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 preparing amplified SNPs useful for quantifying an amount of transplant donor-derived cell-free DNA in a urine sample from a solid organ transplant recipient without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing a urine sample obtained from the transplant recipient;   (b) extracting cell-free DNA from the urine sample, wherein the cell-free DNA comprises transplant donor-derived cell-free DNA and recipient-derived cell-free DNA;   (c) amplifying, in a targeted manner from the cell-free 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 in the panel; and   (e) quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of transplant donor-derived cell-free 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 an amount of transplant donor-derived cell-free DNA above a suitable threshold is indicative of transplant rejection or the risk thereof, and wherein an amount of transplant donor-derived cell-free DNA below a suitable threshold is indicative of transplant tolerance, and 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 organ transplant is selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a cornea transplant, an organ system transplant, a bone marrow transplant, a pancreatic islet cell transplant, a stem cell transplant, a skin tissue transplant, a skin cell transplant, and a xenotransplant. 
     
     
         3 . 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. 
     
     
         4 . The method of  claim 1 , wherein each SNP in the panel is selected to have an overall population minor allele frequency of >0.4, a target population minor allele frequency of >0.4, and the lowest polymerase error rate of the 6 potential allele transitions or transversions, and the genomic distance between each independent SNP is >500 kb. 
     
     
         5 . The method of  claim 1 , wherein the sequencing of the amplified SNPs is performed using a multiplex sequencing platform. 
     
     
         6 . The method of  claim 1 , further comprising testing for the presence of an infectious agent. 
     
     
         7 . The method of  claim 6 , wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites. 
     
     
         8 . The method of  claim 1 , further comprising conducting one or more gene expression profiling assays. 
     
     
         9 . 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 transplant donor-derived cell-free DNA in a urine sample from the transplant recipient without consideration of prior genotype information from the transplant recipient or donor. 
     
     
         10 . A system for carrying out the method of  claim 1 , comprising: one or more processors; and
 memory coupled to the one or more processors for quantifying an amount of transplant donor-derived cell-free DNA in a urine sample from the transplant recipient without consideration of prior genotype information from the transplant recipient or donor.   
     
     
         11 : A method of preparing amplified SNPs useful for quantifying an amount of DNA molecules from transplant donor-derived cell-free DNA in a urine sample from a solid organ transplant recipient without consideration of genotype information from the transplant recipient or donor, the method comprising:
 (a) providing a urine sample obtained from the transplant recipient;   (b) extracting cell-free DNA from the urine sample, wherein the cell-free DNA comprises DNA molecules from the transplant donor and DNA molecules from the recipient;   (c) amplifying, in a targeted manner from the cell-free 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 in the panel; and   (e) quantifying, without consideration of genotype information from the transplant donor or recipient, an amount of DNA molecules from the transplant donor-derived cell-free 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 an amount of DNA molecules from the transplant donor-derived cell-free DNA above a suitable threshold is indicative of transplant rejection or the risk thereof, and wherein an amount of DNA molecules from the transplant donor-derived cell-free DNA below a suitable threshold is indicative of transplant tolerance, and 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 organ transplant is selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a cornea transplant, an organ system transplant, a bone marrow transplant, a pancreatic islet cell transplant, a stem cell transplant, a skin tissue transplant, a skin cell transplant, and a xenotransplant. 
     
     
         13 . 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. 
     
     
         14 . The method of  claim 11 , wherein each SNP in the panel is selected to have an overall population minor allele frequency of >0.4, a target population minor allele frequency of >0.4, and the lowest polymerase error rate of the 6 potential allele transitions or transversions, and the genomic distance between each independent SNP is >500 kb. 
     
     
         15 . The method of  claim 11 , wherein the sequencing of the amplified SNPs is performed using a multiplex sequencing platform. 
     
     
         16 . The method of  claim 11 , further comprising testing for the presence of an infectious agent. 
     
     
         17 . The method of  claim 16 , wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites. 
     
     
         18 . The method of  claim 11 , further comprising conducting one or more gene expression profiling assays. 
     
     
         19 . 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 transplant donor-derived cell-free DNA in comparison to the at least one reference DNA molecule. 
     
     
         20 . The method of  claim 19 , wherein the amount of DNA molecules from the transplant donor-derived cell-free DNA is quantified as copies of DNA molecules per volume. 
     
     
         21 . 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 transplant donor-derived cell-free DNA in a urine sample from the transplant recipient without consideration of prior genotype information from the transplant recipient or donor. 
     
     
         22 . The kit of  claim 21 , further comprising reference DNA molecules. 
     
     
         23 . The kit of  claim 22 , wherein the amount of DNA molecules from the transplant donor-derived cell-free DNA is quantified as copies of DNA molecules per volume. 
     
     
         24 . A system for carrying out the method of  claim 11 , comprising: one or more processors; and memory coupled to the one or more processors for quantifying an amount of DNA molecules from the transplant donor-derived cell-free DNA in a urine sample from the transplant recipient without consideration of prior genotype information from the transplant recipient or donor. 
     
     
         25 . A system for quantifying an amount of transplant donor-derived cell-free DNA in a sample from a transplant recipient of an organ transplant from a transplant donor without consideration of prior genotype information from the transplant recipient or donor, comprising one or more processors; and memory coupled to the one or more processors, the memory encoded with a set of instructions configured to perform a process comprising:
 obtaining measurements of a panel of single nucleotide polymorphisms (SNPs) that were amplified, in a targeted manner, from cell-free DNA derived from a sample from the organ transplant recipient following transplantation and sequenced to provide homozygous or heterozygous allele distribution patterns of the SNPs in the panel; and   quantifying, without consideration of genotype information from the transplant donor or recipient, the amount of transplant donor-derived cell-free 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 an amount of transplant donor-derived cell-free DNA above a suitable threshold is indicative of transplant rejection or the risk thereof, and wherein an amount of transplant donor-derived cell-free DNA below a suitable threshold is indicative of transplant tolerance, and 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.   
     
     
         26 . The system of  claim 25 , wherein the organ transplant is selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a cornea transplant, an organ system transplant, a bone marrow transplant, a pancreatic islet cell transplant, a stem cell transplant, a skin tissue transplant, a skin cell transplant, and a xenotransplant. 
     
     
         27 . The system of  claim 25 , wherein the sample is a urine sample. 
     
     
         28 . The system of  claim 25 , wherein each SNP in the panel is selected to have greater than 0.4 minor allele frequency and low linkage with each other. 
     
     
         29 . The system of  claim 25 , further comprising reagents, controls, instructions for use, and instructions for accessing and using software for testing for the presence of an infectious agent. 
     
     
         30 . The system of  claim 25 , further comprising reagents, controls, instructions for use, and instructions for accessing and using software for conducting one or more gene expression profiling assays.