate concentration.

[0055] FIG. 8B is a graph of the percentage of editing vs. hADARI concentration (nM). FIG. 9 is an illustration of an EndoV-cleaved fluorescence dequenching assay to track A-to-l editing by ADAR1. The substrate is optimal for both hADARI and EndoV.

[0056] FIG. 10 is a gel assay for optimizing the cleavage reaction with S2a derived short hairpin RNA for A-RNA and l-RNA, showing almost quantitative specific cleavage of the redesigned short hairpin l-RNA substrates by human EndoV at the target site.

[0057] FIG. 11 A is a schematic representing the goal of using strategies to enable the human immune system to identify cancer.

[0058] FIG. 11 B is a schematic representing a plate-based assay to quantify A-to-l editing, wherein high editing provides low fluorescence polarization and low editing provides high fluorescence polarization.

[0059] FIG. 11 C is a graph showing fluorescence polarization (mP) vs. EndoV concentration, for both A-RNA+EndoV and l-RNA+EndoV.

[0060] FIG. 12 is a schematic representing the in vitro RNA deamidation of ADAR1.

[0061] FIG. 13A is a schematic representing the degradation of hEndoV by proteinase K. FIG. 13B is a gel assay result showing degradation of hEndoV by proteinase K.

[0062] FIG. 14A is a schematic of FQ_BDF2 (EndoFluor substrate).

[0063] FIG. 14B is a gel assay result showing degradation of A-RNA and l-RNA versions of FQ_BDF2 (EndoFluor substrate) in the presence of Mg, and Mn.

[0064] FIG. 15 is a gel assay result showing degradation of A-RNA and l-RNA versions of FQ_BDF2 (EndoFluor substrate) in the presence of Mn.

[0065] FIG. 16 is a gel assay result showing EndoFluor (deamination / cleavage) on FQ_BDF2. 1 = A-RNA + hEndoV, 2 = A-RNA + hADARI + hEndoV, 3 = l-RNA, 4 = l-RNA + hADARI + hEndoV, 5 = A-RNA + hADARI + hEndoV, 6 = l-RNA + hADARI + hEndoV. Results show that hADARI is heat deactivated after deamination, and hADARI not deactivated after deamination.Docket No.: 021413 / WO

[0066] FIG. 17A is a graph of AF / Fi vs. temperature at reading for various samples. AF = Fluorescence of the sample - Fluorescence of the A-RNA control (A-RNA without hADARI and hEndoV, Fi).

[0067] FIG. 17B is a graph of AF / Fivs. urea concentration. AF = Fluorescence of the sample - Fluorescence of the A-RNA control (A-RNA without hADARI and hEndoV, Fi).

[0068] FIG. 18A is a graph of the cleavage signal with increasing hEndoV concentration for both A-RNA and l-RNA.

[0069] FIG. 18B shows an Endonuclease V-assisted fluorescence dequenching assay with 20 nM 5’-Cy5-BDF2-BHQ-3’ substrate and eEndoV (AD = hADARI, EV = eEndoV, nontreated = after the cleavage reaction, the overnight incubated samples, 2M Urea = overnight incubated samples given urea to a final 2M cone.)

[0070] FIG. 19A is a gel assay showing BDF2 cleavage with 35 nM 5’-FAM labeled BDF2 (A / I-RNA), 750 nM eEndoV, 0.5 mM MnCI2.

[0071] FIG. 19B is a gel assay showing one-pot deamination / cleavage reaction of A-RNA performed with 100 nM RNA, 300 nM hADARI, 300 nM eEndoV.

[0072] FIG. 19C is a gel assay showing one-pot deamination / cleavage reaction of l-RNA performed with 100 nM RNA, 300 nM hADARI, 300 nM eEndoV.

[0073] FIG. 20A is a cleavage assay with 50 nM BDF2 A & l-RNA incubated with eEndoV at different concentrations with / without guide DNAs at 100 nM concentration, 10 mM MgCI2as cofactor.

[0074] FIG. 20B is a cleavage assay with 50 nM BDF2 A & l-RNA incubated with hEndoV282 at different concentrations with / without guide DNAs at 100 nM concentration, 10 mM MgCI2as cofactor.

[0075] FIG. 20C is a cleavage assay with 50 nM BDF2 A & l-RNA incubated with hEndoV309 at different concentrations with / without guide DNAs at 100 nM concentration, 10 mM MgCI2as cofactor.

[0076] FIG. 21A is a graph of an endonuclease cleavage-based fluorescence polarization assay with eEndoV in the presence of guide DNA, G31 at buffer pH 7.5. (A = A-RNA, AD = hADARI, EV = EndoV, 8-azaN RNA = 8-azanebularine RNA, 8-azaN = 8-azanebularine) FIG. 21 B is a graph of an endonuclease cleavage-based fluorescence polarization assay with eEndoV in the absence of guide DNA at pH 7.5. (A = A-RNA, AD = hADARI , EV = EndoV, 8-azaN RNA = 8-azanebularine RNA, 8-azaN = 8-azanebularine).Docket No.: 021413 / WO

[0077] FIG. 21 C is a graph of an endonuclease cleavage-based fluorescence polarization assay with hEndoV309 in the absence of guide DNA at pH close to 9.5. (A = A-RNA, AD = hADARI, EV = EndoV, 8-azaN RNA = 8-azanebularine RNA, 8-azaN = 8-azanebularine)

[0078] DETAILED DESCRIPTION OF THE DISCLOSURE

[0079] The present disclosure is based, at least in part, on the discovery that small molecule inhibitors of hADARI can be effectively, efficiently, and affordably screened with quantitative editing via the EndoV-based fluorescence polarization assays disclosed herein.

[0080] The adenosines in human RNA are frequently edited into Inosines (A-to-l editing) by human adenosine deaminases acting on RNAs (hADARs). This editing plays a crucial role in the normal physiological functions of the cell, including distinguishing normal cellular RNA from viral RNA. Impaired editing is implicated in neurodegenerative diseases and autoimmune disorders. Further, several cancers exploit the hADARI as an immune checkpoint blockade. The hADARI knockdown studies on cancer cells exhibit their sensitization to immunotherapy, making it a suitable biological target for cancer drug development. However, the lack of a suitable high throughput screening (HTS) assay to track the A-to-l editing activity is a major drawback to screening for hADARI inhibitors. Current methods rely on the expression of ADAR to correct a premature stop codon in the mRNA of a fluorescent reporter gene at the target edit site in mammalian cells as a proxy for ADAR activity. This method is laborious, costly, difficult to handle, and time-consuming. In vitro activity assays with recombinant ADARs either report only on RNA binding, but not editing, or require a costly, multi-step RNA sequencing approach to determine the editing percentage. In light of these current limitations, we have developed a convenient, HTS-compatible Endonuclease V (EndoV) based fluorescence polarization assay to determine A-to-l editing quantitatively. The work herein employs EndoV to selectively cleave hADARI deaminated Inosine-containing RNA (l-RNA) carrying a fluorophore at one of the ends near the cleavage site, thus producing a differential fluorescence polarization value corresponding to the editing.

[0081] Even though hADARI is proven as a promising biological target for cancer therapy, the lack of a suitable, convenient assay to determine the A-to-l editing in vitro is a major limitation to screening for small molecule inhibitors / drugs for hADARI by high-throughput screening. The costly HEK cell-dependent editing assays are difficult to handle. They requireDocket No.: 021413 / WO

[0082] careful observation, are prone to growth variation in cells, and are time-consuming. The commercially available ADAR activity assay kits are dependent on the binding activity instead of the editing activity. The developed homogenous, convenient, one-pot EndoV-assisted cleavage assay can produce a direct A-to-l editing-dependent fluorescence polarization signal. Therefore, the developed assay overcomes all the above limitations to develop as a toolkit to track the A-to-l RNA editing in research and development studies. Further, employing the developed assay at HTS solves a critical problem of the lack of commercially valuable, therapeutic hADARI inhibitors.

[0083] Exemplary embodiments describe a cleavage-based assay. In some embodiments, at least one DNA guide is used. In some embodiments, the cleavage assays are developed as fluorescence-based assays, including fluorescence polarization (FP)-based assays and dequenching-based assays. In certain embodiments, the cleavage assay is an FP-based assay and includes usage of DNA guides. These DNA guides can be used, for example, to avoid the non-specific cleavage by EndoV enzymes and / or to increase the signal-to-noise ratio of the assay.

