Method for Detecting Oligonucleotides

Abstract

The present disclosure describes methods for detecting the presence and / or amount of oligonucleotides in a sample. In some embodiments, the present disclosure provides methods for assessing the ability of P-PMO to modulate expression of a target protein, the method comprising performing the method of any one of the preceding claims on a sample obtained from one or more tissues of a subject to which P-PMO has been administered, and comparing the level of PMO derived from P-PMO in the tissue with the level of the target protein in the tissue.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 928,301, filed Oct. 30, 2019, the content of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Many diseases can benefit from treatments involving the administration of oligonucleotides such as antisense oligonucleotides. For example, in disorders such as Duchenne muscular dystrophy (DMD) that have one or more mutations in the dystrophin gene resulting in a non - functional or absent dystrophin protein, the use of oligonucleotides (e.g., antisense oligonucleotides) that “skip” over a predetermined portion of the gene sequence during dystrophin protein production results in a truncated but partially functional dystrophin protein. However, the development of effective oligonucleotides, as well as accurate testing and modeling for predicting effective dosages, are plagued by an unmet need for high - throughput, sensitive, accurate, and reproducible methods for detecting oligonucleotides administered to subjects in need thereof. Such methods are required, for example, to accurately and reproducibly detect and also quantify them in samples from subjects to whom the oligonucleotides have been administered.

Summary of the Invention

Means for Solving the Problems

[0003] The present disclosure is based in part on the development of improved assays that enable sensitive detection of oligonucleotides in samples from subjects to whom the oligonucleotides have been administered.

[0004] In some embodiments, the present disclosure provides a method comprising: (a) incubating a sample comprising an oligonucleotide with an oligonucleotide probe, wherein the oligonucleotide probe hybridizes to the oligonucleotide in the sample; (b) incubating the product of step (a) with a substrate coated with a capture agent, wherein the capture agent binds to the oligonucleotide probe, thereby associating the excess oligonucleotide probe and the hybridized oligonucleotide with the substrate, and optionally washing the plate after the hybridized probe is associated with the substrate; (c) incubating the substrate after step (b) with two or more different single-strand specific nucleases having different specificities for the substrate, wherein the substrate is optionally washed with one or more wash solutions before, during, or after step (c), and the oligonucleotide probe and the hybridized oligonucleotide remain associated with the substrate during one or more wash steps; (d) incubating the substrate after step (c) with a detection agent, wherein the detection agent interacts with the oligonucleotide probe to generate a detectable signal; and (e) detecting the detectable signal.

[0005] In some aspects, the present disclosure provides a method comprising: (a) incubating a sample comprising a phosphorodiamidate morpholino oligonucleotide (PMO) with an oligonucleotide probe, wherein the oligonucleotide probe comprises one or more locked nucleic acid (LNA) residues and hybridizes to the PMO in the sample; (b) incubating the product of step (a) with a substrate coated with a capture agent, wherein the capture agent binds to the oligonucleotide probe, thereby associating the oligonucleotide probe and the hybridized PMO with the substrate; (c) incubating the substrate after step (b) with one or more single-strand specific nucleases, wherein the substrate is optionally washed before, during, or after step (c), and the oligonucleotide probe and the hybridized PMO remain associated with the substrate during one or more washing steps; (d) incubating the substrate after step (c) with a detection agent, wherein the detection agent interacts with the oligonucleotide probe to generate a detectable signal; and (e) detecting the detectable signal.

[0006] In some embodiments, step (c) comprises incubating the substrate with Micrococcus nuclease and Mung Bean nuclease.

[0007] In some embodiments, step (c) comprises incubating the substrate with Micrococcus nuclease and then incubating the substrate with Mung Bean nuclease.

[0008] In some embodiments, the substrate is washed after being incubated with Micrococcus nuclease and before being incubated with Mung Bean nuclease.

[0009] In some embodiments, the sample comprises a phosphorodiamidate morpholino oligonucleotide (PMO).

[0010] In some embodiments, the oligonucleotide probe comprises one or more locked nucleic acid (LNA) residues.

[0011] In some embodiments, the oligonucleotide probe comprises one or more deoxyribonucleic acid (DNA) residues.

[0012] In some embodiments, the oligonucleotide probe comprises a central segment composed of DNA residues flanked by 5'-terminal and 3'-terminal segments each containing LNA residues.

[0013] In some embodiments, the central segment is composed of between 5 and 20 DNA residues.

[0014] In some embodiments, the 5'- and 3'-terminal segments are independently composed of between 2 and 8 LNA residues.

[0015] In some embodiments, step (c) comprises incubating the substrate with only one single-strand specific nuclease.

[0016] In some embodiments, step (c) comprises incubating the substrate with two or more different single-strand specific nucleases.

[0017] In some embodiments, the substrate is sequentially incubated with two or more different single-strand specific nucleases, and the substrate is washed after each incubation.

[0018] In some embodiments, the substrate is incubated with two or more single-strand specific nucleases simultaneously.

[0019] In some embodiments, one or more single-strand specific nucleases include micrococcal nuclease.

[0020] In some embodiments, one or more single-strand specific nucleases include mung bean nuclease.

[0021] In some embodiments, one or more single-strand specific nucleases include micrococcal nuclease and mung bean nuclease.

[0022] In some embodiments, the only single-strand specific nuclease is mung bean nuclease.

[0023] In some embodiments, two or more different nucleases include micrococcal nuclease and mung bean nuclease.

[0024] In some embodiments, the sample is obtained from tissue, and the method further includes quantifying the level of PMO in the tissue based on the level of the detectable signal detected in step (e).

[0025] In some embodiments, the tissue is selected from blood, kidney, liver, gastrointestinal tract, lung, muscle, spleen, brain, spinal cord, or combinations thereof.

[0026] In some embodiments, the blood tissue is plasma or includes it.

[0027] In some embodiments, the muscle tissue is diaphragm, gastrocnemius, tibialis anterior (TA), biceps, heart, and / or quadriceps femoris.

[0028] In some embodiments, the sample includes a PMO containing a sequence of 20-30 consecutive nucleotides and having a nucleotide sequence selected from at least one exon of the mammalian dystrophin gene.

[0029] In some embodiments, at least one exon is selected from 23, 44, 45, 46, 51, or 53.

[0030] In some embodiments, the mammalian dystrophin gene is the human dystrophin gene.

[0031] In some embodiments, the PMO has been previously delivered to a patient, and the PMO is conjugated to a peptide for delivery (P-PMO).

[0032] In some aspects, the disclosure provides a method for evaluating the tissue distribution of a P-PMO, the method comprising performing the method of any one of the preceding claims on one or more samples obtained from one or more tissues of a subject to which the P-PMO has been administered.

[0033] In some embodiments, the method is performed on two or more samples obtained from two or more tissues of the subject.

[0034] In some embodiments, the method is repeated for different P-PMOs.

