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WO2024206999A2 - Procédés et compositions concernant l'utilisation de petits arn en tant que biomarqueurs - Google Patents

Procédés et compositions concernant l'utilisation de petits arn en tant que biomarqueurs Download PDF

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WO2024206999A2
WO2024206999A2 PCT/US2024/022513 US2024022513W WO2024206999A2 WO 2024206999 A2 WO2024206999 A2 WO 2024206999A2 US 2024022513 W US2024022513 W US 2024022513W WO 2024206999 A2 WO2024206999 A2 WO 2024206999A2
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tinyrnas
tinyrna
sample
subject
disease
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WO2024206999A3 (fr
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Kotaro NAKANISHI
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present disclosure provides methods of profiling tinyRNA that can be used in detecting, diagnosing, and/or treating a disease or disorder, including but not limited to viral infections.
  • the present disclosure also provides methods of identifying tinyRNA biomarkers for detecting, diagnosing, and/or treating a disease or disorder, including but not limited to viral infections.
  • a method of associating tinyRNA with a physiological state or condition comprising isolating a sample from a subject, extracting and analyzing a plurality of tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, and determining a physiological state or condition based on the tinyRNA profile as compared to the control.
  • a method of treating a disease or disorder in a subject comprising taking a sample from the subject, extracting and analyzing tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, determining the subject has a disease or disorder based on the tinyRNA profile as compared to the control, and treating the subject with an anti-inflammatory agent, an anti-cancer agent, an antibiotic, an anti-viral, or any combination thereof.
  • a method of determining effectiveness of a treatment protocol or effectiveness of a composition for treatment of a specific disease or disorder in a subject comprising taking a sample from the subject, wherein the subject has received a treatment or a composition for treating the specific disease or disorder, extracting and analyzing tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, and determining the effectiveness of the treatment or the composition from the tinyRNA profile as compared to the control.
  • the tinyRNA is less than 18 nucleotides long. In some embodiments, the tinyRNA is 14 nucleotides longer or shorter.
  • the sample is derived from blood, urine, saliva, plasma, a tissue or a tumor.
  • the physiological state or condition comprises stress, infection, inflammation, cancer, or other disease or disorder.
  • the tinyRNA is associated with an AGO protein.
  • the control is from a healthy subject or comprises a compilation of markers from one or more healthy subjects.
  • the control comprises indicators for a given physiological state.
  • one specific physiological state or condition is analyzed based on the sample. In some embodiments, more than one specific physiological state or condition is analyzed based on the sample.
  • a ratio is determined based on expression level of one or more given tinyRNAs compared to the control.
  • RNA Sequencing (RNA seq) is used to identify tinyRNAs.
  • the tinyRNA has been trimmed in vivo by an exonuclease.
  • the exonuclease is activated upon change of physiological state or condition in the subject.
  • the exonuclease is associated with an AGO protein.
  • the AGO protein comprises AGO1, AGO2, AGO 3, or AGO4.
  • kits comprising one or more reagents for isolating and analyzing one or more tinyRNAs.
  • the one or more tinyRNAs are isolated from a sample derived from a subject.
  • the sample is derived from blood, urine, saliva, plasma, a tissue or a tumor.
  • the kit comprises any combination of an extraction buffer, a storage buffer, a washing buffer, a loading buffer, or a staining buffer.
  • the kit further comprises an agarose solution or a polyacrylamide solution.
  • Figures 1A and 1B show the miRNA and tyRNA biogenesis in human systems.
  • Figure 1A shows the gene of a miRNA is transcribed in the nucleus, processed, and transposed to the cytoplasm. Eventually, the miRNA duplex is loaded into either of four human AGOs to form the RISC (i.e., AGO with miRNA).
  • Figure 1B shows the viral infection induces ISG20. ISG20 trims AGO (WT)-associated miRNAs (top) slowly and NDD-relevant AGO mutant fast (bottom).
  • Figures 2A and 2B show the stability of small RNAs in cells.
  • Figure 2A shows the possible pathways towards AGO-associated tyRNAs.
  • Figure 2B shows the in vivo stability of 14- and 23- nt single-stranded (ss) miR-20a and their siRNA-like duplexes (ds).
  • Figures 3A, 3B, 3C, 3D, and 3E show the ISG20 and other 3′ to 5′ exonucleases trimming AGO-associated miRNAs in HEK293T cells.
  • Figure 3A shows the schematic of the experiment of Figure 3B.
  • Figure 3B shows the accumulation of the tyRNAs upon expression of ISG20.
  • Figure 3C shows the schematic of the experiment setup of Figure 3D.
  • Figure 3D shows the in vivo trimming of FLAG-AGO2-associated miR-20a by ISG20, TREX1, ERI1, PARN, and EXO5 and their catalytic mutants.
  • Figure 3E shows the tyRNA synthesis on four human AGOs by ISG20, TREX1, ERI1, and PARN. Means ⁇ SD. Docket No.103361-460WO1
  • Figures 4A and 4B show that ISG20 trims any miRNAs on any AGOs.
  • Figure 4A shows the in vitro trimming of different miRNAs by ISG20.
  • Figure 4B shows the docking model of ISG20 on the guide-bound AGO3.
  • Figure 5 shows the volcano plot of the gene expression changes after ISG20 expression. Each dot represents an mRNA. The gene whose expression level changed.
  • Figures 6A and 6B show the mutations of NDD-relevant AGO1 ( Figure 6A) and AGO2 ( Figure 6B). The crystal structures indicate that the reported mutations (grey spheres) make the PAZ domain flexible. And thus release the 3′ end of miRNA more readily than their wild type AGOs.
  • Figures 7A, 7B, 7C, and 7D show the schematic of experiments.
  • Figure 7A shows the monitor of the ISG20 levels during the four conditions (i.e., no infection, de novo infection, latency, and reactivation).
  • Figure 7B shows the in vivo trimming of 23-nt miR-20a in the cells with the four conditions. For clarity, treatment with TPA/NaBu of IgG is not shown.
  • Figures 7C and 7D tested the significance of ISG20 (Figure 7C) or other exonuclease (Figure 7D) for tyRNA generation.
  • Figure 8 shows the monitoring of the ISO20 levels during the four conditions (i.e., no infection, de novo infection, latency, and reactivation).
  • Figures 9A and 9B show that the ISG20 generates different lengths of tinyRNAs when 23- nt miR-20a is loaded into the NDD-relevant AGO1 mutants.
  • Figure 9A shows the western blots with antibodies for each protein.
  • Figure 9B shows the in vivo trimming of FLAG-AGO1- associated miR-20a by ISG20.
  • Figure 10 shows the in vitro trimming assay. The accumulation of 11-12 nt RNA species was seem in the mutants, but not in the WT.
  • Figure 11 shows the in vitro trimming assay. FLAG-AGO1(WT), (G199S), and ( ⁇ F180) were programmed with a 23-nt 5′-end radiolabeled miR-20a, followed by incubation with PARN. The reaction was resolved on a denaturing gel.
  • Figures 12A and 12B show the in vivo trimming assays.
  • the ISG20 generates unusual tyRNAs, such as 11 and 16 nt, when 23-nt miR-20a is loaded into AGO1 (Figure 12A) and AGO2 (Figure 12B) NDD-mutants.
  • Figures 13A and 13B show the structural differences between AGO1 ⁇ F180 and AGO1(WT).
  • Figure 13A shows the cryo-electron microscopy structure of AGO1 ⁇ F180 with guide RNA.
  • Figure 13B shows the structures of AGO1 AGO1 ⁇ F180 and WT (PDB ID: 4KXT) are superposed on their PIWI domain.