[0084] In some embodiments, the cleavage assays include one or more denaturation steps. In certain embodiments, denaturation steps include treatment of the reaction mixture with at least one of Proteinase K and Urea. In some embodiments, the cleavage assays rely on quenching the reaction by adding a stop agent (e.g., salt at an effective concentration) and / or by heating the reaction mixture as described herein. In certain embodiments, the cleavage assay is a dequenching-based assay and includes an hours-long (e.g., overnight) incubation of the reaction mixture at or near room temperature prior to obtaining a fluorescence signal from the reaction mixture corresponding to a respective RNA sample.

[0085] Exemplary methods are described herein, including cleavage- and fluorescencebased assays for evaluating synthetic RNA strands and hADARI deaminated RNA, as well as small molecule library screening. Evaluation includes but is not limited to detecting ADAR1 (e.g., hADARI) enzymatic activity in a homogenous format, determining detecting A-to-l editing, quantifying inosine residue presence (e.g., as an amount or concentration, as relatively compared to a control RNA sample and / or as relatively compared to another RNA sample or samples), and quantifying ADAR1 activity (e.g., as an amount or concentration, as relatively compared to a control RNA sample and / or as relatively compared to another RNA sample or samples).Docket No.: 021413 / WO

[0086] Exemplary embodiments describe a cleavage-based assay. In some embodiments, at least one DNA guide is used. In some embodiments, the cleavage assays are developed as fluorescence-based assays, including fluorescence polarization (FP)-based assays and dequenching-based assays. In certain embodiments, the cleavage assay is an FP-based assay and includes usage of DNA guides. These DNA guides can be used, for example, to avoid the non-specific cleavage by EndoV enzymes and / or to increase the signal-to-noise ratio of the assay.

[0087] In some embodiments, the cleavage assays include one or more denaturation steps. In certain embodiments, denaturation steps include treatment of the reaction mixture with at least one of Proteinase K and Urea. In some embodiments, the cleavage assays rely on quenching the reaction by adding a stop agent (e.g., salt at an effective concentration) and / or by heating the reaction mixture as described herein. In certain embodiments, the cleavage assay is a dequenching-based assay and includes an hours-long (e.g., overnight) incubation of the reaction mixture at or near room temperature prior to obtaining a fluorescence signal from the reaction mixture corresponding to a respective RNA sample.

[0088] Exemplary methods are described herein, including cleavage- and fluorescencebased assays for evaluating synthetic RNA strands and hADARI deaminated RNA, as well as small molecule library screening. Evaluation includes but is not limited to detecting ADAR1 (e.g., hADARI) enzymatic activity in a homogenous format, determining detecting A-to-l editing, quantifying inosine residue presence (e.g., as an amount or concentration, as relatively compared to a control RNA sample and / or as relatively compared to another RNA sample or samples), and quantifying ADAR1 activity (e.g., as an amount or concentration, as relatively compared to a control RNA sample and / or as relatively compared to another RNA sample or samples).

[0089] MODULATION AGENTS

[0090] As described herein, gene and / or associated protein expression has been implicated in various diseases, disorders, and conditions. As such, modulation of gene and protein expression can be used for treatment of such conditions. A modulation agent can modulate response, such as by inducing or inhibiting gene and / or protein expression signaling. Modulation can comprise modulating protein expression on cells, modulating the quantity of gene / protein expressing cells, or modulating the quality of gene / protein expressing cells.Docket No.: 021413 / WO

[0091] Modulation agents can be any composition or method that modulates expression on cells. For example, a modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. As another example, the modulation can be the result of gene editing.

[0092] A modulation agent can be an antibody (e.g., a monoclonal antibody). A modulating agent can be an agent that induces or inhibits progenitor cell differentiation into gene / protein expressing cells.

[0093] SIGNAL REDUCTION, ELIMINATION, OR INHIBITION BY SMALL MOLECULE INHIBITORS, SHRNA, SIRNA, ORASOS

[0094] As described herein, a modulation agent can be used for use in various therapies, such as to reduce / elim inate or enhance / increase expression signals. For example, a modulation agent can be a small molecule inhibitor, a short hairpin RNA (shRNA), or a short interfering RNA (siRNA). As another example, RNA (e.g., long noncoding RNA (IncRNA)) can be targeted with antisense oligonucleotides (ASOs) as a therapeutic. Processes for making ASOs targeted to RNAs are well known; see e.g., Zhou et al. 2016 Methods Mol Biol.

[0095] 1402:199-213. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0096] INHIBITING AGENT

[0097] As described herein, inhibitors or antagonists (e.g., antibodies, fusion proteins, small molecules) can reduce or prevent expression / activity / signaling. An inhibiting agent can be any agent that can inhibit activity and / or signaling, decrease protein level, downregulate protein and / or gene expression, or knockdown gene expression.

[0098] As an example, an inhibiting agent can inhibit pathway signaling.

[0099] For example, an inhibiting agent can be an antibody, such as a murine antibody, a humanized murine antibody, or a human antibody.

[0100] As another example, an inhibiting agent can be a fusion protein. For example, the fusion protein can be a decoy receptor for a target protein. Furthermore, the fusion protein can comprise a mouse or human Fc antibody domain fused to the ectodomain of the R1 of interest.

[0101] As another example, an inhibiting agent can be an antisense oligonucleotide (ASO, which has been shown to be a potent and specific inhibitor of various signaling pathways.

[0102] As another example, an inhibiting agent can be an inhibitory protein that serves as an antagonist. For example, the inhibiting agent can be an antagonist viral protein.Docket No.: 021413 / WO

[0103] As another example, an inhibiting agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA).

[0104] As another example, an inhibiting agent can be a single guide RNA (sgRNA).

[0105] Methods for preparing an inhibiting agent (e.g., an agent capable of inhibiting pathway signaling) can comprise construction of a protein / Ab scaffold containing the natural receptor as a neutralizing agent; developing inhibitors of the receptor “down-stream”; or developing inhibitors of the production “up-stream”.

[0106] Inhibition can be performed by genetic modification in a subject or genetically modifying a subject to reduce or prevent expression of a target gene (or genes), such as through the use of CRISPR-Cas9 or analogous technologies, wherein, such modification reduces or prevents expression / activity / signaling.

[0107] Inhibition of agents as described herein can be determined by standard pharmaceutical procedures in assays or cell cultures for determining the ICso. The half maximal inhibitory concentration (ICso) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. The ICso is a quantitative measure that indicates how much of a particular inhibitory substance (e.g., pharmaceutical agent or drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. The biological component could be an enzyme, cell, cell receptor, or microorganism, for example. ICso values are typically expressed as molar concentration. ICso is generally used as a measure of antagonist drug potency in pharmacological research. ICso is comparable to other measures of potency, such as ECso for excitatory drugs. ECso represents the dose or plasma concentration required for obtaining 50% of a maximum effect in vivo. ICso can be determined with functional assays or with competition binding assays.

[0108] CHEMICAL AGENT

[0109] Examples of assay agents / reagents are described herein, and can include pharmaceutically acceptable salts, solvates, polymorphs, tautomers, prodrugs, analogs, stereoisomers thereof, and / or optionally substituted analog thereof.

[0110] The formulas, analogs, and R groups can be optionally substituted or functionalized with one or more groups independently selected from the group consisting of hydroxyl; Ci-walkyl hydroxyl; amine; Ci-wcarboxylic acid; Ci-wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched Ci-walkyl amine; heterocyclyl;Docket No.: 021413 / WO

[0111] heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, 0, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci- alkyl hydroxyl; amine; Ci- carboxyl; Ci- carboxylic acid; Ci-wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched Ci-walkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci-walkyl hydroxyl; amine; Ci-wcarboxylic acid; Ci-wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched Ci- alkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms. Any of the above can be further optionally substituted.

[0112] The term “imine” or “imino”, as used herein, unless otherwise indicated, can include a functional group or chemical compound containing a carbon-nitrogen double bond. The expression “imino compound”, as used herein, unless otherwise indicated, refers to a compound that includes an “imine” or an “imino” group as defined herein. The “imine” or “imino” group can be optionally substituted.

[0113] The term “hydroxyl”, as used herein, unless otherwise indicated, can include -OH. The “hydroxyl” can be optionally substituted.

[0114] The terms “halogen” and “halo”, as used herein, unless otherwise indicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.