[0035] In some aspects, the disclosure provides a method for evaluating the ability of a P-PMO to modulate the expression of a target protein, the method comprising performing the method of any one of the preceding claims on a sample obtained from one or more tissues of a subject to which the P-PMO has been administered, and comparing the level of PMO derived from the P-PMO in the tissue to the level of the target protein in the tissue.

[0036] In some embodiments, the method is performed on two or more samples obtained from two or more tissues of the subject.

[0037] In some embodiments, the method is repeated for different P-PMOs.

[0038] In some embodiments, the target protein is dystrophin.

Brief Description of the Drawings

[0039]

Figure 1

[0040]

Figure 2

[0041]

Figure 3

[0042]

Figure 4-1

Figure 4-2

[0043]

Figure 5

[0044]

Figure 6

Mode for Carrying Out the Invention

[0045] Definitions In this application, unless otherwise apparent from the context, (i) the term "a" can be understood to mean "at least one"; (ii) the term "or" can be understood to mean "and / or"; (iii) the terms "comprising" and "including" can be understood to include the components or steps listed as items, whether presented by themselves or in conjunction with one or more additional components or steps; (iv) the terms "about" and "approximately" can be understood to allow for a standard deviation as understood by those skilled in the art; (v) ranges that include endpoints are provided.

[0046] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of the agent that is the composition or is included in the composition. One of ordinary skill in the art knows the various routes that can be utilized for administration to a subject, such as a human, in appropriate circumstances. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration is bronchial (e.g., by bronchial instillation), intraoral, cutaneous (e.g., one or more of and including topical to the epidermis, intradermal, intercutaneous, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular (intracerebroventricular), within a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by tracheal instillation), vaginal, vitreous, etc. In some embodiments, administration can include only a single dose. In some embodiments, administration can include the application of a fixed number of doses. In some embodiments, administration can include dosing that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period). In some embodiments, administration can include continuous dosing (e.g., infusion) for at least a selected period. In some embodiments, administration can depend on one or more results of a diagnostic or monitoring assay to determine the concentration of a particular administered agent in a sample (e.g., tissue, serum, etc.) from the subject to which the agent was administered.

[0047] Nuclease: As used herein, the term "nuclease" refers to a polypeptide capable of cleaving a bond. In some embodiments, the bond is a phosphodiester bond between nucleotide subunits of a nucleic acid. In some embodiments, the bond is between a peptide and an oligonucleotide. In some embodiments, the nuclease is a single-strand specific nuclease. In some embodiments, the nuclease is a mung bean nuclease. In some embodiments, the nuclease is a micrococcal nuclease.

[0048] Nucleic acid: As used herein, in its broadest sense, refers to an oligonucleotide chain or any compound and / or substance that can be incorporated therein. In some embodiments, a nucleic acid is an oligonucleotide via phosphodiester bonds or a compound and / or substance that can be incorporated therein. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" is RNA or includes it; in some embodiments, "nucleic acid" is DNA or includes it. In some embodiments, "nucleic acid" refers to a nucleic acid molecule. For example, in some such embodiments, a nucleic acid can refer to a polymer of deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form containing nucleotides or analogs thereof. Such nucleic acids can also be referred to by the term "polynucleotide" and / or used interchangeably therewith. In some embodiments, a nucleic acid is partially or wholly single-stranded; in some embodiments, a nucleic acid is partially or wholly double-stranded. In some embodiments, a nucleic acid is, includes, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, includes, or consists of one or more synthetic nucleic acid residues. In some embodiments, a nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, includes, or consists of one or more "locked nucleic acids" known in the art. Alternatively or additionally, in some embodiments, "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, a nucleic acid is or includes phosphorodiamidate morpholino oligonucleotides (PMOs).In some embodiments, the oligonucleotide is phosphorothioate-linked 2'-O-methyl RNA (2’OMeP). In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid has a residue length of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is a complement of a sequence encoding a polypeptide.

[0049] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a polymer of nucleic acids that can be designed in accordance with the intention of the subject in need thereof and / or can be administered to the subject in need thereof. In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO), includes an antisense oligonucleotide (ASO), or functions as an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more nucleic acid bases in length. In some embodiments, the oligonucleotide is a plurality of types of nucleic acids described herein or includes them. In some embodiments, the oligonucleotide is an ASO that can facilitate the generation of a target protein or a functional portion thereof that is not generated in the absence of the ASO when administered to the subject in need thereof. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, the oligonucleotide is conjugated to a peptide (e.g., P-PMO). In some embodiments, the oligonucleotide is a phosphorothioate-linked 2'-O-methyl RNA (2'OMeP).

[0050] Oligonucleotide Probe: As used herein, the term "oligonucleotide probe" refers to a polymer of nucleic acids that can be used in an assay (e.g., a detection assay). In the present disclosure, the oligonucleotide probe may also be referred to as the term "probe" or used interchangeably therewith. In some embodiments, an oligonucleotide probe used in an assay designed to detect the presence or absence of one or more oligonucleotides or candidate oligonucleotides. For example, in some embodiments, the oligonucleotide probe contacts a sample containing the oligonucleotide. In some such embodiments, the methods described herein are used to determine the presence and, where appropriate, the amount of an oligonucleotide designed to be detected by the oligonucleotide probe. In some embodiments, the oligonucleotide probe is a deoxyribonucleotide or ribonucleotide in either single-stranded or double-stranded form, or includes nucleotides or analogs thereof. In some embodiments, the oligonucleotide probe is or includes a plurality of types of nucleic acids described herein. For example, in some embodiments, the oligonucleotide probe is or includes phosphorothioate nucleic acid ("PTO") and DNA (PTO / DNA). In some embodiments, the oligonucleotide probe includes locked nucleic acid and DNA (LNA / DNA). In some such embodiments, PTO or LNA is localized at the 5' and 3' ends of the provided oligonucleotide probe. In some embodiments, the number of PTO or LNA residues at the 5' and 3' ends of the provided oligonucleotide probe is not the same. For example, in some embodiments, the 5' end of the oligonucleotide probe may have 3 LNAs, and its 3' end may have 5 or 6 LNAs. In some embodiments, a particular combination of nucleotides (e.g., LNA / DNA) in the oligonucleotide probe provides one or more advantages in an assay (e.g., a detection assay) compared to other combinations of nucleotides (e.g., PTO / DNA).In some embodiments, the oligonucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleobases in length.

[0051] Single-strand specific nuclease: As used herein, the term "single-strand specific nuclease" refers to a nuclease that preferentially cleaves bonds in single-stranded nucleic acid polymers. In some embodiments, the nucleic acid polymer is an oligonucleotide probe. In some such embodiments, the oligonucleotide probe is present in the mixture in excess, and the single-strand specific nuclease cleaves the probe for washing and / or removal. In some such embodiments, multiple nucleases are used sequentially or simultaneously. In some embodiments, the single-strand specific nuclease is mung bean nuclease. In some embodiments, the single-strand specific nuclease is micrococcal nuclease.