  • the structure of AGO1 ⁇ F180 revealed that the PAZ domain is arranged closer to the N domain compared to the AGO1(WT) structure (top box in Figure 13B).
  • the terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within Docket No.103361-460WO1 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • composition refers to any agent that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition when used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • “Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of'' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination.
  • compositions consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.
  • Docket No.103361-460WO1 A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level.
  • the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction below, above, or in between the given ranges as compared to native or control levels.
  • reduce or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
  • “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician. Docket No.103361-460WO1
  • a “control” is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or "negative.”
  • wild-type refers to the genetic and physical characteristics of the typical form of a species as it occurs in nature. A wild-type or wild type characteristic is conceptualized as a product of the standard “normal” allele at a gene locus, in contrast to that produced by a non- standard “mutant” allele.
  • diagnosis refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms. "Prognosis” may refer to a prediction of how a patient will progress, and whether there is a chance of recovery.
  • Cancer prognosis generally refers to a forecast or prediction of the probable course or outcome of the cancer.
  • cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis in a patient susceptible to or diagnosed with a cancer.
  • Prognosis may also include prediction of favorable responses to cancer treatments, such as a conventional cancer therapy.
  • tissue sample includes any material composed of one or more cells, either individual or in complex with any matrix obtained from a patient.
  • the definition includes any biological or organic material and any cellular subportion, product or by-product thereof.
  • biological fluid refers to a fluid containing cells and compounds of biological origin, and may include blood, stool or feces, lymph, urine, serum, pus, saliva, seminal fluid, tears, urine, bladder washings, colon washings, sputum or fluids from the respiratory, alimentary, circulatory, or other body systems.
  • the "biological fluids" the nucleic acids containing the biomarkers may be present in a circulating cell or may be present in cell-free circulating DNA or RNA.
  • “Expression” as used herein refers to the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce a peptide/protein end product, and ultimately affect a phenotype, as the final effect.
  • the term “genetically modified” refers to a living cell, tissue, or organism whose genetic material has been altered using genetic engineering techniques. The genetic Docket No.103361-460WO1 modification results in an alteration that does not occur naturally by mating and/or natural recombination.
  • Modified genes can be transferred within the same species, across species (creating transgenic organisms), and across kingdoms. New, exogenous genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.
  • a "gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
  • treat include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition.
  • Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of disease or disorder), during early onset (e.g., upon initial signs and symptoms of disease or disorder), or after an established development of disease or disorder.
  • interaction refers to an action that occurs as two or more objects have an effect on one another either with or without physical contact.
  • nucleotide is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA.
  • nucleic acid is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.
  • Docket No.103361-460WO1 The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem.
  • oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA.
  • double-stranded In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.
  • a single-stranded oligonucleotide can exist as a linear molecule without any hydrogen-bonded nucleotides, or can fold three-dimensionally to form hydrogen bonds between individual nucleotides along the single stranded oligonucleotide.
  • polynucleotide refers to a single or double stranded polymer composed of nucleotide monomers.
  • Polynucleotides can be any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides plasmids
  • vectors isolated DNA of any sequence, isolated
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA.
  • the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule.
  • the polynucleotide is composed of nucleotide monomers of generally greater than 100 nucleotides in length and up to about 8,000 or more nucleotides in length.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. Docket No.103361-460WO1
  • a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
  • a “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A.
  • a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
  • guide RNA refer to a specifically designed RNA sequence that recognizes a target nucleic acid of interest and directs an enzyme, including but not limited to an exonuclease enzymes and RNA-induced silencing complex enzymes (such as, for example Argonaute protein (AGOs)) to the target nucleic acid for gene editing.
  • mRNA refers to messenger ribonucleic acid, or single stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is translated by a ribosome in the process of synthesizing a protein. mRNA is created during the process of transcription, where a gene is converted into a primary transcript mRNA (or pre-mRNA).
  • the primary transcript is further processed through RNA splicing to only contain regions that will encode protein.
  • mRNA can also be targeted for epigenetic modifications, such as methylation, to impact mRNA translation, nuclear retention, nuclear export, processing, and splicing.
  • a nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nuclease can possess properties to cause double or single stranded breaks to target nucleic acids. Nucleases are commonly used in gene editing practices to modify a host genome to express or inhibit a target gene.
  • RNAi or RNA interference refers to a process where small RNA molecules, including but not limited to tinyRNA, cityRNA, siRNA, miRNA, and shRNA, can shut down gene expression by binding and blocking the mRNA, protein translation enzymes, or a combination thereof, from performing intended functions.
  • Downstream means in a direction of transcription, the direction of transcription being from a promoter sequence to an RNA-encoding sequence.
  • the direction of transcription is 3′ to 5′.
  • the direction of transcription is 5′ to 3′.
  • Upstream means in a direction opposite the direction of transcription.
  • Upstream and downstream may be used in reference to either strand of a double-stranded DNA molecule even when relative to a sequence on one strand of a double-stranded DNA molecule.
  • complementary refers to the topological compatibility or matching together of interacting surfaces of two molecules (e.g., a probe molecule and its target, particularly a DNA guide molecule and a target RNA molecule).
  • the two molecules e.g., target and its probe
  • the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • the two molecules are complementary if they have sufficiently compatible nucleotide base-pairs such that the two molecules can hybridize.
  • nucleotide molecules e.g., nucleotides, oligonucleotides, polynucleotides, modified nucleotides, etc.
  • nucleotide molecules which have 100% complementarity (e.g., each nucleotide in a sequence of one molecule is the nucleotide base-pair complement of an adjacent nucleotide in a sequence of the second molecule, in sequential order) as well as two or more nucleotide molecules which have less than 100% complementarity but which hybridize under the conditions of the methods disclosed herein.
  • hybridization or “hybridizes” refers to a process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.
  • anneal refers to the process by which a single-stranded nucleic acid sequence pairs by hydrogen bonds to a complementary sequence, forming a double-stranded nucleic acid sequence, including the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured).
  • melting refers to the denaturation of a double-stranded nucleic acid sequence due to high temperatures, resulting in the separation of the double strand into two single strands by breaking the hydrogen bonds between the strands.
  • target refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Docket No.103361-460WO1
  • the term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g.
  • a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g.
  • a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature.
  • nucleic acids e.g. polynucleotides
  • human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g.
  • an expression cassette refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively.
  • an expression cassette comprising a promoter operably linked to a second nucleic acid may include a promoter that is heterologous to the second nucleic acid (e.g.
  • an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid may include a terminator that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation.
  • the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g. polynucleotide) and a terminator operably linked to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation.
  • the expression cassette comprises an endogenous promoter.
  • the expression cassette comprises an endogenous terminator.
  • the expression cassette comprises a synthetic (or non-natural) promoter.
  • the expression cassette comprises a synthetic (or non-natural) terminator.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have Docket No.103361-460WO1 a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below,
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res.25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the Docket No.103361-460WO1 query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
  • codon optimized refers to genes or coding regions of nucleic acid molecules for the transformation of various hosts, refers to the alteration of codons in the gene or coding regions of polynucleic acid molecules to reflect the typical codon usage of a selected organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that selected organism.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is Docket No.103361-460WO1 operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase.
  • operably linked nucleic acids e.g.
  • a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • nucleobase refers to the part of a nucleotide that bears the Watson/Crick base- pairing functionality.