[0115] The term “acetamide”, as used herein, is an organic compound with the formula CH3CONH2. The “acetamide” can be optionally substituted.

[0116] The term “aryl”, as used herein, unless otherwise indicated, include a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The “aryl” can be optionally substituted.Docket No.: 021413 / WO

[0117] The terms “amine” and “amino”, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group. The “amine” or “amino” group can be optionally substituted.

[0118] The term “alkyl”, as used herein, unless otherwise indicated, can include saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straightchain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated Ci -10 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1 -pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1 -hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1 -pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated. The “alkyl” can be optionally substituted.

[0119] The term “carboxyl”, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (-COOH). The “carboxyl” can be optionally substituted.

[0120] The term “carbonyl”, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double-bonded to an oxygen atom (C=O). The “carbonyl” can be optionally substituted.

[0121] The term “alkenyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated. The “alkenyl” can be optionally substituted.

[0122] The term “alkynyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated. The “alkynyl” can be optionally substituted.

[0123] The term “acyl”, as used herein, unless otherwise indicated, can include a functionalDocket No.: 021413 / WO

[0124] group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (-OH) group. The “acyl” can be optionally substituted.

[0125] The term “alkoxyl”, as used herein, unless otherwise indicated, can include O-alkyl groups wherein alkyl is as defined above and 0 represents oxygen. Representative alkoxyl groups include, but are not limited to, -O-methyl, -O-ethyl, -O-n-propyl, -O-n-butyl, -O-n-pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, -O-sec-butyl, -O-isobutyl, -O-tert-butyl, -O-isopentyl, -O-2-methylbutyl, -O-2-methylpentyl, -O-3-methylpentyl, -0-2,2-dimethylbutyl, -0-2,3-dimethylbutyl, -0-2,2-dimethylpentyl, -0-2,3-dimethylpentyl, -0-3,3-dimethylpentyl, -0-2,3,4-trimethylpentyl, -O-3-methylhexyl, -0-2,2-dimethylhexyl, -0-2,4-dimethylhexyl, -0-2,5-dimethylhexyl, -0-3,5-dimethylhexyl, -O-2,4dimethylpentyl, -0-2-methylheptyl, -0-3-methylheptyl, -O-vinyl, -O-allyl, -0-1-butenyl, -0-2-butenyl, -0-isobutylenyl, -0-1 -pentenyl, -0-2-pentenyl, -0-3-methyl-1-butenyl, -O-2-methyl-2-butenyl, -O-2,3-dimethyl-2-butenyl, -0-1 -hexyl, -O-2-hexyl, -0-3-hexyl, -O-acetylenyl, -O-propynyl, -0-1-butynyl, -0-2-butynyl, -0-1 -pentynyl, -0-2-pentynyl and -0-3-methyl-1-butynyl, -0-cyclopropyl, -O-cyclobutyl, -O-cyclopentyl, -O-cyclohexyl, -O-cycloheptyl, -O-cyclooctyl, -0-cyclononyl and -O-cyclodecyl, -O-CH2-cyclopropyl, -O-CH2-cyclobutyl, -O-CH2-cyclopentyl, -O-CH2-cyclohexyl, -O-CH2-cycloheptyl, -O-CH2-cyclooctyl, -0- CH2-cyclononyl, -O-CH2-cyclodecyl, -O-(CH2)2-cyclopropyl, -O-(CH2)2-cyclobutyl, -O-(CH2)2-cyclopentyl, -O-(CH2)2-cyclohexyl, -O-(CH2)2-cycloheptyl, -O-(CH2)2-cyclooctyl, -O-(CH2)2-cyclononyl, or -O-(CH2)2-cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated. The “alkoxyl” can be optionally substituted.

[0126] The term “cycloalkyl”, as used herein, unless otherwise indicated, can include an aromatic, a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 1 to 10 carbon atoms (e.g., 1 or 2 carbon atoms if there are other heteroatoms in the ring), preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1 ,3-cyclohexadienyl, -1 ,4-cyclohexadienyl, -cycloheptyl, -1 ,3-cycloheptadienyl, -1 ,3,5-cycloheptatrienyl, -cyclooctyl, and cyclooctadienyl. The term “cycloalkyl” also can include -lower alky l-cycloalky I, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alkyl-cycloalkyl groups include, but are not limited to, -CH2-cyclopropyl, -CH2-cyclobutyl, -CH2-cyclopentyl, -CH2-Docket No.: 021413 / WO

[0127] cyclopentadienyl, -CH2-cyclohexyl, -CH2-cycloheptyl, or-CH2-cyclooctyl. The “cycloalkyl” can be optionally substituted. A “cycloheteroalkyl”, as used herein, unless otherwise indicated, can include any of the above with a carbon substituted with a heteroatom (e.g., O, S, N).

[0128] The term “heterocyclic” or “heteroaryl”, as used herein, unless otherwise indicated, can include an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of 0, S, and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1 H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e. , at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated. The “heterocyclic” can be optionally substituted.

[0129] The term “indole”, as used herein, is an aromatic heterocyclic organic compound with formula C8H7N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The “indole” can be optionally substituted.

[0130] The term “cyano”, as used herein, unless otherwise indicated, can include a -CN group. The “cyano” can be optionally substituted.

[0131] The term “alcohol”, as used herein, unless otherwise indicated, can include a compound in which the hydroxyl functional group (-OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms. The “alcohol” can be optionally substituted.

[0132] The term “solvate” is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound. Examples of solvates include compounds of the invention in combination with, for example, water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

[0133] The term “mmol”, as used herein, is intended to mean millimole. The term “equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “pg”, as used herein, is intended toDocket No.: 021413 / WO

[0134] mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term "|JL", as used herein, is intended to mean microliter. The term “pM”, as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “°C”, as used herein, is intended to mean degree Celsius. The term “wt / wt”, as used herein, is intended to mean weight / weight. The term “v / v”, as used herein, is intended to mean volume / volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high performance liquid chromatograph. The term “RT”, as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term “N / A”, as used herein, is intended to mean not tested.

[0135] As used herein, the expression “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,T-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or another counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In instances where multiple charged atoms are part of the pharmaceutically acceptable salt, the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound ofDocket No.: 021413 / WO

[0136] the invention, or a salt thereof, that further can include a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0137] MOLECULAR ENGINEERING

[0138] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0139] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0140] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid”, “transgene”, “exogenous polynucleotide” as used herein, each refers to a sequence that originates from a source foreign (e.g., non-native) to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0141] Sequences described herein can also be the reverse, the complement, or the reverse complement of the nucleotide sequences described herein. The RNA goes in the reverse direction compared to the DNA, but its base pairs still match (e.g., G to C). The reverse complementary RNA for a positive strand DNA sequence will be identical to the corresponding negative strand DNA sequence. Reverse complement converts a DNA sequence into its reverse, complement, or reverse-complement counterpart.

[0142] Base Name Bases Represented Complementary Base A Adenine A T

[0143] T Thymidine T A

[0144]

[0145] Docket No.: 021413 / WO

[0146] U Uridine(RNA only) U A

[0147] G Guanidine iG iC

[0148] C Cytidine iC iG

[0149] Y ipYrimidine iC T iR

[0150] R purine A G Y

[0151] iS iStrong(3H bonds) iG C S*

[0152] W Weak(2Hbondsj A T iW*

[0153] K iKeto iT / U G iM

[0154] M iaMino A C iK

[0155] B not A iC G T iV

[0156] D inot C A G T iH

[0157] H not G A C T iD

[0158] V inot T / ll A C G iB

[0159] N illnknown A C G T iN

[0160]

[0161] Complementarity is a property shared between two nucleic acid sequences (e.g., RNA, DNA), such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. Two bases are complementary if they form Watson-Crick base pairs.

[0162] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0163] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce aDocket No.: 021413 / WO

[0164] specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.

[0165] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0166] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes. An operon is a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins.

[0167] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0168] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0169] For a gene to be expressed, its DNA sequence (or polynucleotide sequence) must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventingDocket No.: 021413 / WO

[0170] transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0171] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0172] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0173] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0174] A ribosomal skipping sequence (e.g., 2A sequence such as furin-GSG-T2A) can be used in a construct to prevent covalently linking translated amino acid sequences.

[0175] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilledDocket No.: 021413 / WO

[0176] in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P.