[0052] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments a pre-birth human form). In some embodiments, the subject is afflicted with a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject exhibits no symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics specific to the susceptibility or risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or therapy has been or is to be applied. [Best Mode for Carrying Out the Invention]

[0053] The present disclosure describes a method for detecting an oligonucleotide in a sample from a subject to whom the oligonucleotide has been administered. In particular, the present disclosure describes a method for quantifying and / or identifying one or more oligonucleotides in one or more samples from a subject to whom one or more oligonucleotides have been administered. The methods described herein provide a detection method with unexpected sensitivity, accuracy, and reproducibility compared to previously available assays.

[0054] Detection method The present disclosure provides, inter alia, a method for detecting an oligonucleotide in a given sample. These detection methods are far more sensitive, accurate, and reproducible than any previously available assay for detecting the oligonucleotides described herein. In some embodiments, the assay used for detection of the oligonucleotide comprises the steps of incubating with an oligonucleotide probe, incubating a mixture comprising the oligonucleotide and the oligonucleotide probe with a surface comprising a capture agent, performing one or more digestion steps on the mixture incubated with the surface, and subsequently performing one or more steps comprising contacting the sample with a substrate and a detection agent for detecting and / or quantifying the oligonucleotide in the sample.

[0055] In some embodiments, the sample comprises an oligonucleotide. In some embodiments, the oligonucleotide is a PMO. In some embodiments, the sample comprises serum or tissue to which the oligonucleotide has been added in vitro. In some embodiments, the sample comprises serum or tissue from a subject to whom the oligonucleotide has been administered. In some embodiments, the sample comprises serum or tissue from a subject to whom the oligonucleotide has not been administered.

[0056] In some embodiments, the presence and / or amount of the oligonucleotide is detected using an oligonucleotide probe.

[0057] In some embodiments, incubating a sample containing an oligonucleotide with an oligonucleotide probe results in hybridization between the oligonucleotide probe and a portion of the oligonucleotide.

[0058] In some embodiments, the detection method includes the step of incubating a sample with an oligonucleotide probe. In some embodiments, the sample contains one or more oligonucleotides.

[0059] In some embodiments, the hybridization or incubation includes annealing. In some embodiments, such hybridization or incubation that includes annealing is performed at one or more annealing temperatures. In some such embodiments, the annealing temperature is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 °C.

[0060] In some embodiments, a sample containing an oligonucleotide is subjected to one or more digestion steps prior to incubation with the oligonucleotide probe to cleave and remove peptides from the oligonucleotide and / or the sample. In some embodiments, the one or more digestion steps include a nuclease (e.g., trypsin). In some embodiments, the one or more digestion steps include a plurality of nucleases. In some embodiments, the nuclease is a nuclease that cleaves the bond between the peptide and the oligonucleotide.

[0061] In some embodiments, the detection method includes incubating a sample / oligonucleotide probe mixture with a plate coated with a capture agent. In some such embodiments, the capture agent binds to the oligonucleotide probe (e.g., via a covalently attached label), thereby associating the oligonucleotide probe and the hybridized oligonucleotide with the plate.

[0062] In some embodiments, the detection method includes washing the plate after the sample / oligonucleotide probe mixture has been incubated with the plate coated with the capture agent. In some such embodiments, the washing removes oligonucleotide probes and / or oligonucleotides not associated with the plate.

[0063] In some embodiments, the detection method includes incubating a plate with which one or more oligonucleotide probes are associated (each of which oligonucleotide probes may or may not be hybridized to an oligonucleotide) with one or more single-strand specific nucleases. In some embodiments, the single-strand specific nuclease is Micrococcus nuclease. In some embodiments, the single-strand specific nuclease is mung bean nuclease. In some embodiments, both Micrococcus nuclease and mung bean nuclease are used in the detection methods described herein.

[0064] In some such embodiments, the incubation includes incubation with micrococcal nuclease followed by incubation with mung bean nuclease. In some such embodiments, a step of washing the plate is included between the incubations with micrococcal nuclease and mung bean nuclease. In some embodiments, the incubation includes only micrococcal nuclease. In some embodiments, the incubation includes only mung bean nuclease. In embodiments that include the same nuclease (e.g., mung bean nuclease), any washing step may be included between a first incubation and a second incubation (e.g., incubations that both include the same nuclease).

[0065] In some embodiments, micrococcal nuclease and / or mung bean nuclease preferentially cleave excess single-stranded oligonucleotide probes. In some embodiments, mung bean nuclease has higher specificity for such cleavage than micrococcal nuclease.

[0066] In some embodiments, a substrate incubated with one or more single-strand specific nucleases is further incubated with a detection agent. In some embodiments, when the detection agent is incubated with one or more components, a detectable signal is generated. In some such embodiments, the detection agent interacts with an oligonucleotide probe (e.g., having a covalently attached label) to generate a detectable signal. In some embodiments, a detectable signal is generated only when the oligonucleotide probe hybridizes to an oligonucleotide.

[0067] In some embodiments, a detectable signal is detected. In some embodiments, the detection is colorimetric analysis. In some embodiments, the detection is non-colorimetric analysis. In some such embodiments, the detection is a measurement of optical density or includes it. In some embodiments, the detection is determined using fluorescence detection using a set excitation wavelength and / or emission wavelength.

[0068] In some embodiments, the detectable signal is generated using a probe-specific targeting site. In some embodiments, the probe-specific targeting site includes a conjugated enzyme agent (e.g., an antibody-enzyme conjugate). In some embodiments, the probe-specific targeting site conjugate includes an antibody conjugate. In some embodiments, the antibody conjugate includes an anti-digoxigenin antibody conjugated to an enzyme site (e.g., alkaline phosphatase). In some embodiments, a suitable substrate (e.g., a suitable alkaline phosphatase substrate, e.g., 2’-[2-benzothiazolyl]-6’-hydroxybenzothiazole phosphate [BBTP]) is added to the assay plate and incubated for a suitable time for subsequent reactions to occur to enable the generation of a fluorescence signal. In some embodiments, the suitable period for enabling the generation of the fluorescence signal is less than 2 hours, less than 90 minutes, less than 60 minutes, less than 45 minutes, or less than 30 minutes. In some embodiments, the suitable period for enabling the generation of the fluorescence signal is suitably 30 minutes. In some embodiments, the fluorescence excitation occurs in the range of 400 - 500 nm, 410 - 490 nm, 420 - 480 nm, 430 - 470 nm, 440 - 460 nm, 440 - 450 nm, or approximately 444 nm. In some embodiments, the fluorescence emission detection occurs in the range of 500 - 600 nm, 510 - 590 nm, 520 - 580 nm, 530 - 570 nm, 540 - 560 nm, 550 - 560 nm, or approximately 555 nm. Those skilled in the art will recognize that alternative detection agents and related signal detection methods may be suitable for use in the assays described herein.

