Assessing graft suitability for transplantation

Patent metadata

Field	Value
Publication number	US-12084720-B2
Application number	US-201816954183-A
Country	US
Kind code	B2
Filing date	Dec 14, 2018
Priority date	Dec 14, 2017
Publication date	Sep 10, 2024
Grant date	Sep 10, 2024

Abstract

This invention relates to methods and compositions for assessing the suitability of a graft for transplantation or implantation by measuring total and/or specific cell-free nucleic acids (such as cf-DNA) and/or cell lysis. Specifically, the method comprising obtaining an amount of total cf DNA and/or graft-specific cfDNA released from a potential graft (e.g., ex vivo), e.g., prior to contacting of the potential graft with blood cells of a potential recipient, and/or subsequent to contacting of the potential graft or cells thereof with blood cells from a potential recipient, assessing the amount(s) to determine the suitability of the potential graft for transplantation or implantation.

First claim

The invention claimed is:

1. A method of preparing a preparation of amplified DNA from a sample comprising DNA released from a potential graft and assessing the suitability of the potential graft for transplantation or implantation, comprising:
(a) extracting cell-free DNA (cf-DNA) from the sample, wherein the sample is obtained from a perfusate in contact with the potential graft ex vivo and comprises cf-DNA released from the potential graft subsequent to contacting of the potential graft with blood cells from a potential recipient, wherein the perfusate is a buffered extracellular solution comprising Dextran 40 and human serum albumin, wherein the potential graft is a lung, and wherein the sample is processed with a step that removes contaminating cells from the sample prior to extracting cf-DNA,
(b) amplifying the cf-DNA extracted in (a), and
(c) assessing the amplified DNA to quantify amounts of both total cf-DNA and graft-specific cf-DNA to determine the suitability of the potential graft for transplantation or implantation, and
wherein the method further comprises quantifying short DNA fragments having 75-170 base pairs in length which are indicative of cell apoptosis and long DNA fragments having 500-15,000 base pairs in length which are indicative of cell lysis.

2. The method of claim 1 , wherein the method further comprises obtaining the potential graft.

3. The method of claim 1 , wherein the method further comprises obtaining blood from a potential recipient.

4. The method of claim 1 , wherein the method further comprises obtaining amounts of total cf-DNA and graft-specific cf-DNA at one or more additional time points.

5. The method of claim 1 , wherein the method further comprises comparing the amounts of the total cf-DNA and graft-specific cf-DNA with threshold values of total cf-DNA and graft-specific cf-DNA obtained from one or more additional time points.

6. The method of claim 1 , wherein the potential graft is monitored over time.

7. The method of claim 1 , wherein step (c) comprises quantifying the amounts of total cf-DNA and graft-specific cf-DNA by performing real-time PCR or digital PCR.

8. The method of claim 1 , wherein step (c) comprises quantifying the amounts of the total cf-DNA and graft-specific cf-DNA by performing next-generation sequencing.

9. The method of claim 1 , wherein the amounts of total cf-DNA and graft-specific cf-DNA above a threshold value of total cell-free DNA and graft-specific cf-DNA determined from a different point in time is indicative of decreasing suitability, or unsuitability, of the graft.

10. The method of claim 1 , wherein the amounts of total cf-DNA and graft-specific cf-DNA below a threshold value of total cell-free DNA and graft-specific cf-DNA determined from a different point in time is indicative of increasing suitability, or suitability, of the graft.

11. The method of claim 1 , wherein step (b) comprises performing mismatch PCR amplification to obtain the preparation of amplified DNA.

12. The method of claim 1 , wherein the potential graft is obtained from a different species than human.

13. The method of claim 1 , wherein the potential graft is obtained from a pig.

Assignees

• Natera Inc

Inventors

• Mitchell Aoy Tomita
• Mitchell Michael

Classifications

• C12Q2600/118

  Prognosis of disease development · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title