[0177] 1988. Methods in Enzymology 167, 747-754).

[0178] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0179] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0180] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0181] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can includeDocket No.: 021413 / WO

[0182] but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0183] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0184] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using self-replicating primers, paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0185] "Wild-type" refers to a virus or organism found in nature without any known mutation. Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0186] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and ifDocket No.: 021413 / WO

[0187] necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0188] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0189] “Point mutation” refers to when a single base pair is altered. A point mutation or substitution is a genetic mutation where a single nucleotide base is changed, inserted, or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product — consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect (e.g., synonymous mutations) to deleterious effects (e.g., frameshift mutations), with regard to protein production, composition, and function. Point mutations can have one of three effects. First, the base substitution can be a silent mutation where the altered codonDocket No.: 021413 / WO

[0190] corresponds to the same amino acid. Second, the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid. Or third, the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal. Silent mutations result in a new codon (a triplet nucleotide sequence in RNA) that codes for the same amino acid as the wild type codon in that position. In some silent mutations the codon codes for a different amino acid that happens to have the same properties as the amino acid produced by the wild type codon. Missense mutations involve substitutions that result in functionally different amino acids; these can lead to alteration or loss of protein function. Nonsense mutations, which are a severe type of base substitution, result in a stop codon in a position where there was not one before, which causes the premature termination of protein synthesis and can result in a complete loss of function in the finished protein.

[0191] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0192] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e. , 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions,Docket No.: 021413 / WO

[0193] a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm = 81.5 °C + 16.6(log [Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1 ,5°C for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0194] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

[0195] Conservative Substitutions I

[0196] Side Chain Characteristic Amino Acid

[0197] Aliphatic Non-polar G A P I L V

[0198] Polar-uncharged C S T M N Q

[0199] Polar-charged D E K R

[0200] Aromatic H F WY

[0201] Other N O D E

[0202]

[0203] Conservative Substitutions II

[0204] Side Chain Characteristic Amino Acid

[0205] Non-polar (hydrophobic)

[0206]

[0207] Docket No.: 021413 / WO

[0208] A. Aliphatic: A L I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G

[0209] Uncharged-polar A. Hydroxyl: S TY B. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): K R H Negatively Charged (Acidic): D E Conservative Substitutions III Exemplary Original Residue Substitution Ala (A) Vai, Leu, lie Arg (R) Lys, Gin, Asn Asn (N) Gin, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gin (Q) Asn Glu (E) Asp His (H) Asn, Gin, Lys, Arg Leu, Vai, Met, Ala, He (I) Phe, lie, Vai, Met, Ala, Leu (L) Phe Lys (K) Arg, Gin, Asn Met(M) Leu, Phe, lie

[0210]

[0211] Docket No.: 021413 / WO

[0212] Phe (F) Leu, Vai, He, Ala

[0213] Pro (P) Gly

[0214] Ser (S) Thr

[0215] Thr (T) Ser

[0216] Trp(W) Tyr, Phe

[0217] Tyr (Y) Trp, Phe, Tur, Ser

[0218] He, Leu, Met, Phe,

[0219] Vai (V) Ala

[0220]

[0221] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0222] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif.

[0223] 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0224] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), single guide RNADocket No.: 021413 / WO

[0225] (sgRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon etal. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0226] GENOME EDITING

[0227] As described herein, gene and / or protein expression signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing.

[0228] As described herein, activity, signals, expression, or function can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing (e.g., upregulate, downregulate, overexpress, underexpress, express (e.g., transgenic expression), knock in, knock out, knockdown).

[0229] Processes for genome editing are well known; see e.g., Aldi 2018 Nature Communications 9(1911). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0230] For example, genome editing can comprise CRISPR / Cas9, CRISPR-Cpf1 , TALEN, or ZNFs. Adequate blockage of gene / protein expression / signaling by genome editing can result in protection from autoimmune or inflammatory diseases.

[0231] As an example, clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR-associated (Cas) systems are a new class of genome-editing tools thatDocket No.: 021413 / WO

[0232] target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N)2oNGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR / Cas systems could be useful tools for therapeutic applications to target cells by the removal or addition of signals (e.g., activate (e.g., CRISPRa), upregulate, overexpress, downregulate).

[0233] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

[0234] GENE THERAPY AND GENOME EDITING

[0235] Gene therapies are rapidly advancing and in some embodiments include inserting a functional gene with a viral vector. Improvement to the landscape for gene therapies and gene therapy clinical trials is ongoing (see, e.g., the most recent quarterly data breakdown from Alliance for Regenerative Medicine).

[0236] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof.

[0237] Gene therapy strategies.

[0238] Viral Vectors Strategy

[0239] Retroviruses Retroviruses are RNA viruses transcribing their single-stranded

[0240] genome into a double-stranded DNA copy, which can integrate into host chromosome Adenoviruses (Ad) Ad can transfect a variety of quiescent and proliferating

[0241] cell types from various species and can

[0242]

[0243] mediateDocket No.: 021413 / WO

[0244] robust gene expression

[0245] Adeno-associated Recombinant AAV vectors contain no viral Viruses (AAV) DNA and can carry ~4.7 kb of foreign transgenic material. They

[0246] are replication defective and can replicate only while

[0247] coinfecting with a helper virus

[0248] Non-viral vectors

[0249] plasmid DNA pDNA has many desired characteristics as a (pDNA) gene

[0250] therapy vector; there are no limits on the size or genetic

[0251] constitution of DNA, it is relatively inexpensive to supply,

[0252] and unlike viruses, antibodies are not generated

[0253] against DNA in normal individuals

[0254] RNAi RNAi is a powerful tool for gene specific silencing that

[0255] could be useful as an enzyme reduction therapy or

[0256] means to promote read-through of a premature stop

[0257] codon

[0258]

[0259] Gene therapy can allow for the constant delivery of the enzyme directly to target organs and eliminates the need for weekly infusions. Also, correction of a few cells could lead to the enzyme being secreted into the circulation and taken up by their neighboring cells (cross-correction), resulting in widespread correction of the biochemical defects. As such, the number of cells that must be modified with a gene transfer vector is relatively low.

[0260] Genetic modification can be performed either ex vivo or in vivo. The ex vivo strategy is based on the modification of cells in culture and transplantation of the modified cell into a patient. Cells that are most commonly considered therapeutic targets for monogenic diseases are stem cells. Advances in the collection and isolation of these cells from a variety of sources have promoted autologous gene therapy as a viable option.

[0261] The use of endonucleases for targeted genome editing can solve the limitations presented by the usual gene therapy protocols. These enzymes are custom molecularDocket No.: 021413 / WO

[0262] scissors, allowing cutting DNA into well-defined, perfectly specified pieces, in virtually all cell types. Moreover, they can be delivered to the cells by plasmids that transiently express the nucleases, or by transcribed RNA, avoiding the use of viruses.

[0263] FORMULATION

[0264] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0265] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0266] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc., may also be used.

[0267] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0268] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and aboutDocket No.: 021413 / WO

[0269] 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0270] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0271] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0272] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0273] CELL THERAPY

[0274] Cells generated according to the methods described herein can be used in cellDocket No.: 021413 / WO

[0275] therapy. Cell therapy (also called cellular therapy, cell transplantation, or cytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting T-cells capable of fighting cancer cells via cell-mediated immunity can be used in the course of immunotherapy, grafting stem cells can be used to regenerate diseased tissues, or transplanting beta cells can be used to treat diabetes.

[0276] Stem cell and cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.

[0277] Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogeneic strategy is that unmatched allogenic cell therapies can form the basis of "off the shelf" products.

[0278] Autologous cell therapy or autologous transplantation uses cells that are derived from the subject’s own tissues. It could also involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.

[0279] Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig derived cells can be transplanted into humans. Xenogeneic cell therapies can involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.

[0280] ADMINISTRATION

[0281] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0282] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.Docket No.: 021413 / WO

[0283] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0284] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0285] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.Docket No.: 021413 / WO

[0286] SCREENING

[0287] Also provided are screening methods.

[0288] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.