[0069] In some embodiments, the assays described herein can detect the presence and / or amount of an oligonucleotide in a given sample at a lower detection level than other existing or previously used assays. In some embodiments, the level of the oligonucleotide is below the detection level. In some embodiments, the level of the oligonucleotide detected detects all oligonucleotides contained in a given sample. In some embodiments, the level of the oligonucleotide detected is equal to or less than the amount of oligonucleotide administered to the subject. In some such embodiments, the equal or lesser amount is due to dilution in the subject and not due to the detection level. That is, in some embodiments, the assays described herein that include the oligonucleotide probes described herein can accurately quantify the amount of oligonucleotide present in a given sample.

[0070] Oligonucleotide Design In some embodiments, the present disclosure provides one or more oligonucleotide probes for use in the detection of one or more oligonucleotides. In some embodiments, the one or more oligonucleotide probes are or include one or more types of nucleic acids (e.g., locked nucleic acid and deoxyribonucleic acid). In some such embodiments, the use of the oligonucleotide probes of the present disclosure in the methods described herein results in improved detection of oligonucleotides as compared to previously available detection methods. For example, the use of the methods described herein exhibits improved accuracy, sensitivity, and / or reproducibility in the detection of one or more oligonucleotides in a given sample as compared to previously available methods for detecting oligonucleotides in a sample.

[0071] Oligonucleotide Probe In some embodiments, the oligonucleotide probe is, or comprises, one or more nucleic acids. In some embodiments, the oligonucleotide probe is, or comprises, one or more modified nucleic acids. For example, in some embodiments, the oligonucleotide probe is, or comprises, PTO and DNA nucleic acids. In some embodiments, the oligonucleotide probe is, or comprises, LNA and DNA nucleic acids. In some embodiments, for example, an oligonucleotide probe comprising PTO and DNA or, for example, LNA and DNA is arranged such that PTO or LNA residues are present at the 3' and 5' termini of the oligonucleotide probe and sandwich a central region comprising DNA. In some embodiments, the LNA / DNA oligonucleotide comprises a biotin label at the 3' terminus and / or a digoxigenin label at the 5' terminus.

[0072] Oligonucleotide In some embodiments, the oligonucleotide is, or comprises, PMO. In some embodiments, the oligonucleotide is conjugated to a peptide (e.g., to generate P-PMO). In some embodiments, a sample comprising the oligonucleotide is subjected to one or more digestion steps prior to incubation with the oligonucleotide probe to cleave and remove the peptide from the oligonucleotide and / or the sample. In some embodiments, the oligonucleotide is between about 10 and 100 nucleotides in length. In some embodiments, the oligonucleotide is between about 12 and 85 nucleotides in length. In some embodiments, the oligonucleotide is between about 15 and 60 nucleotides in length. In some embodiments, the oligonucleotide is between about 20 and 50 nucleotides in length. In some embodiments, the oligonucleotide is between about 25 and 35 nucleotides in length.

[0073] Method of using an oligonucleotide detection assay Prior to the insights provided by the present disclosure, related assays for detecting oligonucleotides have had limited reproducibility, accuracy, and / or sensitivity. The methods of the present disclosure are not limited to any particular type of detection of oligonucleotides in any particular situation. For example, in some embodiments, the methods of the present disclosure can be used to monitor a patient's response to the delivery of an oligonucleotide described herein. In some embodiments, the methods of the present disclosure can be used in the evaluation of the efficacy and toxicity of candidate oligonucleotides during research and development.

[0074] The present disclosure provides a detection method that achieves unprecedented sensitivity and also exhibits reproducibility and accuracy. Such methods can be used for any part of the development and use of oligonucleotides, for example, from preclinical candidate screening to monitoring in vivo the levels of oligonucleotides in one or more samples from a patient. In some embodiments, such in vivo monitoring can include, for example, whole serum level monitoring or monitoring the levels in a specific sample from an organ or tissue (such as muscle) to determine the amount of oligonucleotide reaching that organ or tissue. In some embodiments, such in vivo monitoring can include, for example, detecting the levels in a sample from an organ or tissue (such as the kidney, muscle, etc.) to monitor the oligonucleotide burden or concentration in a specific organ or tissue. In some embodiments, the organ or tissue is one or more of blood, plasma, serum, skin, lung, heart, gastrocnemius muscle, biceps muscle, tibialis anterior muscle (TA), quadriceps muscle, diaphragm, central nervous system tissue (such as the brain, spinal cord), or combinations thereof.

[0075] In some embodiments, the present disclosure utilizes improved oligonucleotide probes for detecting oligonucleotides administered to a subject in need thereof. For example, in some embodiments, one or more oligonucleotides are administered to a subject in need thereof. In some such embodiments, one or more oligonucleotide probes can be designed and used to detect one or more oligonucleotides administered to and / or being administered to the subject.

[0076] In some embodiments, the methods of the present disclosure include administering and / or detecting an oligonucleotide or candidate oligonucleotide. In some embodiments, the methods of the present disclosure include administering and / or detecting multiple types of oligonucleotides.

[0077] In some embodiments, detection of the oligonucleotide is performed after administration. In some embodiments, detection is performed about 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, or 72 hours or more after administration. In some embodiments, detection is performed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after administration. In some embodiments, detection is performed at regular intervals (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more). In some embodiments, the administration is the first administration of the oligonucleotide. In some embodiments, the administration is the most recent administration of the oligonucleotide, but does not necessarily have to be the first administration.

[0078] In some embodiments, the sample includes serum and / or tissue from a subject to whom an oligonucleotide has been administered. In some embodiments, the sample includes serum and / or tissue from a subject to whom an oligonucleotide has not been administered (e.g., a control subject that has ingested an oligonucleotide or a placebo oligonucleotide).

[0079] Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is an X-linked recessive muscular dystrophy that causes muscle degeneration / wasting and currently leads to death. DMD affects approximately 1 in 3,500 males. DMD is caused by one or more changes in the dystrophin gene that result in some, most, or all loss of the disrupted reading frame and expression and / or production of the functional dystrophin protein. Dystrophin is an important structural component within muscle tissue, and changes or absence of dystrophin results in abnormal membrane function in muscle cells.

[0080] The dystrophin gene is located on the X chromosome, and males (XY sex chromosomes) are hemizygous for the gene, typically showing more severe symptoms and progressing more rapidly than females. Symptoms are known to include early physical disability and mortality. In some cases, the symptom development trajectory in males can include loss of mobility until adolescence, defects in respiratory and cardiac function until late teens, and death in early adulthood. On the other hand, females (XX sex chromosomes) carrying heterozygous dystrophin mutations typically show a milder phenotype. Changes in the dystrophin gene that preserve the reading frame result in the less severe, non-life-threatening Becker muscular dystrophy (BMD).

