# US 2016/0258017 A1 · Host dna as a biomarker of crohn's disease

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- Number: US 2016/0258017 A1
- Document ID: US20160258017A1
- Status: Published application
- Kind: A1
- Application number: 15/011,314
- Filed: 2016-01-29
- Published: 2016-09-08
- Earliest claimed: 2015-01-30
- Assignees: Enterome S.A.
- Applicants: Enterome
- Inventors: Alessandra Cristina L. CERVINO
- Web page: https://patentgrep.com/patent/US20160258017A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/158: Expression markers
- 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 relates to methods of analysing a sample from a subject having or suspected of having Crohn's disease for the abundance of the subject's nucleic acid (e.g., DNA) in the sample. The invention also relates to methods for measuring abundance of nucleic acid (e.g., DNA) in stool from a human subject having or suspected of having Crohn's Disease (CD). In various embodiments, an in vitro method includes analysing the relative abundance of the host (human) DNA in said sample of stool or nucleic acid extracted or isolated from a stool sample from the host (human); determining the relative abundance of the host (human's) DNA in the sample; and associating the abundance of the host (human) DNA with the host (human) providing the stool sample, or the host (human) providing the stool sample from which the nucleic acid was extracted.

## Claims

### Claim 1

An in vitro method for analyzing a sample from a subject having or suspected of having Crohn's Disease (CD) for the abundance of the subject's DNA in the sample, comprising:
a) Providing a stool sample from said subject;
b) Determining the relative abundance of the subject's DNA in said sample; and;
c) Comparing the relative abundance of the subject's DNA in said sample to a reference value and determining if the relative abundance of the subject's DNA in said sample is higher than the reference value.

### Claim 2

A method for measuring abundance of DNA in stool from a human having or suspected of having Crohn's Disease (CD), said method comprising:
a) providing a sample of stool or nucleic acid extracted from a stool sample from the human;
b) determining the relative abundance of the human's DNA in said sample; and
c) associating said abundance of the human DNA with the human providing the stool sample, or the human providing the stool sample from which the nucleic acid was extracted.

### Claim 3

A method for generating quantitative data for a subject comprising: performing at least one assay to determine the abundance of the subject's DNA in a sample from the subject to generate a first dataset comprising the quantitative data, wherein the quantitative data represents the relative abundance of the subject's DNA in said sample optionally compared to a reference value, wherein the subject has or is at risk of having Crohn's Disease (CD).

### Claim 4

A method for generating a quantitative data set for a subject that has or is at risk of having Crohn's Disease (CD) comprising: performing at least one assay to determine the abundance of the subject's DNA in a sample from the subject to generate a first dataset comprising the quantitative data, wherein the quantitative data represents the relative abundance of the subject's DNA in the sample, optionally compared to a reference value.

### Claim 5 (depends on 3)

The method of claim 3, wherein performance of the assay comprises: providing or obtaining the sample, wherein the sample comprises the subject's DNA; contacting the sample with a detectable reagent; generating a distinct complex between the reagent and the subject's DNA; and detecting the complex to generate the quantitative data.

### Claim 6

An amplification method of measuring the abundance of a target nucleic acid sequence in a sample, comprising:
a) contacting a sample from a subject having or suspected of having Crohn's Disease (CD) comprising a target nucleic acid sequence with a set of oligonucleotide primers, each primer comprising a 5′ end and a 3′ end, wherein a first primer comprises a sequence complementary to a first region in the target nucleic acid sequence, and a second primer comprises a sequence complementary to a second region in the target nucleic acid sequence, wherein the second region is downstream of the first region;
b) further contacting the sample with deoxynucleotide triphosphates (dNTPs), thereby forming a mixture;
c) allowing the first and second oligonucleotide primers to anneal to the target nucleic acid sequence of a) thereby forming an oligonucleotide primer annealed target nucleic acid;
d) exposing the oligonucleotide primer annealed target nucleic acid of step c) to a template-dependent polymerizing agent having 5′ to 3′ polymerization activity under conditions permissive for amplification, and whereby the oligonucleotide primers are extended to produce extension products; and
e) detecting the extension products, thereby measuring the abundance of the target nucleic acid sequence in the sample.