[0289] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0290] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0291] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively largerDocket No.: 021413 / WO

[0292] scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0293] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0294] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0295] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0296] KITS

[0297] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to reagents, precursors, apparatus(es), and equipment as disclosed herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the componentsDocket No.: 021413 / WO

[0298] separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0299] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral nonreacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0300] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0301] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0302] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-Docket No.: 021413 / WO

[0303] readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer program include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0304] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sam brook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0305] Definitions and methods described herein are provided to better define the presentDocket No.: 021413 / WO

[0306] disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0307] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0308] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0309] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or moreDocket No.: 021413 / WO

[0310] steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0311] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0312] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0313] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0314] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0315] EXAMPLES

[0316] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples ofDocket No.: 021413 / WO

[0317] modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0318] Example 1: Endonuclease V-Assisted Fluorescence Polarization Assay To Screen For hADARI Small Molecule Inhibitors

[0319] The adenosines in human RNA are frequently edited into Inosines (A-to-l editing) by human adenosine deaminases acting on RNAs (hADARs) (FIG. 2). This editing plays a crucial role in the normal physiological functions of the cell, including distinguishing normal cellular RNA from viral RNA (FIG. 3). Impaired editing is implicated in neurodegenerative diseases and autoimmune disorders. Further, several cancers exploit the hADARI as an immune checkpoint blockade. The hADARI knockdown studies on cancer cells exhibit their sensitization to immunotherapy, making it a suitable biological target for cancer drug development. However, the lack of a suitable high throughput screening (HTS) assay to track the A-to-l editing activity is a major drawback to screening for hADARI inhibitors (FIG. 4). Current methods rely on the expression of ADAR to correct a premature stop codon in the mRNA of a fluorescent reporter gene at the target edit site in mammalian cells as a proxy for ADAR activity (FIG. 5). This method is laborious, costly, difficult to handle, and timeconsuming. In vitro activity assays with recombinant ADARs either report only on RNA binding, but not editing, or require a costly, multi-step RNA sequencing approach to determine the editing percentage. In light of these current limitations, we have developed a convenient, HTS-compatible Endonuclease V (EndoV) based fluorescence polarization assay to determine A-to-l editing quantitatively (FIG. 6). The work herein employs EndoV to selectively cleave hADARI deaminated Inosine-containing RNA (l-RNA) carrying a fluorophore at one of the ends near the cleavage site, thus producing a differential fluorescence polarization value corresponding to the editing. The assay has several key aspects:

[0320] 1. One-pot two-step assay

[0321] 2. Deaminated RNA cleaved by EndoV

[0322] 3. Fluorescence polarization changes upon EndoV cleavage

[0323] Advantages of the assay include:

[0324] 1. Straightforward and easy to followDocket No.: 021413 / WO

[0325] 2. HTS compatible

[0326] 3. Converts the editing into a fluorescence polarization signal

[0327]

[0328] RNA Substrates

[0329] 1. AUCCGCCAAUUCUUCGAGAAUUAGCGGGU (SEQ ID NO: 1)

[0330] 2. UGUCAUUAGACGUUCAGUUAGUACCACCAAUGACA (SEQ ID NO: 2) Human ADAR1 (SEQ ID NO: 3) MNPRQGYSLSGYYTHPFQGYEHRQLRYQQPGPGSSPSSFLLKQIEFLKGQLPEAPVIGK QTPSLPPSLPGLRPRFPVLLASSTRGRQVDIRGVPRGVHLRSQGLQRGFQHPSPRGRSL PQRGVDCLSSHFQELSIYQDQEQRILKFLEELGEGKATTAHDLSGKLGTPKKEINRVLYSL AKKGKLQKEAGTPPLWKIAVSTQAWNQHSGWRPDGHSQGAPNSDPSLEPEDRNSTSV SEDLLEPFIAVSAQAWNQHSGWRPDSHSQGSPNSDPGLEPEDSNSTSALEDPLEFLDM AEIKEKICDYLFNVSDSSALNLAKNIGLTKARDINAVLIDMERQGDVYRQGTTPPIWHLTDK KRERMQIKRNTNSVPETAPAAIPETKRNAEFLTCNIPTSNASNNMVTTEKVENGQEPVIKL ENRQEARPEPARLKPPVHYNGPSKAGYVDFENGQWATDDIPDDLNSIRAAPGEFRAIME MPSFYSHGLPRCSPYKKLTECQLKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQ WINGREFPPAEAGSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAE SQTPTPSATSFFSGKSPVTTLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFP SVSAPSKKVAKQMAAEEAMKALHGEATNSMASDNQPEGMISESLDNLESMMPNKVRKIG ELVRYLNTNPVGGLLEYARSHGFAAEFKLVDQSGPPHEPKFVYQAKVGGRWFPAVCAHS KKQGKQEAADAALRVLIGENEKAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLT GSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGWVSLGTGNRCVKGD SLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLY ISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPT WDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICC RVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGT RGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGL KD M G YG N Wl S KP Q E E KN F YLC P V

[0331] E. coll EndoV (SEQ ID NO: 4) MDLASLRAQQIELASSVIREDRLDKDPPDLIAGADVGFEQGGEVTRAAMVLLKYPSLELVE YKVARIATTMPYIPGFLSFREYPALLAAWEMLSQKPDLVFVDGHGISHPRRLGVASHFGLLDocket No.: 021413 / WO

[0332] VDVPTIGVAKKRLCGKFEPLSSEPGALAPLMDKGEQLAWVWRSKARCNPLFIATGHRVSV DSALAWVQRCMKGYRLPEPTRWADAVASERPAFVRYTANQP

[0333] Human Endonuclease V 282 (SEQ ID NO: 5) MALEAAGGPPEETLSLWKREQARLKAHVVDRDTEAWQRDPAFSGLQRVGGVDVSFVKG DSVRACASLWLSFPELEVVYEESRMVSLTAPYVSGFLAFREVPFLLELVQQLREKEPGLM PQVLLVDGNGVLHHRGFGVACHLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTR GDSFPLLGDSGTVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQA DICSREHIRKSLG LPGPPTPRSPKAQRPVACPKGDSGESSALC

[0334] Human Endonuclease V 309 (SEQ ID NO: 6) MALEAAGGPPEETLSLWKREQARLKAHVVDRDTEAWQRDPAFSGLQRVGGVDVSFVKG DSVRACASLWLSFPELEVVYEESRMVSLTAPYVSGFLAFREVPFLLELVQQLREKEPGLM PQVLLVDGNGVLHHRGFGVACHLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTR GDSFPLLGDSGTVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQA DICSREHIRKSLGLPGPPTPRSPKAQRPVACPKGDSGESSGEGQPPQDHSPGPRTAPRP GSQEQAGKDWQ

[0335] Guide DNA Strands

[0336] (5’-TGTCATTGGTGGTACTAACTGAA-3’, SEQ ID NO: 7)

[0337] 5’-TGTCATTGGTGGTACTAACTGAACGTCTAAT-3’, SEQ ID NO: 10)

[0338] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGACA-3’ (SEQ ID NO: 11) 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGAC-3’ (SEQ ID NO: 12)

[0339] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGA-3’ (SEQ ID NO: 13)

[0340] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATG-3’ (SEQ ID NO: 14)

[0341] 5’-TGTCATTGGTGGTACTAACTGAACCACTAAT-3’ (SEQ ID NO: 15) Experimental Data

[0342] One-pot deamination / cleavage assay

[0343] The assay conditions for the one-pot deamination / cleavage assay are as follows. The in vitro deamination was performed in 15 mM Tris. HCI, 26 mM KCI, 40 mM Potassium Glutamate, 1.5 mM EDTA, 4% glycerol, 0.003% NP40, pH 7.4 buffer with 100 nM 5’-FAM, 3’-Cy5 labeled RNA substrate 2 (UGUCAUUAGACGUUCAGUUAGUACCACCAAUGACA,Docket No.: 021413 / WO

[0344] SEQ ID NO: 2) and 300 nM hADARI at 30 °C for 1.5 hours. The hADARI was deactivated by heating at 90 °C for 5 minutes, followed by adding manganese chloride and E. coli EndoV (either purified or the commercially available NEB EndoV) to 5 mM and 300 nM concentrations. The reaction samples were mixed with 50% formamide, denatured at 80 °C for 5 mins, and analyzed by running on 15% polyacrylamide gel.