  • a polynucleotide sequence is “heterologous” to a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined Docket No.103361-460WO1 ionic strength pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C., or, 5 ⁇ SSC, 1% SDS, incubating at 65° C., with wash in 0.2 ⁇ SSC, and 0.1% SDS at 65° C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1 ⁇ SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Exemplary conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Docket No.103361-460WO1 Methods It has been discovered that microRNAs (miRNAs) are trimmed to 14 nucleotides (nt) or shorter by several 3′->5′ exonucleases. These exonucleases are known to be induced upon stress, viral infection, and cancers. Therefore many tinyRNAs unique to the specific disease are in the patient's blood samples.
  • tinyRNAs are not considered as biomarkers because such short RNAs have been thought to be just degraded RNAs.
  • RNA-seq of tinyRNAs within the blood samples can determine which miRNAs are abundant or deficient in the patient compared with healthy controls.
  • tinyRNAs also referred to herein as tyRNAs
  • the present disclosure provides methods of profiling tinyRNAs that can be used in detecting, diagnosing, and/or treating a disease or disorder, including but not limited to viral infections.
  • the present disclosure also provides methods of identifying tinyRNA biomarkers for detecting, diagnosing, and/or treating a disease or disorder, including but not limited to viral infections.
  • a method of associating tinyRNA with a physiological state or condition comprising isolating a sample from a subject, extracting and analyzing a plurality of tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, and determining a physiological state or condition based on the tinyRNA profile as compared to the control.
  • a method of treating a disease or disorder in a subject comprising taking a sample from the subject, extracting and analyzing tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, determining the subject has a disease or disorder based on the tinyRNA profile as compared to the control, and treating the subject with an anti-inflammatory agent, an anti-cancer agent, an antibiotic, an anti-viral, or any combination thereof.
  • a method of determining effectiveness of a treatment protocol or effectiveness of a composition for treatment of a specific disease or disorder in a subject comprising taking a sample from the subject, wherein the subject has received a treatment or a composition for treating the specific disease or disorder, extracting and analyzing tinyRNAs from the sample, thereby forming a tinyRNA profile, comparing the tinyRNAs extracted in the preceding step to a control, and determining the effectiveness of the treatment or the composition from the tinyRNA profile as compared to the control.
  • Docket No.103361-460WO1 In some embodiments, the tinyRNA is less than 18 nucleotides long.
  • the tinyRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides. In some embodiments, the tinyRNA is 14 nucleotides longer or shorter. In some embodiments, the method comprises an AGO-associated guide RNA that comprises 17 nucleotides or shorter. In some embodiments, the AGO-associated guide RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides. In some embodiments, the sample is derived from blood, urine, saliva, plasma, a tissue or a tumor. In some embodiments, the tyRNAs do not cause gene silencing. In some embodiments, the tyRNAs cause gene silencing in the presence of a Booster construct.
  • the Booster constructs are auxiliary RNA molecules capable of carrying and loading the desired tyRNAs into endogenous AGOs.
  • the Booster constructs provide a tyRNA loading system for sufficient tyRNA loading into endogenous AGOs.
  • the physiological state or condition comprises stress, infection, inflammation, cancer, or other disease or disorder.
  • Exemplary physiological states or conditions include, but are not limited to neurological diseases/disorders, cancer, infectious diseases (such as, for example bacterial infection, viral infections, fungal infections, and parasitic infections), cardiovascular diseases, respiratory diseases, congenital diseases/disorders, gastrointestinal diseases, metabolic diseases, or any combinations thereof.
  • the neurological disease/disorder includes, but is not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo-facio-skeletal syndrome, Leigh syndrome, Prion diseases, monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt-Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, and other related diseases.
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • Friedreich ataxia Lewy body disease
  • spinal muscular atrophy Alpers’ disease
  • Batten disease Cerebro-oculo-facio-skeletal syndrome
  • Leigh syndrome Prion diseases
  • monomelic amyotrophy multiple system atrophy
  • the cancer includes, but is not limited to acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chord
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), Docket No.103361-460WO1 hypereosinophilic syndrome (HES))
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e
  • the infectious disease includes, but is not limited to common cold, influenza (including, but not limited to human, bovine, avian, porcine, and simian strains of influenza), measles, acquired immune deficiency syndrome/human immunodeficiency virus (AIDS/HIV), anthrax, botulism, cholera, campylobacter infections, chickenpox, chlamydia infections, cryptosporidosis, dengue fever, diphtheria, hemorrhagic fevers, Escherichia coli (E.
  • influenza including, but not limited to human, bovine, avian, porcine, and simian strains of influenza
  • measles including, but not limited to human, bovine, avian, porcine, and simian strains of influenza
  • AIDS/HIV acquired immune deficiency syndrome/human immunodeficiency virus
  • anthrax botulism
  • cholera campylobacter infections
  • chickenpox chlamydia infections
  • coli infections, ehrlichiosis, gonorrhea, hand-foot-mouth disease, hepatitis A, hepatitis B, hepatitis C, legionellosis, leprosy, leptospirosis, listeriosis, malaria, meningitis, meningococcal disease, mumps, pertussis, polio, pneumococcal disease, paralytic shellfish poisoning, rabies, rocky mountain spotted fever, rubella, salmonella, shigellosis, small pox, syphilis, tetanus, trichinosis (trichinellosis), tuberculosis (TB), typhoid fever, typhus, west nile virus, yellow fever, yersiniosis, and zika.
  • the bacterial infection is caused by the group of bacteria consisting of M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellular, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.
  • avium subspecies paratuberculosis Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria Docket No.103361-460WO1 ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, other Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus py
  • the viral infection is caused by the group of virus consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.
  • virus consisting of Herpes Simplex virus- 1, Herpes
  • the fungal infection is caused by the group of fungi consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.
  • the parasitic infection is caused by the group of parasitic organisms consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Schistosoma mansoni, other Schistosoma species, and Entamoeba histolytica.
  • the cardiovascular disease includes, but is not limited to coronary artery disease, high/low blood pressure, cardiac arrest/heart failure, congestive heart failure, congenital heart defects/diseases (including, but not limited to atrial septal defects, atrioventricular septal defects, coarctation of the aorta, double-outlet right ventricle, d-transposition of the great arteries, Ebstein anomaly, hypoplastic left heart syndrome, and interrupted aortic arch), arrhythmia, peripheral artery disease, stroke, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathies, hypertensive heart disease, pulmonary heart disease, cardiac Docket No.103361-460WO1 dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, eosinophilic myocarditis, valvular heart diseases, rheumatic heart diseases, and other related cardiovascular diseases.
  • coronary artery disease high/low blood pressure
  • the respiratory disease includes, but is not limited to asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pneumonia, bronchitis (chronic or acute bronchitis), emphysema, cystic fibrosis/bronchiectasis, pleural effusion, acute chest syndrome, acute respiratory distress syndrome, asbestosis, aspergilosis, severe acute respiratory syndrome (including, but not limited to SARS-CoV-1 and SARS-CoV-2), respiratory syncytial virus (RSV), middle eastern respiratory syndrome (MERS), mesothelioma, pneumothorax, pulmonary arterial hypertension, pulmonary hypertension, pulmonary embolism, sarcoidosis, sleep apnea, and other respiratory diseases.