### Claim 7

A vessel having a reaction solution, the reaction solution comprising:
a) A polymerizing agent that synthesizes or amplifies nucleic acid;
b) a sample comprising stool or DNA extracted or isolated from stool of a subject having or suspected of having Crohn's Disease (CD);
c) oligonucleotide primers or oligonucleotide probe designed to specifically hybridize to a target sequence of the subject's DNA;
d) deoxynucleotide triphosphates (dNTPs); and
e) buffers or other agents permissive for detection, replication or amplification of the target sequence.

### Claim 8 (depends on 1)

The method of claim 1, wherein the sample comprises stool or nucleic acid extracted or isolated from stool.

### Claim 9 (depends on 1)

The method of claim 1, wherein the abundance of the subject's DNA is determined by quantitation of a genomic DNA sequence.

### Claim 10 (depends on 1)

The method of claim 1, wherein the abundance of the subject's DNA is determined by quantitation of a non-transcribed region or locus of genomic DNA.

### Claim 11 (depends on 1)

The method of claim 1, wherein the abundance of the subject's DNA is determined by quantitation of a single copy per haploid genomic DNA sequence.

### Claim 12 (depends on 1)

The method of claim 1, wherein the abundance of the subject's DNA is determined by quantitative polymerase chain reaction (qPCR).

### Claim 13 (depends on 1)

The method of claim 1, further comprising classifying the subject or human, based upon the relative abundance of the DNA in said sample being higher or lower than said reference value.

### Claim 14

(canceled)

### Claim 15 (depends on 1)

The method of claim 1, further comprising classifying the subject or human, if the relative abundance of the DNA in said sample is higher than the reference value and the amount of calprotectin in the sample is higher than a second reference value.

### Claim 16 (depends on 1)

The method of claim 1, further comprising determining the amount of calprotectin in the sample.

### Claim 17 (depends on 1)

The method of claim 1, wherein the sample is not from a subject that has colon cancer or a bacterial infection.

### Claim 18 (depends on 1)

The method of claim 1, wherein the sample is not from a subject that has a clostridium difficile infection.

### Claim 19 (depends on 1)

The method of claim 1, wherein the method is not for diagnosis of colon cancer or a bacterial infection.

### Claim 20 (depends on 1)

The method of claim 1, wherein the method is not for diagnosis of a clostridium difficile infection.

### Claim 21

(canceled)

### Claim 22 (depends on 1)

The method of claim 1, wherein the subject has one or more of the following symptoms: mucosal inflammation, mucosal ulcerations, or enhanced level of inflammation markers such as platelet count, mean platelet volume, erythrocyte sedimentation rate (ESR), serum thrombopoietin, serum erythropoietin, C-reactive protein, orosomucoid (α1-acid glycoprotein), TNFα, Interleukins (e.g., IL1, IL2, IL6, IL8, IL10, IL15) as, lactoferrin or calprotectin.

### Claim 23 (depends on 1)

The method of claim 1, wherein the subject has been diagnosed with Crohn's Disease (CD).

### Claim 24 (depends on 1)

The method of claim 1, wherein the subject has been or is currently being treated for Crohn's Disease (CD).

### Claim 25 (depends on 1)

The method of claim 1, wherein the subject has Crohn's Disease in an unstable state.

### Claim 26 (depends on 1)

The method of claim 1, wherein the subject has Crohn's Disease in a stable state.

### Claim at position 27

-30. (canceled)

### Claim 31 (depends on 1)

An in vitro method for testing the efficiency of a treatment in a subject suffering from CD, or to evaluate the response of a patient to a treatment, comprising the following steps:
a) diagnosing the activity of CD before and after the administration of a treatment, according to the method of claim 1, and
b) concluding that the treatment is efficient in said subject if the state before the administration of the treatment was unstable but becomes stable upon administration of the treatment.

### Claim at position 29

-38. (canceled)

### Claim 39 (depends on 1)

The method of claim 1, wherein said subject is a human patient.

### Claim 40 (depends on 1)

The method of claim 1, wherein said relative abundance is the relative amount of host DNA as compared with the total amount of DNA present in said sample.

### Claim 41

(canceled)

### Claim 42 (depends on 41)

The method of claim 41, wherein said abundance is measured by using at least one nucleic acid fragment selected from the group of nucleic acid fragments of sequence SEQ ID NO:1 to SEQ ID NO:6, variants thereof and complementary sequences thereof.

### Claim 43

(canceled)