[0345] Cleavage assay with human EndoV

[0346] The cleavage reactions with 50 nM 5’-FAM labeled substrate 2 (UGUCAUUAGACG UUCAGUUAGUACCACCAAUGACA, SEQ ID NO: 2) and accessory guide DNA is TGTCATTGGTGGTACTAACTGAA (SEQ ID NO: 7) at different human EndoV concentrations were performed in 10 mM Tris. HCI, 50 mM KCI, 5% glycerol, 1 mM DTT, 10 mM MgCl2, pH 7.5 at 37 °C for 1 hour. The reactions were then mixed with 50% formamide and the samples were analyzed by 15% polyacrylamide gel.

[0347] Results

[0348] Deamination PCR experiments were performed with three RNA substrates, S1 (FIG.

[0349] 7 A), S2 (FIG. 7B), and S3 (FIG. 7C). The incorporation of G instead of A was observed in PCR of all substrates, which corresponds to Inosines in Sanger Sequencing. All three substates were found to be highly edited at their major edit sites (S1a: quantitative, S2a: 92%, S3a: 91%).

[0350] Identification of hADARI substrate RNA candidates

[0351] The human ADAR1p150 isoform was purchased from BPS Bioscience. The in vitro deamination of the RNA substrates was carried out using hADARI. Samples containing 50 nM RNA substrate and 500 nM hADARI were incubated in 15 mM Tris. HCI, 26 mM KCI, 40 mM Potassium Glutamate, 0.003 % NP-40, 0.5 mM EDTA, pH 7.5 buffer at 30 °C for 2 hours. For the hADARI concentration-dependent editing assay, the 20 nM HER1 RNA was incubated with different concentrations of hADARI (0.5 nM, 1.0 nM, 5 nM, 10 nM, 25 nM, 50 nM, and 100 nM) in the same buffer at 30 °C for 3 hours. The reactions were quenched by heat deactivating hADARI at 95 °C for 10 mins. The RNA was extracted from each reaction, reverse-transcribed into cDNA, PCR amplified and sequenced through Sanger sequencing.

[0352] The chromatograms were analyzed using SnapGene software. The percentage of editing was calculated using the formula:

[0353] % of editing = (G) / (A+G) x 100Docket No.: 021413 / WO

[0354] An hDAR1 concentration-dependent editing assay was performed with S1 to determine the optimized enzyme-to-substrate ratio (FIG. 8). It was found that a 1:1 molar ratio of substrate-to-enzyme was optimal for the assay at 20 nM substrate concentration. Next, a one-pot deamination / cleavage reaction with S2a derived short hairpin RNA was performed, and a gel assay was performed with A-RNA and l-RNA. It was found that the S2a substrate is optimal for both hADARI and EndoV (FIG. 9). Then experiments were performed to optimize the cleavage reaction with S2a derived short hairpin RNA. It was found that almost quantitative specific cleavage of the redesigned short hairpin l-RNA substrates by human EndoV at the target site with concentrations as low as 0.5 pM EndoV (FIG. 10).

[0355] Further work in Example 1 includes optimizing the FP assay for a better signal-to-noise ratio, transferring the assay to high-throughput screening, and validation of identified hits through cellular assays. With regard to the high-throughput assay, a multi-well plate can performed, wherein high editing provides low fluorescence polarization and low editing provides high fluorescence polarization.

[0356] Example 2: Endonuclease V-Assisted Fluorescence Polarization Assay: Integration of Proteinase K, Fluorophore-Quencher RNA Substrate, and Plate Readers

[0357] Example 2 includes fluorescence assisted assay development to screen for ADAR1 inhibitors and exploring the substrate specificity of hEndoV. The goals of Example 2 include:

[0358] 1. Fabrication and use of the fluorophore quencher RNA substrate (FQ_BDF2) for the final fluorescence dequenching assay.

[0359] 2. Confirm the EndoV degradation by proteinase K after the RNA cleavage reaction.

[0360] 3. Cleavage assay on FQ_BDF2 substrate.

[0361] 4. Optimize the reaction conditions to make cleaved RNA band fall off from the RNA strand.

[0362] 5. Proof-of-concept for the EndoFluor on the plate reader.

[0363] To confirm hEndoV degradation by proteinase K (FIG. 13A), degradation assays were performed, which showed that proteinase K actively degrades hEndoV in all tested conditions at RT (FIG. 13B). Then, the hEndoV cleavage on FQ_BDF2 (EndoFluor substrate) was tested (FIG. 14). It was shown that double-labeling is affecting the hEndoV cleavage to a large extent, leaching out Mn with EDTA after the cleavage reaction maintains the RNA integrity, and manganese is a better cofactor compared to magnesium for the cleavageDocket No.: 021413 / WO

[0364] reaction on the new hairpin under the tested conditions. Therefore, the hEndoV cleavage on FQ_BDF2 in the presence of Manganese was further tested (FIG. 15). Cleavage of l-RNA by hEndoV in the presence of Mn was observed, but that reaction conditions need to be further optimized for the cleavage reaction.

[0365] Next, EndoFluor (deamination / cleavage) was tested on FQ_BDF2 (FIG. 16), and it was found that EndoFluor worked on the double-labeled hairpin, and that the cleavage reaction can be performed without deactivating hADARI. Then, EndoFluor (deamination / cleavage) signal was tested on a plate reader. After deamination, the samples were heat-denatured, hEndoV was added, followed by proteinase K degradation (FIG. 17A). Then the same samples were incubated with increasing concentrations of urea, incubated for 10 minutes at RT and the fluorescence signal was recorded (FIG. 17B). It was found that there is a strong fluorescence dequenching signal for the A-RNA incubated with hADARI and hEndoV that stands as proof of concept for the EndoFluor assay, and that the cleaved band can come off either at high temperature or at RT with added urea. Finally, the cleavage signal was tested with increasing hEndoV concentration (FIG. 18A).

[0366] Further development in Example 2 includes:

[0367] 1. Test the FQ_BDF2 cleavage with all three EndoVs under different conditions (EcEndoV, hEndoV 282, hEndoV 309).

[0368] 2. Generate the EndoFluor response with hADARI titration and with varied A-to-l concentrations of synthetic RNA.

[0369] 3. Develop a correlational % hADARI inhibition curve from the EndoFluor signal 4. Use 8-azanebularine RNA sequences to use as a positive control for inhibition.

[0370] 5. Perform EndoFluor with proper positive and negative controls.

[0371] Fluorescence Dequenching Assay

[0372] The fluorescence dequenching assays were carried out in the cleavage assay buffer. The RNA was refolded down the temperature gradient in cleavage assay buffer before starting any reactions. For the deamination reaction, 20 nM RNA was incubated with 20 nM hADARI in 10 mM Tris. HCI, 50 mM KCI, 1 mM DTT, 5% glycerol, pH 7.5 in a total 50 pL reaction volume at 37oC for 1.5 hours. Manganese was added to a final 2 mM concentration in 100 pL volume followed by different concentrations of eEndoV (0 nM, 50 nM, 250 nM, 500 nM in 100 pL). The reactions volume was adjusted to a final 100 pL volume using 2x reaction buffer and nuclease-free water. The reaction mixtures were incubated at 37 °C for 1 hour.Docket No.: 021413 / WO

[0373] The reaction mixtures were further incubated at room temperature for 18 hours. When urea was used, it was added to a final 2M concentration. A 30 pL each of the reaction mixtures was transferred to a 384-well plate and the fluorescence intensity values were recorded on the BioTek Cytation 5 instrument.

[0374] Endonuclease V-based Fluorescence Dequenching Assay (EndoFluor)

[0375] To test the robustness of our one-pot deamination / cleavage approach, we performed a fluorescence dequenching assay using Cy5-BHQ3-labeled BDF2 RNA (FIG. 18B). The in vitro deamination followed by EndoV cleavage produced the desired fluorescence dequenching signal. This embodiment’s approach requires an overnight incubation of the reaction mixture at room temperature before taking the fluorescence readings.