[0081] The dystrophin gene is a large one with 79 exons, and the most common genetic changes in DMD include genomic deletions of one or more exons. Most commonly, such deletions include regions around or within and / or at the 5' end of exons 44 - 55 of the gene. Currently, there is no known cure for DMD, but in addition to the previous use of corticosteroids, certain antisense-oligonucleotide-based therapeutic agents have been approved for use in patients with DMD.

[0082] Treatment based on exon skipping induced by oligonucleotides acts through targeted exon skipping during the splicing process that generates functional dystrophin mRNA translated into a functional protein. This process uses a complex multi-particle cellular mechanism that brings adjacent exon-intron junctions in pre-mRNA close to each other, enables cleavage of the phosphodiester bond present at the end of the intron, and ultimately splices the exons together. As is well known currently in the art, the use of a given antisense oligonucleotide (ASO) can facilitate the bypass or removal of a given error in the transcribed pre-mRNA molecule from the mature coding mRNA transcript when spliced. For example, skipping of out-of-frame mutations in the dystrophin gene can result in restoration of the reading frame and production of a functional (albeit truncated) dystrophin protein.

[0083] There are various types of oligonucleotides based on DNA and / or RNA backbones or analogs thereof, as well as oligonucleotide conjugates to various types of peptides for improved cell penetration. Nevertheless, there remains an unmet need for the ability to accurately and sensitively detect such oligonucleotides in samples from patients. When treatment is to involve the use of oligonucleotides, it is extremely important that accurate, sensitive, and reproducible detection of those oligonucleotides be possible. The techniques provided by this disclosure provide accurate, sensitive, and reproducible detection of such oligonucleotides.

[0084] This disclosure is further illustrated by the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure in any way.

Examples

[0085] The present disclosure exemplifies a method for detecting one or more oligonucleotides in a sample.

[0086] Example 1: Oligonucleotide Detection Assay This example describes an exemplary assay for the detection of oligonucleotides in a sample. The assay of the present disclosure exceeds and significantly improves, surprisingly, the detection ability beyond any currently available assay used for detecting oligonucleotides. The assay described herein exhibits improved accuracy, sensitivity, and reproducibility compared to other available detection methods.

[0087] In this example, the oligonucleotide was first provided in a composition containing a peptide and an oligonucleotide as a peptide phosphorodiamidate morpholino oligonucleotide (P-PMO).

[0088] Probe Sequences and Design This assay used locked nucleic acid (LNA) / DNA oligonucleotide probes for the detection of oligonucleotides. The LNA / DNA oligonucleotide probes were designed using LNA bases at the 5' and 3' ends of each oligonucleotide probe and compared with phosphorothioate (PTO) / DNA oligonucleotide probes as shown in Figure 2 (for an explanation of PTO / DNA probes, see, for example, Burki et al., 2015, Nucleic Acid Therapeutics, 25(5), pp. 275-84).

[0089] Exemplary Sequences and Oligonucleotides In this example, the oligonucleotides were derived from an antisense oligonucleotide conjugated with a peptide and an antisense oligonucleotide not conjugated with a peptide. Specifically, as shown in Figure 2a, a 25-mer PMO antisense sequence against mouse dystrophin exon 23 (M23D) having the sequence (5'-GGCCAAACCTCGGCTTACCTGAAAT-3'; SEQ ID NO: 1) was designed in-house and ordered from a commercial oligonucleotide supplier. For the oligonucleotides conjugated with a peptide, peptide-PMO ("P-PMO") was synthesized by conjugation of M23D PMO with one of two cell-penetrating peptides (peptide A or B for generating P-PMO A and / or P-PMO B), each of which contains 18 amino acids).

[0090] Oligonucleotide probe Complementary oligonucleotide probes were designed for each of P-PMO A and P-PMO B and ordered from Exiqon (LNA / DNA probes; Woburn, MA) or IDT (PTO / DNA probes; Coralville, IA). As shown in Figure 2a, the oligonucleotide probes for M23D PMO were a “cleaved” 19-mer LNA / DNA probe (3’-TTTGGAGCCGAATGGACTT-5’; SEQ ID NO: 2; LNA bases highlighted in bold and underlined in Figure 2a) and a “full-length” 25-mer PTO / DNA probe (3’-CCGGTTTGGAGCCGAATGGACTTTA-5’; SEQ ID NO: 3; PTO bases highlighted in bold in Figure 2a). Both probes were tested during assay development, and after finalizing the assay methodology, the cleaved probe was used for all subsequent assays (e.g., quantitative assays, such as in vivo detection and quantification of PMO levels, etc.). Both oligonucleotide probes were dual-labeled with biotin at the 3’ end and digoxigenin at the 5’ end. Lyophilized oligonucleotide probes were suspended in nuclease-free water at 10 μM to prepare stock solutions. PMO and P-PMO were suspended in distilled water at a 10 mg / ml stock. Probes were aliquoted and stored at -20 °C, and PMO and P-PMO were aliquoted and stored at -70 °C. All probes, PMO, and P-PMO were heated at 65 °C for 15 minutes, briefly vortexed, and then ELISA was initiated.

[0091] ELISA-based detection assay PMO or P-PMO serial dilutions for standard curves and quality controls in 5.0% mouse serum, and 5.0 mg / ml or 1.0 mg / ml tissue homogenates were prepared using 1xTE buffer supplemented with 0.1% v / v Triton® X-100 with an initial concentration of 51.2 nM PMO / P-PMO. Serum samples were diluted 20-fold and tissues were homogenized and diluted to 5.0 mg / ml or 1.0 mg / ml tissue homogenates with the same buffer. 200 μL of calibrator solutions, quality controls, and / or samples were treated overnight at 37 °C in a deep 1.0-mL 96-well plate with a thermomixer C (600 rpm) with 20 μL of 40 mg / ml trypsin (purchased as a lyophilized powder from Sigma-Aldrich) to cleave the peptide component and release the PMO. 2.5 nM oligonucleotide probe in 40 μL of hybridization buffer (with 0.1% serum) was added to each well. The plate was then sealed and incubated at 65 °C for 15 min (600 rpm) and cooled at room temperature for 15 min. The plate was then incubated at 50 °C for 30 min (500 rpm) to hybridize the oligonucleotide probe to the PMO. 150 microliters of the hybridized solution was then transferred to a plate coated with NeutrAvidin (washing buffer: 50 mM Tris-HCl, 150 mM sodium chloride, pH 7.6, pre-washed using 0.1% v / v Tween®-20 - the same washing buffer was used for all subsequent washing steps) and incubated at 37 °C for 30 min to bind the biotin-labeled probe to the plate coated with NeutrAvidin. The plate was then washed three times and 150 μL of 0.2 U / μl micrococcal nuclease (in 50 mM Tris-HCL; pH 8.2, 200 mM NaCl, 5 mM CaCl, and 0.1 mg / mL bovine serum albumin) was added to each well and incubated at 37 °C for 1.5 h (150 rpm).The plate was then washed three times and 120 μL of 0.3 U / μL mung bean nuclease (in 30 mM NaCl; pH 8.2, 50 mM sodium acetate, 1 mM ZnSO4; pH 5.0) was added to each well and incubated at 37 °C for 1.5 h (150 rpm). The plate was washed three times and anti-digoxigenin antibody conjugated to alkaline phosphatase was added at a dilution of 1:5,000 in SuperBlock (TBS) blocking buffer with 0.25% v / v Tween®-20, incubated at 37 °C for 30 min and washed three times. Then, 125 μL of AttoPhos substrate was added to each well, the plate was sealed in aluminum foil and incubated at 37 °C for 30 min (150 rpm). The fluorescence intensity at 444 nm excitation and 555 nm emission was measured by a Molecular Service SpectraMax MT5 microplate reader.

