Non-invasive diagnosis of graft rejection in organ transplant patients

• US 8,703,652 B2
• Filed: 11/05/2010
• Issued: 04/22/2014
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Quake, Stephen R., Snyder, Thomas M., Valantine, Hannah
• Primary Examiner(s): Leavitt, Maria
• Assistant Examiner(s): Bunker, Amy M
• Application Number:
  US13/508,318
• Publication Number:
  US 20120295810A1
• Time in Patent Office: 1,264 Days
• Field of Search: 506/9, 435/6.12, 435/6.11
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16H 20/40 relating to mechanical, radiation or invasive therapies, e.g. surgery, laser therapy, dialysis or acupuncture
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems

Abstract

The disclosure provides methods, devices, compositions and kits for diagnosing or predicting transplant status or outcome in a subject who has received a transplant. The methods comprise determining the presence or absence of one or more nucleic acids from a donor transplant, wherein said one or more nucleic acids from said donor are identified based on a predetermined marker profile, and diagnosing or predicting transplant status or outcome based on the presence or absence of said one or more nucleic acids.

Claims

1. A method for detecting transplant rejection, graft dysfunction, or organ failure, the method comprising:

(a) providing a sample comprising cell-free nucleic acids from a subject who has received a transplant from a donor;

(b) obtaining a genotype of donor-specific polymorphisms or a genotype of subject-specific polymorphisms, or obtaining both a genotype of donor-specific polymorphisms and subject-specific polymorphisms, to establish a polymorphism profile for detecting donor cell-free nucleic acids, wherein at least one single nucleotide polymorphism (SNP) is homozygous for the subject if the genotype comprises subject-specific polymorphisms comprising SNPs;

(c) multiplex sequencing of the cell-free nucleic acids in the sample followed by analysis of the sequencing results using the polymorphism profile to detect donor cell-free nucleic acids and subject cell-free nucleic acids; and

(d) diagnosing, predicting, or monitoring a transplant status or outcome of the subject who has received the transplant by determining a quantity of the donor cell-free nucleic acids based on the detection of the donor cell-free nucleic acids and subject cell-free nucleic acids by the multiplexed sequencing, wherein an increase in the quantity of the donor cell-free nucleic acids over time is indicative of transplant rejection, graft dysfunction or organ failure, and wherein sensitivity of the method is greater than 56% compared to sensitivity of current surveillance methods for cardiac allograft vasculopathy (CAV).

2. The method of claim 1, wherein the polymorphism profile comprises one or more genetic variations selected from single nucleotide polymorphisms (SNPs), variable number of tandem repeats (VNTRs), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, insertion elements, insertions, repeats, or deletions.

3. The method of claim 1, wherein the cell-free nucleic acids are deoxyribonucleic acid (DNA).

4. The method of claim 1, wherein the multiplexed sequencing comprises shotgun sequencing.

5. The method of claim 1, wherein the multiplexed sequencing occurs in a single container.

6. The method of claim 1, wherein the multiplexed sequencing comprises sequencing at least ten different nucleic acids.

7. The method of claim 1 further comprising administering an immunosuppressive drug.

8. The method of claim 1, wherein the diagnosing, predicting, or monitoring transplant status or outcome comprises treating a transplant rejection in a subject in need thereof.

9. The method of claim 1, wherein the diagnosing, predicting, or monitoring transplant status or outcome comprises determining, modifying, or maintaining an immunosuppressive regimen.

10. The method of claim 1 further comprising conducting an assay selected from:

digital polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), array, or any combination thereof.

11. The method of claim 1, wherein the polymorphism profile comprises at least one single nucleotide polymorphism.

12. The method of claim 1, wherein the sample is blood or serum.

13. The method of claim 1, wherein the sample is urine or stool.

15. The method of claim 1, wherein the transplant is a heart transplant or kidney transplant.

16. The method of claim 1, wherein the cell-free nucleic acids are ribonucleic acid (RNA).

14. The method of 1, wherein the transplant is selected from the group consisting of:

kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, pancreas after kidney transplant, and simultaneous pancreas-kidney transplant.