[0376] Example 3: Endonuclease V-Assisted Fluorescence Polarization Assay: Testing Multiple EndoVs, Condition and Control Optimization

[0377] Experimental Data

[0378] RNA substrates

[0379] BDF2_A = 5’-UGUCAUUAGACGUUCAGUUAGUACCACCAAUGACA-3’ (SEQ ID NO: 2) BDF2J = 5’-UGUCAUUIGACGUUCAGUUAGUACCACCAAUGACA-3’ (SEQ ID NO: 8, n = inosine)

[0380] The 8-azaN RNA (control)

[0381] 5’-AUCCGCCAAUUCUUCGAGAAUU(8-azanebularine)GCGGGU-3’ (SEQ ID NO: 9, n = 8-azanebularine)

[0382] Guide DNA Strands

[0383] (5’-TGTCATTGGTGGTACTAACTGAA-3’, SEQ ID NO: 7)

[0384] 5’-TGTCATTGGTGGTACTAACTGAACGTCTAAT-3’, SEQ ID NO: 10)

[0385] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGACA-3’ (SEQ ID NO: 11) 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGAC-3’ (SEQ ID NO: 12)

[0386] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATGA-3’ (SEQ ID NO: 13)

[0387] 5’-TGTCATTGGTGGTACTAACTGAACCACTAATG-3’ (SEQ ID NO: 14)

[0388] 5’-TGTCATTGGTGGTACTAACTGAACCACTAAT-3’ (SEQ ID NO: 15)

[0389] Human ADAR1 (SEQ ID NO: 3)Docket No.: 021413 / WO

[0390] MNPRQGYSLSGYYTHPFQGYEHRQLRYQQPGPGSSPSSFLLKQIEFLKGQLPEAPVIGK QTPSLPPSLPGLRPRFPVLLASSTRGRQVDIRGVPRGVHLRSQGLQRGFQHPSPRGRSL PQRGVDCLSSHFQELSIYQDQEQRILKFLEELGEGKATTAHDLSGKLGTPKKEINRVLYSL AKKGKLQKEAGTPPLWKIAVSTQAWNQHSGWRPDGHSQGAPNSDPSLEPEDRNSTSV SEDLLEPFIAVSAQAWNQHSGWRPDSHSQGSPNSDPGLEPEDSNSTSALEDPLEFLDM AEIKEKICDYLFNVSDSSALNLAKNIGLTKARDINAVLIDMERQGDVYRQGTTPPIWHLTDK KRERMQIKRNTNSVPETAPAAIPETKRNAEFLTCNIPTSNASNNMVTTEKVENGQEPVIKL ENRQEARPEPARLKPPVHYNGPSKAGYVDFENGQWATDDIPDDLNSIRAAPGEFRAIME MPSFYSHGLPRCSPYKKLTECQLKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQ WINGREFPPAEAGSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAE SQTPTPSATSFFSGKSPVTTLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFP SVSAPSKKVAKQMAAEEAMKALHGEATNSMASDNQPEGMISESLDNLESMMPNKVRKIG ELVRYLNTNPVGGLLEYARSHGFAAEFKLVDQSGPPHEPKFVYQAKVGGRWFPAVCAHS KKQGKQEAADAALRVLIGENEKAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLT GSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVWSLGTGNRCVKGD SLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLY ISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPT WDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICC RVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGT RGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGL KDMGYGNWISKPQEEKNFYLCPV

[0391] E. coll EndoV (SEQ ID NO: 4) MDLASLRAQQIELASSVIREDRLDKDPPDLIAGADVGFEQGGEVTRAAMVLLKYPSLELVE YKVARIATTMPYIPGFLSFREYPALLAAWEMLSQKPDLVFVDGHGISHPRRLGVASHFGLL VDVPTIGVAKKRLCGKFEPLSSEPGALAPLMDKGEQLAWVWRSKARCNPLFIATGHRVSV DSALAWVQRCMKGYRLPEPTRWADAVASERPAFVRYTANQP

[0392] Human Endonuclease V 282 (SEQ ID NO: 5) MALEAAGGPPEETLSLWKREQARLKAHVVDRDTEAWQRDPAFSGLQRVGGVDVSFVKG DSVRACASLWLSFPELEVVYEESRMVSLTAPYVSGFLAFREVPFLLELVQQLREKEPGLM PQVLLVDGNGVLHHRGFGVACHLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTR GDSFPLLGDSGTVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQA

[0393]

[0394] Docket No.: 021413 / WO

[0395] DICSREHIRKSLGLPGPPTPRSPKAQRPVACPKGDSGESSALC

[0396] Human Endonuclease V 309 (SEQ ID NO: 6) MALEAAGGPPEETLSLWKREQARLKAHVVDRDTEAWQRDPAFSGLQRVGGVDVSFVKG DSVRACASLWLSFPELEVVYEESRMVSLTAPYVSGFLAFREVPFLLELVQQLREKEPGLM PQVLLVDGNGVLHHRGFGVACHLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTR GDSFPLLGDSGTVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQA DICSREHIRKSLGLPGPPTPRSPKAQRPVACPKGDSGESSGEGQPPQDHSPGPRTAPRP GSQEQAGKDWQ

[0397] Endonuclease V -based Fluorescence Polarization Assay (EndoFluor)

[0398] To test the employability of EndoV enzymes for the development of an ADAR1 activity assay, cleavage assays were performed using synthetic hairpin RNAs (5’-FAM-BDF2_A, 5’-FAM-BDF2_I) (FIG. 19A). On confirming inosine-specific cleavage, a pot deaminationcleavage reaction was performed that showed the desired cleavage, representing a successful deamination reaction by hADARI and cleavage by eEndoV (FIG. 19B and 19C). All tested enzymes (eEndoV, hEndoV282, hEndoV309) exhibited a robust inosine-specific cleavage efficiency (FIG. 20). The cleavage properties of these enzymes are largely affected by the metal ions used in the cleavage assay. The eEndoV showed a non-specific 5’-end cleavage of the double-strand ends in the presence of manganese (FIG. 19A). This can be reduced either by opening the duplex to create a 5’ -overhang or by changing the cofactor to magnesium (FIG. 20A). Both changes can also be introduced at once. In comparison, human EndoVs have shown less off-target cleavage effect (FIG. 20B). Initial experiments with double-labeled substrates revealed a superior inosine-specific cleavage activity for hEndoVs at pH 9.5 with magnesium. Therefore, the reactions were performed at pH 7.5 and 9.5 to demonstrate the feasibility of the assay across different conditions.

[0399] The 35-nucleotide BDF2 substrate, upon successful deamination and cleavage in a homogeneous assay, should exhibit decreased fluorescence polarization. The experiments with eEndoV and hEndoV309 showed optimistic results for the assay (EndoFluor) development. As expected, eEndoV at pH 7.5 with MnCl2 as cofactor showed a drastic decrease in fluorescence polarization (FP) value, representing its non-specific cleavage activity at its 5’-end (FIG. 21 B). Again, in conjunction with gel-based cleavage assay data, the introduction of the guide DNA suppressed this non-specific cleavage (FIG. 21 A).Docket No.: 021413 / WO

[0400] Promisingly, the method was able to show the inhibition of hADARI deamination by 8-azanebularine-containing RNA as an easily readable FP signal. Furthermore, the data also support the claims that 8-azanebularine and ZYS-1 cannot inhibit the hADARI deamination directly. To simplify the method using mild conditions with less off-target cleavage, the EndoFluor was finally performed with hEndoV309 at pH 9.5 using magnesium as a cofactor. Again, EndoFluor was able to show a clear difference between the hADARI -inhibited vs noninhibited samples. Even though there is room for improvement for the signal-to-noise ratio, the method reported the first HTS-compatible hADARI activity assay that can track the deamination.

[0401] Materials and Methods

[0402] The FAM-labeled short RNA oligos were purchased through IDT. The recombinant hADARI p150 was purchased from BPS Bioscience. The eEndoV, hEndoV282, and hEndoV309 were cloned into the pET28a vector through Gibson assembly. These proteins were purified in-house through metal affinity chromatography. The 8-azanebularine RNA was ordered from the University of Utah peptide core facility.

[0403] Cleavage assays with synthetic hairpin substrates

[0404] The RNA was refolded down the temperature gradient in cleavage assay buffer before starting any reactions. The cleavage reactions were performed at either 35 or 50 nM substrate concentration (BDF2_A, BDF2_I) with or without guide DNA (5’-TGTCATTGGTGGTACTAACTGAA-3’, SEQ ID NO: 7) at different EndoV concentrations in 10 mM Tris. HCI, 50 mM KCI, 5% glycerol, 1 mM DTT, 10 mM MgCI2or 0.5 mM MnCI2, pH 7.5 at 37 °C in a 20 pL final volume for 1 hour. When guide DNA was added, the reaction mixtures were incubated at 50 °C for 10 minutes before the addition of EndoV. The reaction mixtures were then denatured with 10 pL of 50% formamide at 80 °C for 5 minutes. A 15 pL sample each was run on 15% polyacrylamide gel at 180V for 75 minutes.