[0092] PTO / DNA vs. LNA / DNA oligonucleotide probes

[0093] When compared in the ELISA-based detection assay of this example, the PTO / DNA oligonucleotide probe yielded a minimal fluorescence signal when the oligonucleotide was present at less than 64 pM (see Figures 2b and 2c). Surprisingly, in contrast, the LNA-containing oligonucleotide probe yielded a significantly higher fluorescence signal than the PTO / DNA oligonucleotide probe, including at low oligonucleotide concentrations. The signal-to-noise ratio and limit of quantification were also improved using the LNA-containing oligonucleotide probe compared to the PTO-containing oligonucleotide probe. For example, the lower limit of quantification ("LLOQ") for the PTO / DNA oligonucleotide probe was less than 8 pM (S:N < 2; see Table 1 below), which is consistent with past reports for this type of probe (Burki et al., 2015, Nucleic Acid Therapeutics, 25(5), pp. 275-84). In contrast, an oligonucleotide at a concentration of 2 pM was detected using the LNA / DNA oligonucleotide probe, and a steady S:N was observed (S:N = 5.2).

[0094] Annealing and digestion This example also describes a defined procedural modification to an existing assay that surprisingly and significantly improved accuracy, reproducibility, and decreased the limit of detection of oligonucleotides in samples to picomolar concentrations.

[0095] In this example, both the annealing temperature and digestion conditions were varied and analyzed (see Figures 2d and 2e). When the oligonucleotide annealing temperature was increased compared to that of the existing assay, an improvement in signal was observed (Figure 2d). The signal intensity remained unchanged in the presence or absence of a second digestion step using mung bean (MB) nuclease (Figure 2e). However, the S:N was improved when residual single-stranded oligonucleotides were digested in the presence of mung bean (MB) nuclease, particularly at pH 5 (see Table 2 below).

[0096] The detection assay was quantified in mouse serum. The accuracy and reproducibility of the standard curve were analyzed using at least eight prepared standards (calibrators) made by spiking in known concentrations of PMO diluted in 5% mouse serum. Figures 3a and 3b show the reproducibility and accuracy of the detection assay using P-PMO standards prepared in four different sera and run in two independent assays. Quality control samples were considered acceptable if the back-calculated concentration had a CV of less than 25%. Tables 3 and 4 show the assay accuracy in different serum preparations using different P-PMOs. The CV was generally less than 20%, except for the detection of P-PMO A at the LLOQ of 1 pM (29.7%). The inter-batch variation was less than 20% between assays, except for P-PMO A at 1 pM (20.3%).

[0097] The detection assay described in this example was also verified using several mouse tissues. The assay detects and measures the level of PMO after trypsin digestion (which cleaves the peptide ("P") from the PMO). The P-PMO level is estimated through detection of the PMO level determined by an assay as described herein. The low PMO uptake in tissues and the peptide conjugation to generate P-PMO are known to facilitate uptake into tissues; thus, most of the PMO detected in tissues is presumed to be due to uptake mediated by P-PMO. Table 5 shows the average detection values obtained at the LLOQ across these tissues listed in the table. The variation was less than 25% for all tissues analyzed, regardless of which P-PMO was used, across the assays.

[0098] Dynamic detection using in vivo samples In CD1 mice, the serum levels of P-PMO A (by measurement of PMO) were measured over a 2-week period. P-PMO levels were determined using an oligonucleotide probe to detect the level of PMO conjugated to its delivery peptide. Figure 4a shows an initial serum concentration of 2.3 μM (- / + 0.2 μM) after 5 minutes; the left and right panels show the same data, but the right panel has a split x-axis).

[0099] The detected P-PMO A concentration decreased rapidly over the first 8 hours, at which time the detected concentration was 3.3 nM (- / + 1.1 nM). The P-PMO A serum levels remained relatively stable for 1 week, at which time the detected concentration was 1.2 nM (- / + 0.38 nM). After 2 weeks, the detected concentration was 609 pM (- / + 327 pM). Similar data were observed when analyzing P-PMO B (Figure 4b).

[0100] As shown in Figures 4c, 4d, 4e, 4f, 4g, 4h, 4i, and 4j, P-PMO levels in several tissues were also determined. In the target tissues (heart, diaphragm, gastrocnemius, tibialis anterior (TA)), detectable P-PMO concentrations decreased initially (at the 0.5 - 2 hour time point). Thereafter, the P-PMO concentrations detected in these tissues remained relatively constant for at least 3 days. P-PMO levels remained detectable (and quantifiable) throughout the study. Higher concentrations of P-PMO were detected in the first-pass organs (e.g., lungs, kidneys). Over time and 3 days after dosing, the P-PMO concentration decreased steadily. Clearance in the kidneys was slow and consistent with the effects of a known class of oligonucleotides (e.g., ASO). Both P-PMO A and P-PMO B showed similar drug levels in serum and all tissues at each time point analyzed.

[0101] To test the persistence of the oligonucleotides, serum and various tissues were also analyzed two weeks after the last dosing. Serum levels for both mice treated with PMO and P-PMO were measured within the range of 1 nM. At this time point, the overall oligonucleotide levels in the tissues were similar (Figure 5a).

[0102] To account for the differences in the doses administered, the drug levels (e.g., P-PMO A and the corresponding PMO not conjugated to the peptide) were normalized to the total number of moles dosed. Figure 5b demonstrates that the number of moles of P-PMO dosed was 1 / 43 of the number of moles of PMO not conjugated to the peptide, yet the P-PMO was detected at much higher concentrations in serum and tissues than the PMO not conjugated to the peptide. Furthermore, the oligonucleotide concentration was at least 60-fold higher in several target tissues after P-PMO treatment compared to treatment with the PMO not conjugated to the peptide.

