Methods of monitoring immunosuppressive therapies in a transplant recipient

Abstract

The present disclosure relates to methods of monitoring the status of an allograft in a transplant recipient, as well as to methods of monitoring and adjusting immunosuppressive therapies being administered to the transplant recipient.

What is claimed is: 
     
         1 . A method of quantifying an amount of non-self DNA in a transplant recipient of pancreatic islet cells from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs,   (c) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (d) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns, wherein individual genotyping of the donor and the recipient across the genome as a whole or portions thereof to determine which allele of each SNP in the panel belongs to the donor and the recipient is not performed.   
     
     
         2 . The method of  claim 1 , wherein the DNA is cell-free DNA. 
     
     
         3 . The method of  claim 1 , wherein the DNA is cell-bound DNA. 
     
     
         4 . The method of  claim 2 , wherein an amount of non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         5 . The method of  claim 3 , wherein an amount of non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         6 . The method of  claim 1 , wherein each SNP in the panel is selected to have greater than 0.4 minor allele frequency and low linkage with each other. 
     
     
         7 . The method of  claim 1 , further comprising testing for the presence of an infectious agent. 
     
     
         8 . The method of  claim 7 , wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites. 
     
     
         9 . The method of  claim 1 , further comprising conducting one or more gene expression profiling assays. 
     
     
         10 . A kit for carrying out the method of  claim 1 , comprising: primers, reagents, controls for targeted single nucleotide polymorphism (SNP) amplification and sequencing, instructions for use, and instructions for accessing and using software for quantifying an amount of non-self DNA in a transplant recipient of pancreatic islet cells from a transplant donor. 
     
     
         11 . A method of quantifying an amount of DNA molecules from non-self DNA in a transplant recipient of pancreatic islet cells from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs,   (c) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (d) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of DNA molecules from non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns, wherein individual genotyping of the donor and the recipient across the genome as a whole or portions thereof to determine which allele of each SNP in the panel belongs to the donor and the recipient is not performed.   
     
     
         12 . The method of  claim 11 , wherein the DNA is cell-free DNA. 
     
     
         13 . The method of  claim 11 , wherein the DNA is cell-bound DNA. 
     
     
         14 . The method of  claim 12 , wherein an amount of DNA molecules from non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of DNA molecules from non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         15 . The method of  claim 13 , wherein an amount of DNA molecules from non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of DNA molecules from non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         16 . The method of  claim 11 , wherein each SNP in the panel is selected to have greater than 0.4 minor allele frequency and low linkage with each other. 
     
     
         17 . The method of  claim 11 , further comprising testing for the presence of an infectious agent. 
     
     
         18 . The method of  claim 17 , wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites. 
     
     
         19 . The method of  claim 11 , further comprising conducting one or more gene expression profiling assays. 
     
     
         20 . The method of  claim 11 , further comprising adding at least one reference DNA molecule to the sample before the amplifying step and quantifying an amount of DNA molecules from the non-self DNA in comparison to the at least one reference DNA molecule. 
     
     
         21 . The method of  claim 20 , wherein the amount of DNA molecules from the non-self DNA is quantified as copies of DNA molecules per volume. 
     
     
         22 . A kit for carrying out the method of  claim 11 , comprising: primers, reagents, controls for targeted single nucleotide polymorphism (SNP) amplification and sequencing, instructions for use, and instructions for accessing and using software for quantifying an amount of DNA molecules from non-self DNA in a transplant recipient of pancreatic islet cells from a donor. 
     
     
         23 . A method of preparing amplified SNPs useful for quantifying an amount of non-self DNA in a transplant recipient of pancreatic islet cells from a transplant donor without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing DNA from a sample obtained from the recipient post-transplantation;   (b) extracting DNA from the sample, wherein the DNA comprises non-self DNA and recipient-derived DNA;   (c) amplifying, in a targeted manner from the DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs;   (d) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs (SNP allele distribution patterns) in the panel; and   (e) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of non-self DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns, wherein individual genotyping of the donor and the recipient across the genome as a whole or portions thereof to determine which allele of each SNP in the panel belongs to the donor and the recipient is not performed.   
     
     
         24 . The method of  claim 23 , wherein the DNA is cell-free DNA. 
     
     
         25 . The method of  claim 23 , wherein the DNA is cell-bound DNA. 
     
     
         26 . The method of  claim 24 , wherein an amount of non-self cell-free DNA above a predetermined threshold indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease, and an amount of non-self cell-free DNA below a predetermined threshold indicates a status of engraftment, expansion and/or persistence of the transplanted cells. 
     
     
         27 . The method of  claim 25 , wherein an amount of non-self cell-bound DNA above a predetermined threshold and/or increasing or stable over a time interval indicates a status of engraftment, expansion, and/or persistence of the transplanted cells, and an amount of non-self cell-bound DNA below a predetermined threshold and/or decreasing over a time interval indicates a status of exhaustion, contraction, loss of persistence, transplanted cell rejection, disease relapse, and/or graft vs. host disease. 
     
     
         28 . The method of  claim 23 , further comprising testing for the presence of an infectious agent. 
     
     
         29 . The method of  claim 28 , wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites. 
     
     
         30 . The method of  claim 23 , further comprising conducting one or more gene expression profiling assays.