[0405] One-pot deamination / cleavage assay

[0406] The RNA was refolded down the temperature gradient in cleavage assay buffer before starting any reactions. The in vitro deamination was performed in 15 mM Tris. HCI, 26 mM KCI, 40 mM Potassium Glutamate, 1.5 mM EDTA, 4% glycerol, 0.003% NP40, pH 7.4 buffer with 100 nM BDF2 RNA and 300 nM hADARI at 30 °C for 1.5 hours. The hADARI was deactivated by heating at 90 °C for 5 minutes, followed by adding manganese chloride andDocket No.: 021413 / WO

[0407] E. coli EndoV (either purified or the commercially available NEB EndoV) to 5 mM and 300 nM concentrations. The reaction samples were mixed with 50% formamide, denatured at 80 °C for 5 mins, and analyzed by running on 15% polyacrylamide gel at 180V for 75 minutes. Fluorescence Polarization Assay

[0408] The FP assays were carried out in the cleavage assay buffer. The RNA was refolded down the temperature gradient in cleavage assay buffer before starting any reactions. For the deamination reaction, 60 nM RNA was incubated with 60 nM hADARI in 10 mM Tris. HCI, 50 mM KCI, 1 mM DTT, 5% glycerol, 10% DMSO, pH 7.5 in a total 35 pL reaction volume at 37°C for 1.5 hours. The reaction components were added to a final reaction volume of 70 pL - 600 nM guide DNA (5-TGTCATTGGTGGTACTAACTGAACGTCTAAT-3’, SEQ ID NO: 10), 5 mM MnCb, 1x reaction buffer, except the EndoV. The RNA was allowed to reanneal with guides at 50 °C for 10 minutes. The EndoV was then added to a final 300 nM concentration and incubated at 37 °C for 1 hour. For each 70 pL reaction mixture, two volumes of 5M NaCI i.e. , 140 pL, was added and incubated at room temperature for 6 hours. The FP values were recorded on the BioTek Cytation 5 instrument. For heat-denatured ADAR1 control, 600 nM enzyme was mixed with an equal amount of 2x reaction buffer, incubated at 90 °C for 10 minutes. For all other controls (8-azaN RNA, 8-azaN, ZYS-1), 10x stocks were made either in NF-water or DMSO. The controls were incubated with hADARI for 30 minutes before adding the substrate RNA.

[0409] For reactions with hEndoV309, the deamination reaction conditions are the same. There is no guide DNA addition / annealing step. After the deamination step, one-fourth volume of 2x buffer 10 mM Tris. HCI, 50 mM KCI, 1 mM DTT, 5% glycerol, pH 12.08, was added to bring the final reaction buffer close to pH 9.5. Then MgCl2 was added to a final 10 mM concentration, followed by nuclease-free water and hEndoV309 to a final 750 nM concentration.

[0410] CLAIMS

[0411] What is claimed is:

[0412] 1. A method to detect the A-to-l editing or deamination activity of ADAR, the method comprising:

Claims

Docket No.: 021413 / WOproviding an RNA sample, wherein the RNA sample comprises a fluorescent probe; adding a DNA guide to the RNA sample;adding an endonuclease V (EndoV) and at least one of Mg and Mn to the RNA sample to form a reaction mixture;adding salt to the reaction mixture; anddetecting and quantifying a fluorescence-based signal from the fluorescent probe, wherein a strength of the fluorescence-based signal is based on a concentration of inosine residues in the RNA sample.

2. The method of claim 1, wherein the fluorescent probe comprises an RNA hairpin probe.

3. The method of claim 2, wherein the RNA hairpin probe comprises a fluorophore.

4. The method of claim 1, wherein providing the RNA sample is selected from: providing an RNA oligomer mixture comprising RNA oligomers containing one or more inosine residues; andproviding an RNA oligomer mixture comprising RNA oligomers and hADARI, wherein the hADARI incorporates inosine into each RNA oligomer via hADARI -mediated adenosine-to-inosine editing.

5. The method of claim 1, wherein the DNA guide is selected from an unlabeled DNA guide and a labeled DNA guide.

6. The method of claim 5, wherein the labeled DNA guide comprises at least one of a fluorophore, a quencher, and a large functional group.

7. The method of claim 1, wherein the EndoV is a histidine-tagged, maltose-binding protein-containing fusion protein.

8. The method of claim 1 , wherein the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV).

9. The method of claim 8, wherein the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

10. The method of claim 1, wherein adding the EndoV cleaves the RNA oligomer into RNA fragments, and wherein the RNA fragments subsequently dissociates from the RNA oligomer, resulting in a detectable change to the fluorescence signal of the fluorescent probe.

11. The method of claim 1 , wherein the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.Docket No.: 021413 / WO12. A method of detecting A-to-l editing or deamination activity of ADAR in a high-throughput inhibitor screen, the method comprising:adding hADARI to each candidate molecule of a small molecule library to form a respective candidate mixture;adding an RNA hairpin probe to each respective candidate mixture;adding a DNA guide to each respective candidate mixture;adding EndoV and at least one of Mg and Mn to each candidate mixture;adding a stop agent to each candidate mixture; andreading an RNA fluorescence-based signal of each candidate mixture such that a fluorescence signal of each candidate mixture indicates the hADARI activity of each respective candidate molecule of the small molecule library.

13. The method of claim 12, wherein the RNA hairpin probe comprises a fluorophore.

14. The method of claim 12, wherein the small molecule library is provided as a homogeneous plate assay.

15. The method of claim 12, wherein the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV).

16. The method of claim 15, wherein the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

17. The method of claim 12, wherein the stop agent comprises a salt.

18. The method of claim 12, wherein the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

19. A method to detect A-to-l editing or deamination activity of ADAR, the method comprising:providing an RNA sample comprising a fluorescent probe and a quencher probe; adding an endonuclease V (EndoV) and at least one of Mg and Mn to the RNA sample to form a reaction mixture; anddetecting and quantifying a fluorescence-based signal from the fluorescent probe, wherein a strength of the fluorescence-based signal is based on a concentration of inosine residues in the RNA sample.Docket No.: 021413 / WO20. The method of claim 19, wherein providing the RNA sample is selected from: providing an RNA oligomer mixture comprising RNA oligomers containing one or more inosine residues; andproviding an RNA oligomer mixture comprising RNA oligomers and hADARI, wherein the hADARI incorporates inosine into each RNA oligomer via hADARI -mediated adenosine-to-inosine editing.

21. The method of claim 19, wherein the EndoV is a histidine-tagged, maltose-binding protein-containing fusion protein.

22. The method of claim 19, wherein the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV).

23. The method of claim 22, wherein the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

24. The method of claim 19, wherein adding the EndoV cleaves the RNA oligomer into RNA fragments, and wherein the RNA fragments subsequently dissociate from the RNA oligomer, resulting in a detectable change to the fluorescence signal of the fluorescent probe.

25. The method of claim 19, wherein the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

26. A method of detecting A-to-l editing or deamination activity of ADAR in a high-throughput inhibitor screen, the method comprising:adding hADARI to each candidate molecule of the small molecule library to form a respective candidate mixture;adding an RNA hairpin probe to each respective candidate mixture;adding EndoV and at least one of Mg and Mn to each respective candidate mixture; and reading an RNA fluorescence of each candidate mixture such that a fluorescence signal of each candidate mixture indicates the hADARI activity of each respective candidate molecule of the small molecule library.

27. The method of claim 26, wherein the RNA hairpin probe comprises a fluorophore and a quencher.

28. The method of claim 26, wherein the small molecule library is provided as a homogeneous plate assay.Docket No.: 021413 / WO29. The method of claim 26, wherein the EndoV is a histidine-tagged, maltose-binding protein-containing fusion protein.

30. The method of claim 26, wherein the EndoV is selected from an E. coli Endonuclease V (eEndoV) and a human Endonuclease V (hEndoV).

31. The method of claim 30, wherein the EndoV is selected from eEndoV according to SEQ ID NO: 4, hEndoV 282 according to SEQ ID NO: 5, and hEndoV 309 according to SEQ ID NO: 6.

32. The method of claim 26, wherein the fluorescence signal is selected from fluorophore-labeled RNA fragments visualized by gel electrophoresis, Forster resonance energy transfer (FRET), fluorescence enhancement, fluorescence polarization, and microscale thermophoresis.

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