[0103] These data demonstrate the unexpected sensitivity of this assay for detecting oligonucleotides for at least two weeks after the last dosing. Evaluation of oligonucleotide levels after even longer periods following administration may be clinically useful, for example, if oligonucleotide levels can correlate with efficacy. Thus, to determine the sensitivity of the detection assay described in this example, PMO levels in serum and several muscle tissues were monitored for 13 weeks after drug administration (e.g., after oligonucleotide administration, e.g., after P-PMO administration). Figure 6a shows that P-PMO B was detectable in the serum of mice dosed at 6 or 12 mg / kg for up to 4 weeks after administration, but was no longer detectable within the assay's detection limit by 8 weeks after administration. At doses of 20 and 40 mg / kg, P-PMO B was still detectable in serum at 8 weeks after administration, but was no longer detectable by 13 weeks. Analysis of certain exemplary skeletal muscles (6b heart, 6c diaphragm, 6d TA, and 6e quadriceps femoris) showed detectable oligonucleotides in all tissues for up to 13 weeks (the duration of the study) after administration.

[0104] The advantage of the assay of the present disclosure compared to any other available assay is demonstrated by the increased sensitivity that allows for a reliable and accurate quantification of P-PMO levels at 13 weeks after administration using the methods described herein for detection at levels that were previously undetectable by other assays. The average P-PMO concentrations detected at 13 weeks after administration were 3 - 10 picomoles / g in the heart; 1 - 90 picomoles / g in the diaphragm; 1.8 - 15.8 picomoles / g in the TA; and 0.6 - 5.7 picomoles / g in the quadriceps femoris.

[0105] Importantly, the extremely low concentrations of oligonucleotides detected in the assays described herein could not be detected or quantified at such low levels in other previously described assays (Burki et al., 2015, Nucleic Acid Therapeutics, 25(5), pp. 275-84), demonstrating the superiority of the assays of the present disclosure. Also, when data from two different in vivo studies (represented by the data in FIGS. 4 and 6) were compared with data obtained using an ELISA assay with in vitro samples (such as those in FIG. 1, etc.), the results were the same, demonstrating the reproducibility of the assay(s) of the present disclosure. The data for week 1 for the heart, TA, and diaphragm were compared (between the studies shown in FIGS. 4 and 6 at a dose of 6 mg / kg), and there were some individual outliers (two each for TA and diaphragm), but the data (excluding outliers) were overall equivalent between the studies and were reproducible. The levels from the two different studies were as follows at the week 1 time point for each tissue assayed, as represented by the data shown in FIGS. 4 and 6: heart, 36 vs 55 picomoles / g; diaphragm, 42 vs 43 picomoles / g; TA, 15 vs 23 picomoles / g. Table

Table 1

Table 2

Table 3

Table 4

Table 5

[0106] Equivalents The present invention has been described in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate and not limit the scope of the present invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. In certain embodiments, for example, the following items are provided. (Item 1) (a) Incubating a sample containing an oligonucleotide with an oligonucleotide probe, wherein the oligonucleotide probe hybridizes with the oligonucleotide in the sample, the incubating step; (b) Incubating the product of step (a) with a substrate coated with a capture agent, wherein the capture agent binds to the oligonucleotide probe, thereby associating excess oligonucleotide probe and hybridized oligonucleotide with the substrate, and optionally washing the plate after the hybridized probe is associated with the substrate, the incubating step; (c) Incubating the substrate after step (b) with two or more different single-strand specific nucleases having different specificities for the substrate, wherein the substrate is optionally washed with one or more washing solutions before, during or after step (c), and the oligonucleotide probe and hybridized oligonucleotide remain associated with the substrate during one or more washing steps, the incubating step; (d) Incubating the substrate after step (c) with a detection agent, wherein the detection agent interacts with the oligonucleotide probe to generate a detectable signal, the incubating step; and (e) Detecting the detectable signal A method comprising. (Item 2) (a) Incubating a sample containing a phosphorodiamidate morpholino oligonucleotide (PMO) with an oligonucleotide probe, wherein the oligonucleotide probe contains one or more locked nucleic acid (LNA) residues and hybridizes with the PMO in the sample, the incubating step; (b) Incubating the product of step (a) with a substrate coated with a capture agent, wherein the capture agent binds to the oligonucleotide probe, thereby associating the oligonucleotide probe and hybridized PMO with the substrate, the incubating step; (c) A step of incubating the substrate after step (b) with one or more single-strand specific nucleases, wherein the substrate is optionally washed before, during or after step (c), and the oligonucleotide probe and the hybridized PMO remain associated with the substrate during one or more washing steps; the step of incubating; (d) A step of incubating the substrate after step (c) with a detection agent, wherein the detection agent generates a detectable signal by interacting with the oligonucleotide probe; the step of incubating; and (e) A step of detecting the detectable signal A method comprising. (Item 3) The method according to item 1, wherein step (c) comprises incubating the substrate with micrococcal nuclease and mung bean nuclease. (Item 4) The method according to item 3, wherein step (c) comprises incubating the substrate with the micrococcal nuclease and then incubating the substrate with the mung bean nuclease. (Item 5) The method according to item 4, wherein the substrate is washed after being incubated with the micrococcal nuclease and before being incubated with the mung bean nuclease. (Item 6) The method according to item 1, wherein the sample comprises phosphorodiamidate morpholino oligonucleotide (PMO). (Item 7) The method according to item 1, wherein the oligonucleotide probe comprises one or more locked nucleic acid (LNA) residues. (Item 8) The method according to any one of the preceding items, wherein the oligonucleotide probe comprises one or more deoxyribonucleic acid (DNA) residues. (Item 9) The method according to item 2 or 7, wherein the oligonucleotide probe comprises a central segment composed of DNA residues sandwiched between a 5'-terminal segment and a 3'-terminal segment each containing LNA residues. (Item 10) The method according to item 9, wherein the central segment is composed of 5 to 20 DNA residues. (Item 11) The method according to item 10 or 11, wherein the 5'- and 3'-terminal segments are independently composed of 2 to 8 LNA residues. (Item 12) Step (c) of the method according to item 2, comprising incubating the substrate with only one single-strand specific nuclease. (Item 13) Step (c) of the method according to item 2, comprising incubating the substrate with two or more different single-strand specific nucleases. (Item 14) The method according to item 13, wherein the substrate is sequentially incubated with the two or more different single-strand specific nucleases, and the substrate is washed after each incubation. (Item 15) The method according to item 13, wherein the substrate is incubated simultaneously with the two or more single-strand specific nucleases. (Item 16) The method according to item 2, wherein the one or more single-strand specific nucleases include micrococcal nuclease. (Item 17) The method according to item 2, wherein the one or more single-strand specific nucleases include mung bean nuclease. (Item 18) The method according to item 2, wherein the one or more single-strand specific nucleases include micrococcal nuclease and mung bean nuclease. (Item 19) The method according to item 11, wherein the only one single-strand specific nuclease is mung bean nuclease. (Item 20) The method according to item 13 or 14, wherein the two or more different nucleases include micrococcal nuclease and mung bean nuclease. (Item 21) The method according to item 1 or 2, wherein the sample is obtained from tissue, and the method further comprises a step of quantifying the level of PMO in the tissue based on the level of the detectable signal detected in step (e). (Item 22) The method according to item 21, wherein the tissue is selected from blood, kidney, liver, gastrointestinal tract, lung, muscle, spleen, brain, spinal cord, or a combination thereof. (Item 23) The method according to item 22, wherein the blood tissue is plasma or serum, or contains the same. (Item 24) The method according to item 22, wherein the muscle tissue is diaphragm, gastrocnemius, biceps, tibialis anterior (TA), heart, and / or quadriceps femoris. (Item 25) The method according to item 1 or 2, wherein the sample contains a PMO comprising a sequence of 20 to 30 consecutive nucleotides and has a nucleotide sequence selected from at least one exon of the mammalian dystrophin gene. (Item 26) The method according to item 25, wherein at least one exon is selected from exon 23, 44, 45, 46, 51, or 53. (Item 27) The method according to item 25 or 26, wherein the mammalian dystrophin gene is the human dystrophin gene. (Item 28) The method according to any one of items 2 to 27, wherein the PMO has been delivered to the patient in the past and the PMO was conjugated to a peptide for delivery (P-PMO). (Item 29) A method for evaluating the tissue distribution of P-PMO, the method comprising performing the method according to any one of the preceding items on one or more samples obtained from one or more tissues of a subject to which the P-PMO has been administered. (Item 30) The method according to item 29, wherein the method is performed on two or more samples obtained from two or more tissues of the subject. (Item 31) The method according to item 29 or 30, wherein the method is repeated for different P-PMOs. (Item 32) A method for evaluating the ability of P-PMO to regulate the expression of a target protein, the method comprising performing the method according to any one of the preceding items on a sample obtained from a tissue of a subject to which the P-PMO has been administered, and comparing the level of PMO derived from the P-PMO in the tissue with the level of the target protein in the tissue. (Item 33) The method according to item 32, wherein the method is performed on two or more samples obtained from two or more tissues of the subject. (Item 34) The method according to item 32 or 33, wherein the method is repeated for different P-PMOs. (Item 35) The method according to any one of items 32 to 34, wherein the target protein is dystrophin.