  • COPD chronic obstructive pulmonary disease
  • pulmonary fibrosis pneumonia
  • bronchitis chronic or acute bronchitis
  • emphysema cystic fibrosis/bronchiect
  • the congenital disease includes, but is not limited to albinism, amniotic band syndrome, anencephaly, Angelman syndrome, Barth syndrome, chromosomal abnormalities (including, but not limited to abnormalities to chromosome 9, 10, 16, 18, 20, 21, 22, X chromosome, and Y chromosome), cleft lip/palate, club foot, congenital adrenal hyperplasia, congenital hyperinsulinism, congenital sucrase-isomaltase deficiency (CSID), cystic fibrosis, De Lange syndrome, fetal alcohol syndrome, first arch syndrome, gestational diabetes, Haemophilia, heterochromia, Jacobsen syndrome, Katz syndrome, Klinefelter syndrome, Kabuki syndrome, Kyphosis, Larsen syndrome, Laurence-Moon syndrome, macrocephaly, Marfan syndrome, microcephaly, Nager’s syndrome, neonatal jaundice, neurofibromatosis, Noonan syndrome, Pallister-Killian syndrome, Pierre
  • the gastrointestinal disease includes, but is not limited to heartburn, irritable bowel syndrome, lactose intolerance, gallstones, cholecystitis, cholangitis, anal fissure, hemorrhoids, proctitis, colon polyps, infective colitis, ulcerative colitis, ischemic colitis, Crohn’s disease, radiation colitis, celiac disease, diarrhea (chronic or acute), constipation (chronic or acute), diverticulosis, diverticulitis, acid reflux (gastroesophageal reflux (GER) or gastroesophageal reflux disease (GERD)), Hirschsprung disease, abdominal adhesions, achalasia, acute hepatic porphyria (AHP), anal fistulas, bowel incontinence, centrally mediated abdominal pain syndrome (CAPS), clostridioides difficile infection, cyclic vomiting syndrome (CVS), dyspepsia, eosinophilic gastroente
  • the metabolic disease includes, but is not limited to diabetes mellitus Type I, diabetes mellitus Type II, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe syndrome, metachromatic leukodystrophy, Niemann-Pick syndrome, phenylketonuria (PKU), Tay-Sachs disease, Wilson’s disease, hemachromatosis, mitochondrial disorders or diseases (including, but not limited to Alpers Disease; Barth syndrome; beta.- oxidation defects:carnitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency: Complex V deficiency; cytochrome c oxidase (COX) deficiency, LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM
  • the tiny RNA is associated with an AGO protein.
  • the control is from a healthy subject or comprises a compilation of markers from Docket No.103361-460WO1 one or more healthy subjects.
  • the control comprises indicators for a physiological state of any preceding aspect.
  • one specific physiological state or condition is analyzed based on the sample.
  • more than one specific physiological state or condition is analyzed based on the sample.
  • a ratio is determined based on expression level of one or more given tinyRNAs compared to the control.
  • the tinyRNAs of any preceding aspect are detected and/or identified using at least one sequencing technique including, but not limited to RNA-seq, high-throughput sequencing, and next generation sequencing.
  • the tiny RNA of any preceding aspect has been trimmed in vivo by an exonuclease.
  • the tinyRNA of any preceding aspect has been trimmed ex vivo by an exonuclease.
  • the exonuclease is activated upon change of physiological state or condition in the subject.
  • the exonuclease is associated with an AGO protein.
  • the AGO protein comprises AGO1, AGO2, AGO 3, or AGO4.
  • the exonuclease comprises ISG20, or a mutant thereof.
  • mutant refers a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250).
  • a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
  • “mutant”, “variant”, and “derivative” can be used interchangeably herein.
  • a kit comprising one or more reagents for isolating and analyzing one or more tinyRNAs.
  • the one or more tinyRNAs are isolated from a sample derived from a subject.
  • the sample is derived from blood, urine, saliva, plasma, a tissue or a tumor.
  • the kit comprises any combination of an extraction buffer, a storage buffer, a washing buffer, a loading buffer, or a staining buffer.
  • the kit further comprises an agarose solution or a polyacrylamide solution.
  • the one or more tinyRNAs are isolated using a purification technique such as, for example a column-based purification method or a size-dependent Docket No.103361-460WO1 purification method.
  • the one or more tinyRNAs are amplified prior to analyses.
  • nucleic acid polymerization and amplification techniques are known in the art, including reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.
  • RT reverse transcription
  • PCR polymerase chain reaction
  • q-PCR quantitative PCR
  • NASBA nucleic acid sequence-base amplification
  • ligase chain reaction multiplex ligatable probe amplification
  • IVT in vitro transcription
  • strand displacement amplification strand displacement amplification
  • TMA transcription-mediated amplification
  • RNA Erberwine amplification
  • An exemplary PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species.
  • An exemplary reaction includes three steps: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
  • Exemplary PCR reactions may include 20 or more cycles of denaturation, annealing, and elongation.
  • the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps.
  • a reverse transcription reaction (which produces a complementary cDNA sequence) is performed prior to PCR reactions.
  • Reverse transcription reactions include the use of, e.g., an RNA-based DNA polymerase (reverse transcriptase) and a primer.
  • two or more tinyRNAs or nucleic acids are amplified in a single reaction volume or multiple reaction volumes.
  • one or more tinyRNA or nucleic acids may be used as a normalization control or a reference nucleic acid for normalization. Normalization may be performed in separate or the same reaction volumes as other amplification reactions.
  • One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least one miRNA of interest and at least one reference nucleic acid in one reaction volume by using more than one pair of primers and/or more than one probe.
  • the primer pairs may comprise at least one amplification primer that uniquely binds each nucleic acid, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.
  • Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of tinyRNAs for diagnostic, prognostic, and therapeutic applications.
  • a single combined reaction for q-PCR may be used to: (1) decreased risk of experimenter error, (2) reduce assay-to-assay variability, (3) decrease risk of target or product contamination, and (4) increase assay speed.
  • the qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase.
  • a "hot start" approach may be used to maximize assay performance (U.S. Patents 5,411,876 and 5,985,619).
  • the components for a reverse transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S.
  • RNA samples of interest can be screen nucleic acids or RNA isolated from samples of interest and a related reference such as, but not limited to a normal adjacent tissue (NAT) samples.
  • a panel of amplification targets may be chosen for real-time RT-PCR quantification.
  • the panel of targets includes one or more tinyRNA described herein.
  • the selection of the panel or targets can be based on the results of microarray expression analyses, such as with mzTVanaTM miRNA Bioarray VI (Ambion), Human miRNA Microarrays (V3) (Agilent), miRLinkTM Arrays (Asuragen), or any other suitable microarray.
  • Software tools such as NormFinder (Andersen et al., 2004) may be used to determine targets for normalization with the targets of interest and tissue sample set.
  • the cycle threshold (Ci) value (a log value) for the microRNA of interest is subtracted from the geometric mean Ci value of normalization targets.
  • Fold change can be determined by subtracting the dCi normal reference (N) from the corresponding dCi sample being evaluated (T), producing a ddCi (T-N) value for each sample.
  • the average ddCi(T-N) value across all samples is converted to fold change by 2ddCt.
  • the representative p-values are determined by a two-tailed paired Student's t-test from the dCi values of sample and normal reference. Docket No.103361-460WO1 Also contemplated are methods for using digital PCR.
  • Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA.
  • the key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR.
  • PCR carries out one reaction per single sample.
  • dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts.
  • RNA Seq For example in digital PCR, a sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. (The capture or isolation of individual nucleic acid molecules has been effected in micro well plates, capillaries, the dispersed phase of an emulsion, and arrays of miniaturized chambers, as well as on nucleic acid binding surfaces.) The partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution.
  • each part will contain "0" or “1” molecules, or a negative or positive reaction, respectively.
