# US 11,028,440 B2 · Methods for identifying and using small RNA predictors > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,028,440 B2 - Document ID: US11028440B2 - Status: Granted - Kind: B2 - Application number: 16/029,036 - Filed: 2018-07-06 - Published: 2018-10-25 - Granted: 2021-06-08 - Earliest claimed: 2017-01-23 - Assignees: Gatehouse Bio, Inc. - Applicants: Srnalytics, Inc. - Inventors: David Salzman - Web page: https://patentgrep.com/patent/US11028440B2 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 2600/158: Expression markers - C12Q 2600/178: miRNA, siRNA or ncRNA - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The invention provides a method for identifying or detecting small RNA (sRNA) predictors of a disease or a condition. The method comprises identifying one or more sRNA sequences that are present in one or more samples of an experimental cohort, and which are not present across a comparator cohort; and optionally identifying one or more sRNA sequences that are present in one or more samples of a comparator cohort, and which are not present across an experimental cohort. In contrast to identifying dysregulated non-coding RNAs (such as miRs that are up- or down-regulated), the invention identifies sRNAs that are binary predictors, that is, present in one cohort (e.g., an experimental cohort) and not another (e.g., a comparator cohort). Further, by quantifying reads for individual sequences (e.g., iso-miRs), without consolidating reads to annotated reference sequences, the invention unlocks the diagnostic utility of miRs and other sRNAs. ## Claims ### Claim 1 A method for identifying small RNA (sRNA) predictors for neurodegenerative disease, comprising: (1) providing sRNA sequencing data from an experimental cohort and a comparator cohort of solid tissue or biological fluid samples, the sRNA sequencing data being generated by a sequencing-by-synthesis sequencing platform; wherein the samples of the experimental cohort and the samples of the comparator cohort are from patients having the presence or absence, respectively, of a neurodegenerative disease; wherein the sRNA sequencing data comprises sequence reads having adaptor sequences at 5′- and 3′-ends, and sRNA sequences are defined from the sequence reads by deletion of the adaptor sequences so as to identify variations at the 3′- and 5′-ends of the sRNA sequences, the sRNA sequences being defined without consolidating sequence reads to a reference sequence; (2) selecting sRNA sequences that are present in at least 10% of the samples in the experimental cohort and which are not present in any samples of the comparator cohort, thereby identifying positive sRNA predictors, and wherein at least one selected sRNA sequence is an isomiR; and (3) detecting the presence and absence of the selected positive sRNA predictors in RNA extracted from samples of a validation cohort using a quantitative or qualitative PCR assay that is specific for the sRNA predictors, wherein the PCR assay employs a stem loop primer and a fluorescently-labeled probe; thereby identifying sRNA predictors for the neurodegenerative disease. ### Claim 2 (depends on 1) The method of claim 1, wherein the biological samples are biological fluid samples selected from blood, serum, plasma, urine, saliva, or cerebrospinal fluid. ### Claim 3 (depends on 1) The method of claim 1, wherein the experimental cohort and the comparator cohort each have at least 50 samples. ### Claim 4 (depends on 3) The method of claim 3, wherein the experimental cohort and the comparator cohort each have at least 100 samples. ### Claim 5 (depends on 1) The method of claim 1, wherein the neurodegenerative disease is selected from Alzheimer's Disease, Parkinson's Disease, Huntington's Disease. ### Claim 6 (depends on 1) The method of claim 1, wherein sRNA sequences are selected that have a sequence read count of at least 20 in the majority of samples that are positive for the sRNA sequence. ### Claim 7 (depends on 6) The method of claim 6, wherein sRNA sequences are selected that have a sequence read count of at least 50 in the majority of samples that are positive for the sRNA sequence. ### Claim 8 (depends on 1) The method of claim 1, wherein at least one sRNA sequence is selected that is present in at least 20% of samples in the experimental cohort. ### Claim 9 (depends on 8) The method of claim 8, wherein from 2 to 50 sRNA sequences are selected and validated using the quantitative or qualitative PCR assay. ### Claim 10 (depends on 9) The method of claim 9, wherein from 4 to 15 sRNA sequences are selected and validated using the quantitative or qualitative PCR assay. ### Claim 11 (depends on 9) The method of claim 9, wherein the quantitative or qualitative PCR assay is real time PCR. ### Claim 12 (depends on 1) The method of claim 1, wherein the isomiR has a non-templated variation at the 3′-end and/or 5′ end. ### Claim 13 (depends on 1) The method of claim 1, wherein the isomiR has a templated variation at the 3′ end and/or 5′ end. ### Claim 14 (depends on 1) The method of claim 1, wherein about 100% of the sequence reads have adaptor sequences deleted so as to identify variations at the 3′- and 5′-ends of the sRNA sequences. ### Claim 15 (depends on 1) The method of claim 1, wherein at least one of the selected sRNA sequences is present in at least 30% of samples in the experimental cohort. ### Claim 16 (depends on 1) A method for evaluating a subject for a neurodegenerative disease, comprising: providing a biological sample from the subject, and identifying the presence or absence of the sRNA predictors identified according to the method of claim 1, wherein the sRNA predictors are detected using a PCR assay employing a stem loop primer and a fluorescently-labeled probe, wherein the presence of one or more sRNA predictors in the sample is indicative of the presence of the neurodegenerative disease, and wherein one or more of the sRNA predictors are isomiRs. ### Claim 17 (depends on 16) The method of claim 16, wherein the neurodegenerative disease Huntington's Disease. ### Claim 18 (depends on 16) The method of claim 16, wherein the neurodegenerative disease is Alzheimer's Disease. ### Claim 19 (depends on 16) The method of claim 16, wherein the neurodegenerative disease is Parkinson's Disease. ### Claim 20 (depends on 16) The method of claim 16, wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid. ### Claim 21 (depends on 20) The method of claim 20, wherein the biological fluid is cerebrospinal fluid. ### Claim 22 (depends on 16) The method of claim 16, wherein presence or absence of at least five sRNA predictors are tested in the sample.