Claims

**Claim 1**: A method for determining the presence of an oligonucleotide in a sample, comprising the following steps: (a) incubating a sample containing an oligonucleotide with an oligonucleotide probe, wherein the oligonucleotide probe hybridizes with the oligonucleotide in the sample, the step of incubating; (b) incubating the product of step (a) with a substrate coated with a capture agent, wherein the capture agent binds to the oligonucleotide probe, thereby associating the oligonucleotide probe and the hybridized oligonucleotide with the substrate, the step of incubating; (c) incubating the substrate after step (b) with two or more different single-strand specific nucleases having different specificities, wherein the two or more different single-strand specific nucleases include micrococcal nuclease and mung bean nuclease, the step; (d) incubating the substrate after step (c) with a detection agent, wherein the detection agent interacts with the oligonucleotide probe to generate a detectable signal, the step of incubating; and (e) detecting the detectable signal A method comprising the above steps. **Claim 2** The method according to claim 1, wherein the oligonucleotide in the sample comprises a phosphorodiamidate morpholino oligonucleotide (PMO), the oligonucleotide probe comprises one or more locked nucleic acid residues, and the PMO in the sample hybridizes with the oligonucleotide probe. **Claim 3** The method according to claim 1, wherein step (c) comprises incubating the substrate with micrococcal nuclease and then incubating the substrate with mung bean nuclease. **Claim 4** The method according to claim 3, wherein the substrate is washed after being incubated with micrococcal nuclease and before being incubated with mung bean nuclease. **Claim 5** The method according to claim 1, wherein the sample comprises a phosphorodiamidate morpholino oligonucleotide (PMO). **Claim 6** The method according to claim 1, wherein the oligonucleotide probe comprises one or more locked nucleic acid residues.

7. The method according to claim 1, wherein the oligonucleotide probe comprises one or more deoxyribonucleic acid (DNA) residues.

8. The method according to claim 2, wherein the oligonucleotide probe comprises a central segment composed of 5 to 20 DNA residues sandwiched between 5′-terminal and 3′-terminal segments each independently composed of 2 to 8 locked nucleic acid residues.

9. The method according to claim 2, wherein the substrate is sequentially incubated with the two or more different single-strand-specific nucleases, and the substrate is washed after each incubation.

10. The method according to claim 2, wherein the substrate is incubated simultaneously with the two or more different single-strand-specific nucleases.

11. The method according to claim 2, wherein the sample is obtained from a tissue, and the method further comprises quantifying the level of PMO in the tissue based on the level of the detectable signal detected in step (e).

12. The method according to claim 11, wherein the tissue is selected from blood, kidney, liver, gastrointestinal tract, lung, muscle, spleen, brain, spinal cord, or a combination thereof.

13. The method according to claim 12, wherein the blood is plasma or serum, or contains the same.

14. The method according to claim 12, wherein the muscle is diaphragm, gastrocnemius, biceps brachii, biceps femoris, tibialis anterior (TA), heart, and / or quadriceps femoris.

15. The method according to claim 2, wherein the sample comprises a PMO containing a sequence of 20 to 30 consecutive nucleotides and having a nucleotide sequence selected from at least one exon of the mammalian dystrophin gene.

16. The method according to claim 15, wherein at least one exon is selected from exon 23, 44, 45, 46, 51, or 53.

17. The method according to claim 16, wherein the mammalian dystrophin gene is the human dystrophin gene.

18. The method according to claim 2, wherein the PMO has been delivered to a patient in the past, and the PMO is a peptide phosphorodiamidate morpholino oligonucleotide (P-PMO) for delivery. A method for evaluating the tissue distribution of a peptide phosphorodiamidate morpholino oligonucleotide (P-PMO), said method comprising performing the method according to claim 2 on one or more samples obtained from one or more tissues of a subject to which said P-PMO has been administered.

20. The method according to claim 19, wherein the method is performed on two or more samples obtained from two or more tissues of the subject.

21. The method according to claim 19, wherein the method is repeated for different P-PMOs.

22. A method for evaluating the ability of a P-PMO to modulate the expression of a target protein, said method comprising performing the method according to any one of claims 2 to 21 on a sample obtained from a tissue of a subject to which the P-PMO has been administered, and comparing the level of PMO derived from the P-PMO in the tissue with the level of the target protein in the tissue.

23. The method according to claim 22, wherein the method is performed on two or more samples obtained from two or more tissues of the subject.

24. The method according to claim 22, wherein the method is repeated for different P-PMOs.

25. The method according to claim 22, wherein the target protein is dystrophin.

26. The method according to claim 1, wherein the substrate is washed with one or more washing solutions before, during or after step (c), and the oligonucleotide probe and the hybridized oligonucleotide remain associated with the substrate during one washing step or a plurality of washing steps.

27. The method according to claim 2, wherein the substrate is washed before, during or after step (c), and the oligonucleotide probe and the hybridized PMO remain associated with the substrate during one washing step or a plurality of washing steps.

Images