  • nucleic acids may be quantified by counting the regions that contain PCR end-product, positive reactions. In conventional PCR, the number of PCR amplification cycles is proportional to the starting copy number. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and therefore provides absolute quantification.
  • the expression of one or more biomarkers may be measured by a variety of techniques that are well known in the art.
  • assays include, but are not limited to, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-point PCR, multiplex end-point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA), branched DNA (bDNA) assay, rolling circle amplification (RCA), single molecule hybridization detection, invader assay, and/or Bridge Litigation Assay.
  • HPA hybridization protection assay
  • bDNA branched DNA
  • RCA rolling circle amplification
  • a nucleic acid microarray may be used to quantify the differential expression of a plurality of biomarkers.
  • Microarray analysis may be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology (Santa Clara, CA) or the Microarray System from Incyte (Fremont, CA).
  • single- stranded nucleic acids e.g., miRNAs, cDNAs or oligonucleotides
  • the arrayed sequences may be then hybridized with specific nucleic acid probes from the cells of interest.
  • Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
  • the RNA may be amplified by in vitro transcription and labeled with a marker, such as biotin.
  • the labeled probes may then be hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove the non-specifically bound probes, the chip may be scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
  • the raw fluorescence intensity data in the hybridization files may be preprocessed with the robust multichip average (RMA) algorithm to generate expression values.
  • RMA robust multichip average
  • Quantitative real-time PCR may also be used to measure the differential expression of a plurality of biomarkers.
  • the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction.
  • the amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of miRNA.
  • the reaction may be performed in the presence of a fluorescent dye, such as SYBR Green, which binds to double stranded DNA.
  • the reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified.
  • a non-limiting example of a fluorescent reporter probe is a TaqMan® probe (Applied Biosystems, Foster City, CA).
  • the fluorescent reporter probe fluoresces when the quencher is removed during the PCR extension cycle.
  • Multiplex qRT-PCR may be performed by using multiple gene-specific reporter probes, each of which contains a different fluorophore. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. To minimize errors and reduce any sample-to-sample variation, qRT-PCR may be performed using a reference standard. The ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
  • Luminex® multiplexing microspheres may also be used to measure the differential expression of a plurality of biomarkers.
  • These microscopic polystyrene beads are internally color- coded with fluorescent dyes, such that each bead has a unique spectral signature (of which there are up to 100). Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest (i.e., biomarker tinyRNAs). The target, in turn, is also tagged with a fluorescent reporter.
  • the beads are then incubated with the sample containing the targets, of which up 100 may be detected in one well.
  • the small size/surface area of the beads and the three dimensional exposure of the beads to the targets allows for nearly solution-phase kinetics during the binding reaction.
  • the captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay.
  • the data from the acquisition files may be converted into expression values using means known in the art. In situ hybridization may also be used to measure the differential expression of a plurality of biomarkers.
  • This method permits the localization of miRNAs of interest in the cells of a tissue section.
  • the tissue may be frozen, or fixed and embedded, and then cut into thin sections, which are arrayed and affixed on a solid surface.
  • the tissue sections are incubated with a labeled antisense probe that will hybridize with a tinyRNAs of interest.
  • the hybridization and washing steps are generally performed under highly stringent conditions.
  • the probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be detected and visualized under a microscope. Multiple tinyRNAs may be detected simultaneously, provided each antisense probe has a distinguishable label.
  • the hybridized tissue array is generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for each biomarker. The number of biomarkers whose expression is measured in a sample may vary. Since the risk score is based upon the differential expression of the biomarkers, a higher degree of accuracy should be attained when the expression of more biomarkers is measured; however, a large number of biomarkers in the gene signature would hamper the clinical usefulness.
  • RNA arrays or miRNA probe arrays which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and are positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters. Representative methods and apparatus for preparing a microarray have been described, for example, in U.S.
  • Some embodiments involve the preparation and use of tinyRNA arrays or probe arrays, which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of tinyRNA molecules or precursor tinyRNA molecules and that are positioned on a support or support material in a spatially separated organization.
  • tinyRNA arrays or probe arrays which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of tinyRNA molecules or precursor tinyRNA molecules and that are positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates Docket No.103361-460WO1 or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g.
  • Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of miRNA- complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
  • array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass, metal, plastic, and silicon. Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
  • the labeling and screening methods are not limited by with respect to any parameter except that the probes detect tinyRNAs; consequently, methods and compositions may be used with a variety of different types of miRNA arrays. Representative methods and apparatuses for preparing a microarray have been described, for example, in U.S.
  • the arrays can be high density arrays, such that they contain 2, 20, 25, 50, 80, 100, or more, or any integer derivable therein, different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more, or any integer or range derivable therein, different probes.
  • the probes can be directed to targets in one or more different organisms or cell types.
  • the oligonucleotide probes may range from 5 to 50, 5 to 45, 10 to 40, 9 to 34, or 15 to 40 nucleotides in length.
  • the Docket No.103361-460WO1 oligonucleotide probes are 5, 10, 15, 20, 25, 30, 35, 40 nucleotides in length, including all integers and ranges there between.
  • the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm2.
  • the surface area of the array can be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm2.
  • a person of ordinary skill in the art could readily analyze data generated using an array.
  • Such protocols are disclosed herein or may be found in, for example, WO 9743450 ; WO 03023058 ; WO 03022421 ; WO 03029485 ; WO 03067217 ; WO 03066906 ; WO 03076928 ; WO 03093810 ; WO 03100448A1.
  • the population of target nucleic acids may be contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al.
  • nucleic acids may be labeled by adding labeled nucleotides (one- step process) or adding nucleotides and labeling the added nucleotides (two-step process).
  • nucleic acids are labeled by catalytically adding to the nucleic acid an already labeled nucleotide or nucleotides.
  • One or more labeled nucleotides can be added to miRNA molecules. See U.S Patent 6,723,509.
  • an unlabeled nucleotide or nucleotides may be catalytically added to a tinyRNA, and the unlabeled nucleotide is modified with a chemical moiety that enables it to be subsequently labeled.
  • the chemical moiety is a reactive amine such that the nucleotide is an amine-modified nucleotide.
  • amine-modified nucleotides are well known to those of skill in the art, many being commercially available such as from Ambion, Sigma, Jena Bioscience, and TriLink.
  • the issue for labeling miRNA is how to label the already existing molecule.
  • embodiments concern the use of an Docket No.103361-460WO1 enzyme capable of using a di- or tri-phosphate ribonucleotide or deoxytibonucleotide as a substrate for its addition to a miRNA.
  • it involves using a modified di- or tri-phosphate ribonucleotide, which is added to the 3′ end of a tinyRNA.
  • the source of the enzyme is not limiting.
  • sources for the enzymes include yeast, gram negative bacteria such as E. coli, Lactococcus lactis, and sheep pox virus.
  • Enzymes capable of adding such nucleotides include, but are not limited to, poly(A) polymerase, terminal transferase, and polynucleotide phosphorylase.
  • a ligase is contemplated as not being the enzyme used to add the label, and instead, a non-ligase enzyme is employed. Terminal transferase catalyzes the addition of nucleotides to the 3′ terminus of a nucleic acid.
  • Polynucleotide phosphorylase can polymerize nucleotide diphosphates without the need for a primer.
  • Labels on tinyRNAs may be colorimetric (includes visible and UV spectrum, including fluorescent), luminescent, enzymatic, or positron emitting (including radioactive). The label may be detected directly or indirectly. Radioactive labels include125I, P, P, and S. Examples of enzymatic labels include alkaline phosphatase, luciferase, horseradish peroxidase, and [3- galactosidase. Labels can also be proteins with luminescent properties, e.g., green fluorescent protein and phycoerythrin.
