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Detection and quantification of donor cell-free DNA in the circulation of organ transplant recipients

Inactive Publication Date: 2016-04-28
CHRONIX BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a method for detecting transplant rejection in a patient by using a combination of SNPs with a high minor allele frequency in the recipient and donor. The method involves amplifying cfDNA from the recipient and donor, and identifying the homozygous SNPs in each. These SNPs are then used as biomarkers to monitor the integrity of the graft. The method is sensitive and accurate, and can be performed using a small amount of DNA from the recipient.

Problems solved by technology

The only limitation of such a method is the amount of DNA that is interrogated, which is mainly driven by the volume of blood that is analyzed.

Method used

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  • Detection and quantification of donor cell-free DNA in the circulation of organ transplant recipients
  • Detection and quantification of donor cell-free DNA in the circulation of organ transplant recipients
  • Detection and quantification of donor cell-free DNA in the circulation of organ transplant recipients

Examples

Experimental program
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Effect test

example 1

Quantification of Donor Cell-Free DNA

Methods

SNP Assays

[0072]SNPs were selected from public databases considering those which show a known and validated minor allelic frequency of >40% in Caucasians and >45% over all reported ethnicities. As a next step, SNP that are in or directly adjacent to a repetitive element were eliminated. The remaining SNPs were then investigated for their usefulness in a probe hydrolysis assay. This was done in silico by using thermodynamic calculations (Schlitz & von Ahsen, Biotechniques 27:1218-1222, 1224, 1999) to optimize the binding differences for the two probes that hybridize to the two alleles at the desired temperature of 65° C. at standard PCR buffer conditions (e.g. 0.18 mol / L salt and 0.5 μmol / L DNA / primer). Because the slope of a dsDNA probe melting curve is mainly dependent on the enthalpy of the probes (Marky & Breslauer, Biopolymers 26:1601-1620, 1987), the latter dominates the selection for a maximized difference of free Gibbs energy betwee...

example 2

Further Analysis of cfDNA-Quantification of GcfDNA as Copies / mL

[0085]Where the ratio of graft to host cfDNA has analytical advantages by eliminating disturbing variables, such as DNA extraction efficiency, variablities in host cfDNA may obfuscate the view on the engrafted organ. The early phase after transplant was used as model to compare the percentage or absolute plasma concentration of GcfDNA is a more valuable graft integrity measure.

Materials and Methods

[0086]Blood samples from patients after liver (LTx), heart (HTx) and kidney (KTx) were drawn according to IRB approved protocols. Samples (288) from 23 LTx were included for evaluation of the potency to measure copy numbers of GcfDNA in the initial post-operative phase. For the cfDNA extraction investigations, pools from normal volunteers were used.

[0087]EDTA-whole blood was drawn and processed within 4 hours. For LTx patients, cfDNA tubes (9 mL) Streck Inc. were used for a subset of samples. Extraction of cfDNA from 1-2.5 ml o...

example 4

Use of GcfDNA SNP Analysis to Optimize Immunosuppressive Therapy

[0092]Immunosuppression minimization requires tools to assess the minimal necessary exposure in individual patients. Drug concentrations and conventional markers are not precise predictors for this purpose. Therefore, in the present study a new practical and cost-effective method for determination of graft-derived cell-free DNA (GcfDNA) was investigated as a sensitive marker of graft injury after liver transplantation (LTx).

[0093]Methods: GcfDNA was quantified (n=171) using droplet digital PCR assay in N=12 adult patients predominantly during the early phase (days 8-30) after LTx to determine the amount of graft DNA. Values obtained in patients with various causes of graft dysfunction (i.e. hepatitis C infection [HCV+], cholestasis, low tacrolimus concentrations, and rejection) were compared to a published cut-off (10%) from a historical control group (N=10) of stable adult LTx patients without any clinical or laborator...

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Abstract

This invention provides methods, compositions, and kits relating to detecting donor cell-free DNA in the circulation of an organ transplant recipient for the early identification of transplant rejection

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority benefit to U.S. provisional application No. 61 / 828,553, filed May 29, 2013, which application is herein incorporated by reference.BACKGROUND OF THE INVENTION[0002]Using modern molecular biological techniques the detection of trace amounts of divergent genetic material in a single sample is feasible. This has potential applications for a number of conditions such as prenatal diagnosis, tumor diagnosis, and detection of transplant rejection. An increase of heart donor DNA in the circulation of stable heart transplant recipients during rejection episodes has been reported (Snyder, et al., Proc Natl Acad Sci USA 108:6229-6234, 2011). However, to be clinically useful the method used for the detection of graft DNA must not only be specific and sensitive, it must also have a rapid turn-around-time and be economically feasible to perform. The methods described to date are extremely time consuming and expensive to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/156C12Q2600/106C12Q2600/172C12Q2600/16G01N33/6854G01N2800/245C12Q1/6881
Inventor SCHUTZ, EKKEHARDBECK, JULIA
Owner CHRONIX BIOMEDICAL
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