  • the colorimetric and fluorescent labels contemplated for use as conjugates include, but are not limited to, Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum DyeTM; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.
  • Alexa Fluor dyes such as BODIPY FL
  • Cascade Blue
  • dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIP
  • fluorescently labeled ribonucleotides are available from Molecular Probes, and these include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5- UTP, and BODIPY TR-14-UTP.
  • Other fluorescent ribonucleotides are available from Amersham Biosciences, such as Cy3-UTP and Cy5-UTP.
  • fluorescently labeled deoxyribonucleotides include Dinitrophenyl (DNP)-1 1-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FF-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488- 7-OBEA-dCTP, Alexa Fluor 546-16-OBEA
  • nucleic acids may be labeled with two different labels.
  • fluorescence resonance energy transfer FRET
  • the label may not be detectable per se, but indirectly detectable or allowing for the isolation or separation of the targeted nucleic acid.
  • the label could be biotin, digoxigenin, polyvalent cations, chelator groups and the other ligands, include ligands for an antibody.
  • a number of techniques for visualizing or detecting labeled nucleic acids are readily available.
  • Such techniques include, microscopy, arrays, Fluorometry, Light cyclers or other real time PCR machines, FACS analysis, scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT, antibody-based detection methods (Westerns, immunofluorescence, immunohistochemistry), histochemical techniques, HPLC (Griffey et al., 1997), spectroscopy, capillary gel electrophoresis (Cummins et al., 1996), spectroscopy; mass spectroscopy; radiological techniques; and mass balance techniques.
  • FRET fluorescent resonance energy transfer
  • a person of ordinary skill in the art is well aware of ways of visualizing, identifying, and characterizing labeled nucleic acids, and accordingly, such protocols may be used as part of some embodiments.
  • tools that may be used also include fluorescent Docket No.103361-460WO1 microscopy, a BioAnalyzer, a plate reader, Storm (Molecular Dynamics), Array Scanner, FACS (fluorescent activated cell smier), or any instrument that has the ability to excite and detect a fluorescent molecule.
  • the one or more tinyRNAs are analyzed using a technique including, but not limited to gel-based techniques (such as, for example agarose gel and polyacrylamide gel), polymerase chain reactions (PCRs), sequencing methods (such as, for example RNA seq, next generation sequencing, and high throughput sequencing).
  • gel-based techniques such as, for example agarose gel and polyacrylamide gel
  • PCRs polymerase chain reactions
  • sequencing methods such as, for example RNA seq, next generation sequencing, and high throughput sequencing.
  • Methods can be used to detect differences in tinyRNA expression or levels between two samples, or a sample and a reference (e.g., a tissue or other biological reference or a digital reference representative of a non-cancerous state).
  • Specifically contemplated applications include identifying and/or quantifying differences between tinyRNA from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, or between two differently treated samples (e.g., a pretreatment versus a posttreatment sample). Also, tinyRNA may be compared between a sample believed to be susceptible to a particular therapy, disease, or condition and one believed to be not susceptible or resistant to that therapy, disease, or condition. A sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal relative to that disease or condition.
  • Phenotypic traits include symptoms of a disease or condition of which a component is or may or may not be genetic or caused by a hyperproliferative or neoplastic cell or cells, such as nodules or tumors. Phenotypic traits also include characteristics such as longevity, morbidity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity. In certain embodiments, tinyRNA profiles may be generated to evaluate and correlate those profiles with pharmacokinetics.
  • tinyRNA profiles may be created and evaluated for patient tumor and blood samples prior to the patient's being treated or during treatment to determine if there are miRNAs whose expression correlates with the outcome of treatment. Identification of differential miRNAs can lead to a diagnostic assay involving them that can be used to evaluate tumor and/or blood samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical trial. If a tinyRNA profile is determined to be correlated with drug efficacy or drug toxicity that determination may be relevant to whether that patient is an appropriate patient for receiving the drug or for a particular dosage of the drug.
  • a diagnostic assay can be created based on the profiles that doctors can use to identify individuals with a disease or a prognostic assay to determine or identify those individuals who are at risk to develop a disease or condition such as metastasis.
  • treatments can be designed based on tinyRNA profiling. Examples of such methods and compositions are described in the U.S. Provisional Patent Application entitled “Methods and Compositions Involving miRNA and miRNA Inhibitor Molecules" filed on May 23, 2005.
  • this disclosure entails measuring expression of one or more biomarkers in a sample of cells from a subject.
  • the expression information may be obtained by a lab, a technician, a device, or a clinician.
  • the pattern or signature of expression in each sample may then be used to generate a risk score for prognosis or classification of a physiological state or condition, such as predicting cancer survival or recurrence.
  • the level of expression of a biomarker may be increased or decreased in a subject relative to other subjects with or without the physiological characteristic or condition.
  • the expression of a biomarker may be higher in long-term survivors than in short-term survivors. Alternatively, the expression of a biomarker may be higher in short-term survivors than in long- term survivors.
  • Example 1 TinyRNAs as Biomarkers
  • Epstein-Barr virus (EBV) is known to damage the brain and central nervous system (CNS) directly and indirectly through infected lymphocytes. These viruses are latent in healthy conditions but reactivate in immunocompromised individuals with severe consequences.
  • miRNAs microRNAs
  • nt miRNAs 20 ⁇ 23-nucleotide (nt) miRNAs are loaded into four Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs) to repress the translation of mRNAs complementary to their miRNAs.
  • AGO Argonaute
  • RISCs RNA-induced silencing complexes
  • AGO-associated miRNAs were discovered to be trimmed to 14-nt or shorter tinyRNAs (tyRNAs) by three 3′ ⁇ 5′ exonucleases: interferon- stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and enhanced RNAi 1 (ERI1).
  • ISG20 is highly expressed during EBV replication.
  • TREX1 three prime repair exonuclease 1
  • ERI1 enhanced RNAi 1
  • ISG20 is highly expressed during EBV replication.
  • a dual-luciferase reporter assay demonstrated that 14-nt tyRNAs and their parental miRNAs ( ⁇ 22nt) have different target specificities.
  • the tyRNAs are determined in EBV-infected cells and it was validated that the herpesvirus infection enhances tyRNA generation in neurodevelopmental disorder (NDD) patients whose AGO has specific single-point mutations.
  • NDD neurodevelopmental disorder
  • the successful outcome provided what is believed to be the first comprehensive data sets of tyRNAs as new biomarkers.
  • tyRNAs generated during the different stages of EBV infection To obtain a comprehensive profile of tyRNAs in EBV-infected cells, the tyRNA level is quantified in the four different stages of EBV-infected cells: 1) no infection, 2) de novo infection, 3) latency, and 4) lytic reactivation.
  • Akata-4E3 (EBV-) and BJAB Burkitt lymphoma cells (EBV-) are used for no infection and de novo infection
  • Akata-BX1 (EBV+) and BJAB- BX1 (EBV+) cells are used for latency and reactivation.
  • each of the endogenous four AGOs i.e., AGO1, AGO2, AGO3, and AGO4 are immunopurified with their specific antibody 12, 24, 36, and 48 hours later.
  • AGO-associated 5′ end-labeled miR-20a are resolved on a denaturing gel to quantify the conversion percentage from miRNA to tyRNA.
  • the mRNA and protein levels of ISG20 are monitored. The results reveal the timing of tyRNA accumulation and the correlation with the ISG20 expression level. Accordingly, the endogenous AGO-associated RNAs are extracted at the determined best timing from all cell lines across the four different stages, followed by RNA-seq.
  • the successful outcome of this aim provides a profile of tyRNAs that are upregulated and downregulated in the different infection stages.
  • EBV infection-induced tyRNAs whose generation is enhanced in NDD-related AGO mutants. It was contemplated that children with a specific AGO mutation develop NDD after viral infection including EBV.
  • AGOs recognize the miRNA 3′ end and protect it from the cellular 3′ ⁇ 5′ exonucleases. Based on previous crystal structures, it was surmised that NDD-relevant AGO mutants easily release the 3′ end from the PAZ domain and expose it to ISG20, TREX1, and ERI1.
  • the resultant tyRNAs change the gene expression pattern in the neural cells and lymphocytes in NDD patients.
  • pCAGEN vector encoding FLAG-tagged NDD-relevant AGO1 or AGO2 mutant are transfected into SH-SY5Y and Ntera-2 cells, as well as the EBV-infected cells.
  • the generated tyRNAs are extracted from the immunoprecipitated AGO mutants with anti-FLAG affinity agarose beads, followed by RNA-seq.
  • the total RNA is sequenced to understand how the mRNA levels change upon tyRNA generation.
  • the long-term goal is to provide a foundation to establish a new frontier in understanding how herpesvirus infection generates tyRNAs and thus dysregulates gene expression.
  • the determined tyRNAs serve as biomarkers and make previously identified miRNA biomarkers more informative.
  • Epstein-Barr virus is known to cause neural diseases by directly infecting neurons or indirectly via infected B-lymphocytes.
  • EBV infection induces neuroinflammation and demyelination, promotes the proliferation, degeneration, and necrosis of glial cells, promotes proliferative disorders of B- and T-lymhocytes, and develops central nervous system diseases.
  • the pathological mechanism of EBV-invoked neural diseases is an open question being investigated from the various aspects currently known. Such a situation indicates a demand for completely new insights to understand the mechanism of EBV-invoked neural diseases.
  • MicroRNAs miRNAs
  • MiRNAs More than 2,000 microRNAs (miRNAs) have been reported in humans as of 2019.
  • MiRNAs fall within a range of Docket No.103361-460WO1 19 ⁇ 23 nucleotides (nt) because precursor miRNAs (pre-miRNAs) are processed by Dicer, a molecular ruler which generates size-specific miRNA duplexes (Figure 1A). After these duplexes are loaded into Argonaute (AGO) proteins, one strand is ejected while the remaining guide strand and AGO form the RNA-induced silencing complex (RISC) ( Figure 1A).
  • AGO Argonaute
  • RISC RNA-induced silencing complex
  • AGOs and Dicers are the primary factors of the anti-viral response in insects and plants, none of their human counterparts have been thought to serve as the primary defense machinery because vertebrates have developed an innate immune system. Therefore, the significance of miRNAs in the anti-viral response has been controversial. In this context, it was recently discovered that AGO- associated 20 ⁇ 23-nt miRNAs are trimmed to 14-nt or shorter tinyRNAs (tyRNAs) by interferon- stimulated gene 20 kDa protein (ISG20) (Sim GK, A. C.; Park, M.
  • RNA sequencing (RNA-seq) studies identified AGO-associated 10 ⁇ 18- nucleotide tyRNAs in plants and vertebrates that mapped onto tRNAs and miRNAs. How these tyRNAs are synthesized remains unknown.
  • RNA-seq RNA sequencing
  • ss miR-20a or their small interfering RNA (siRNA)-like duplex (ds) were transfected into HEK293T cells. Only the guide RNA was radiolabeled at its 5′ end. Both 14- and 23-nucleotide ssRNAs and the 14-nucleotide siRNA-like duplex were degraded ( Figure 2B), showing that neither has a chance of being loaded into AGOs efficiently. In contrast, the 23- nucleotide siRNA-like duplex remained 19 ⁇ 23 nucleotides, and no tyRNA was detected, indicating that a detectable level of tyRNAs is not generated in normal conditions.
  • siRNA small interfering RNA
  • ERI1 is known to negatively regulate global miRNA abundance in mouse lymphocytes, but the mechanism remains unclear.
  • Poly(A)-specific ribonuclease (PARN) shortened the 23-nt guide down to 19 nt, whereas exonuclease 5 (EXO5) did not trim the guide at all (Lanes 8 and 10 in Figure 3D).
  • PARN Poly(A)-specific ribonuclease
  • EXO5 did not trim the guide at all
  • the experiment was repeated with their catalytically dead mutants to confirm that the observed trimming was due to their exonuclease activity.
  • none of the mutants generated tyRNA (Lanes 3, 5, 7, and 9 in Figure 3D), proving that the catalytic center of the exonucleases is essential for tyRNA generation.
  • ISG20, TREX1, and ERI1 also generated tyRNAs from AGO1-, AGO3-, and AGO4- associated miR20a (Figure 3E).
  • the susceptivity of different miRNAs to the 3′ ⁇ 5′ exonucleases was investigated in vitro.
  • FLAG-tagged AGOs were programmed with a 5′-end radiolabeled 23-nt miR-20a (SEQ ID NO: 1), 21-nt let-7a (SEQ ID NO: 2), 22-nt miR-16 (SEQ ID NO: 3), or 23-nt miR-19b (SEQ ID NO: 4), followed by incubation with ISG20.
  • Gly199 plays the essential role in twisting the stalk uniquely, and the mutation changes the position of the PAZ domain against the rest of the AGO.
  • 22% have a missense mutation of G199S (Figure 6A).
  • Another study reported 13 germline AGO2 mutants, including Gly201 corresponding to Gly199 of AGO1, relevant to NDDs (Lessel D, et al. Germline AGO2 mutations impair RNA interference and human neurological development. Nat Commun. 2020;11(1):5797. Epub 2020/11/18. doi: 10.1038/s41467-020-19572-5. PubMed PMID: 33199684; PMCID: Docket No.103361-460WO1 PMC7670403).
  • tyRNAs are upregulated and downregulated during EBV infection and reactivation and when tyRNAs are accumulated allows for the use of tyRNAs as biomarkers for EBV infection-driven neurological diseases.
  • the present disclosure provides the tyRNA expression profile in different EBV infection stages. It was contemplated that each tyRNA is upregulated or downregulated depending on the infection status. The approach to testing this determines AGO-associated tyRNAs by RNA-seq. Herein, AGO-associated 15-nt or shorter RNAs are defined as tyRNAs. The rationale is that its successful completion contributes a fundamental element to knowledge base, without which the significance of tyRNAs in herpesvirus-infection diseases cannot be understood.
  • tyRNA-expression is correlated with the mRNA and protein levels of ISG20.
  • Experimental Design Identifying the best timing of tyRNA accumulation and its correlation with ISG20 levels. The following two experiments are performed in parallel, each of which determines the expression level of ISG20 or the tyRNA generation over a period of time after de novo viral infection, latency, and reactivation.
  • lymphoma cell lines in Table 1 are infected with EBV. Docket No.103361-460WO1
  • TPA tetradecanoyl phorbol acetate
  • NaBu sodium butyrate
  • anti-IgG treatment respectively.
  • the cells are lysed, followed by quantification of the mRNA and protein levels of ISG20 with qRT-PCR (TaqMan Gene Expression Assays, 4331182) and western blot (anti-ISG20 antibody, 22097-1-AP, from ProteinTech) respectively ) Figure 7A and Figure 3D).
  • miR-20a is trimmed slightly slower than let-7a, miR-16, and miR-19a ( Figure 4A). Therefore, an incubation time required for converting miR-20a to 13 ⁇ 14 nt must be long enough to convert the majority of miRNAs to tyRNAs.
  • the same experiment is repeated with transfection of a siRNA targeting the ISG20 mRNA (siISG20) as a negative control ( Figure 7C).
  • siRNA targeting the ISG20 mRNA siISG20
  • Figure 7C Determine tyRNAs generated during de novo EBV infection, latency, and reactivation.
  • the results of the above experimental design determines the best timing to maximize tyRNA yield.
  • AGO-associated tyRNAs are purified from EBV-infected cells but without transfection of the siRNA-like duplex of miR-20a ( Figure 8).
  • RNA-seq Three replicates for RNA-seq. are made.
  • the results reveal the relative timing between ISG20 induction and the tyRNA generation.
  • the 23-nt 5′-end radiolabeled miR-20a is trimmed to 13 ⁇ 14 nt only when the cells are infected with EBV. Change in the tyRNA expression is barely observed when siISG20 is transfected. Such results demonstrate that ISG20 is the essential factor in generating tyRNAs. Then, the results determine the tyRNA-expression profile upon EBV infection and reactivation.
  • RNA-seq data shows the differences in the trimming susceptibility between miRNAs.
  • the successful outcome provides the first tyRNA profile as biomarkers.
  • the experiment prioritizes using Akata and BJAB cell lines but also start the EBV-infection experiments using SH-SY5Y (CRL-226, ATCC) and Ntera-2 (CRL-1973, ATCC) cells.
  • SH-SY5Y CL-226, ATCC
  • Ntera-2 CL-1973, ATCC
  • pCAGEN-FLAG-AGO1 wild type
  • the FLAG-AGO WT and mutants are immunoprecipitated with anti-FLAG affinity agarose beads, followed by extracting the associated RNAs ( Figure 3D).
  • the cDNA library of the extracted RNAs which is a mixture of miRNAs and tyRNAs, are compiled and submitted to Novogene for sequencing. The sequence data are analyzed. To make sure that the observed difference in tyRNA profile between the WT and mutants is due to their accessibility of AGO-associated miRNAs to ISG20 ( Figure 1B), the experiment is repeated with siISG20.
  • Example 2 AGO1 Mutants It was contemplated that microRNAs (miRNAs) are trimmed by ISG20 (interferon- stimulated gene 20 kDa) differently depending on whether they are loaded into AGO1 wild type (WT) or one of two mutants, G199S and ⁇ F180, found in patients with a neurodevelopmental disorder (NDD). Maltose-binding protein (MBP) or ISG20 in HEK293T cells was co-expressed with FLAG-AGO1 WT or either of the NDD-relevant mutants ( Figure 9A). A siRNA-like duplex of 23-nt miR-20a, whose guide strand is radiolabeled at its 5′ end, was also co-transfected.
  • MBP Maltose-binding protein
  • ISG20 in HEK293T cells was co-expressed with FLAG-AGO1 WT or either of the NDD-relevant mutants (Figure 9A).
  • the FLAG-AGO1 WT and mutants were immunoprecipitated with anti-FLAG beads, and those AGO1-associated RNAs were resolved on a denaturing gel.
  • the miR-20a loaded into the FLAG- AGO1 WT was trimmed to 14 nt (Lane 2 of Figure 9A), whereas those loaded into the AGO1 mutants were trimmed to two lengths, one of which is shorter than 14 nt (Lanes 4 and 6 of Figure 9B). This distinct difference shows that ISG20 generates different lengths of tinyRNAs when miRNAs are loaded into the AGO1 mutant. This experiment was repeated three times to confirm this result.
  • Example 3 In vitro trimming assay revealed AGO1 F180Del has a different guide-binding capacity The present example provides confirmation of 23-nt miR-20a trimming in HEK293T cells by AGO1 mutants.
  • RNA concentration See Table 10). Labeling reactions: 37°C in PCR tube for 1 hour. 65°C heat inactivation for 20 minutes. Using 25uL total reaction volumes to avoid diluting with water when purifying. Docket No.103361-460WO1 Used 1uL of PNK even if reaction ratio is off slightly.
  • RNA May need to heat PNK buffer to about 37°C to resuspend salts.
  • Purification of labeled RNA 1. Prepare prepacked G-25 column by vortexing the suspended beads, and spinning at 2600 ⁇ g for 1 minute to drain into waste tube. 2. Make reaction to 25uL and apply directly to the beads (not the wall of tube!) 3. Spin at 2600 ⁇ g for 1 minute into new collection tube. Scintillation Counter 1. 2mL scintillation fluid + 1uL RNA sample 2. Load samples into correct bin with 32P. 3. Assured the switch was flipped so no orange marking was shown and the third metal piece in the back was exposed to count the bin. In Vitro Trimming Assay 1.
  • Formed RISCs by incubating 20 pmol of spiked guide, 200 pmol AGO, and 1x trimming master mix for 1 hr. at 37 C. 5.
  • the in vivo trimming assay showed that interferon-stimulated gene 20 kDa (ISG20), a 3′ ⁇ 5′ exonuclease, trimmed 23-nt miR-20a differently when the miRNA was loaded into AGO1 (WT) and neurodevelopmental disorder (NDD) mutants ( Figures 9A and 9B).
  • AGO1 (WT)- Docket No.103361-460WO1 associated 23-nt miR-20a was trimmed to 13 ⁇ 14 nt.
  • AGO1-NDD mutant-associated 23-nt miR-20a was trimmed to much shorter than 14 nt (unusual guide trimming).
  • ISG20 trimmed AGO1-NDD mutant- associated 23-nt miR-20a differently from AGO1 (WT)-associated one ( Figures 9A and 9B).
  • WT AGO1
  • Figures 9A and 9B To determine the precise lengths of unusual tyRNAs, the same experiment was repeated and ran the trimmed tyRNAs with 10 ⁇ 23-nt ladders.
  • ISG20 was expressed in HEK293T cells with FLAG-AGO1 WT or either NDD-relevant mutant, G199S or ⁇ F180 ( Figure 12).
  • the FLAG-AGO1 (WT) and mutants were immunoprecipitated with anti-FLAG beads, and those AGO1-associated RNAs were resolved on a denaturing gel.23-nt miR-20a was trimmed to about 11 nt when loaded into AGO1-NDD mutants, G199S and ⁇ F180. Their counterpart Argonaute2 (AGO2) NDD-mutants, G201V and ⁇ F182, were recently reported by the Kreienkamp and Meister groups. Herein, it was examined whether ISG20 also trims 23-nt miR-20a differently when loaded into those AGO2-NDD mutants and AGO2 (WT).
  • RNA Labeling reaction parameters for 5′ OH let-7a SEQUENCES 1.
  • SEQ ID NO: 1 – 23-nt miR-20a UAAAGUGCUUAUAGUGCAGGUAG 2.
  • SEQ ID NO: 2 – 21-nt let-7a UGAGGUAGUAGGUUGUAUAGU 3.
  • SEQ ID NO: 3 – 22-nt miR-16 UAGCAGCACGUAAAUAUUGGCG 4.

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Abstract

La présente invention concerne des procédés de profilage de petits ARN pendant diverses étapes d'une infection virale, et des procédés de détection, de diagnostic et/ou de traitement de sujets atteints d'infections virales sur la base d'un profilage de petits ARN.
PCT/US2024/022513 2023-03-30 2024-04-01 Procédés et compositions concernant l'utilisation de petits arn en tant que biomarqueurs Pending WO2024206999A2 (fr)

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