Method for identifying kidney allograft rejection genes in urine and utility of making those measurements
AssigneeNatera, Inc.
InventorsAlain MIR, Alvin CHON, Aaron BRUNS, Swee Seong WONG
Life of this patent
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Abstract
The present disclosure provides methods for preparation and analysis of biological samples of kidney transplant recipients, wherein the methods comprise nucleic acids or protein derived from a urine sample of the kidney transplant recipient, and measuring amounts of nucleic acids or protein expressed from pre-selected target genes indicative of kidney disease states. These methods enable assessment of kidney transplant rejection and kidney transplant rejection states such as T-cell mediated rejection (TCMR), antibody-mediated rejection (ABMR), or a mixed TCMR and ABMR disease state.
Claims89
A method of preparing a composition of nucleic acids derived from a urine sample of a kidney transplant recipient useful for determination of kidney transplant rejection or risk of kidney transplant rejection comprising:
- (a) extracting nucleic acids from the urine sample of the kidney transplant recipient, wherein the extracted nucleic acids comprise one or more mRNAs of target genes and/or miRNAs associated with kidney transplant rejection;
- (b) preparing a composition of nucleic acids from the extracted nucleic acids in step (a) by isolating mRNAs and/or miRNAs and removing contaminating molecules, optionally wherein preparing the composition comprises reverse transcribing complementary DNA (cDNA) from the nucleic acids extracted in step (a);
- (c) measuring an amount of the one or more mRNAs and/or miRNAs, and generating one or more transplant rejection scores from the measured amount of the one or more mRNAs and/or miRNAs, wherein the one or more transplant rejection scores provide a quantitative value of kidney transplant rejection risk or the presence of kidney transplant rejection.
The method of any one of the preceding claims, comprising collecting the urine sample from the transplant recipient at different times and generating the one or more transplant rejection scores from each urine sample.
The method of any one of the preceding claims, wherein the kidney transplant recipient is administered a treatment for the determined kidney disease state or kidney transplant rejection.
The method of any one of the preceding claims, further comprising:
- (i) measuring an amount of donor-derived cell-free DNA in a sample obtained from the transplant recipient, extracting cell-free DNA from the sample obtained from the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA;
- (ii) performing targeted amplification of the extracted DNA at 50-50,000 target loci in a single reaction volume;
- (iii) sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads and measuring the amount of donor-derived cell-free DNA based on the sequencing reads, generating a transplant rejection score indicating transplant rejection based on whether the measured amount of donor-derived cell-free DNA or a function thereof exceeds a cutoff threshold of cell-free DNA amount that indicates transplant rejection, wherein transplant rejection is determined based on both the one or more transplant rejection scores from the measured amount of one or more mRNAs and/or miRNAs and the transplant rejection score determined based on the measured amount of donor-derived cell-free DNA.
The method of any one of the preceding claims, wherein measuring the amount of mRNAs and/or miRNAs comprises amplification of at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target nucleic acid molecules, from 2-10, 200-100, 50-500, or 50-2000 target nucleic acid molecules, using at least at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 100 target RNA molecules, from 2-10, 200-100, 50-500, or 50-2000 pairs of forward and reverse PCR primers.
The method of any one of the preceding claims, wherein the one or more mRNAs and/or miRNAs are determined by text mining databases.
The method of any one of the preceding claims, wherein the amount of one or more mRNAs and/or miRNAs is measured relative to a housekeeping gene.
A method of preparing a composition of nucleic acids derived from a urine sample of a kidney transplant recipient useful for determination of kidney transplant rejection or risk of kidney transplant rejection comprising:
- (a) extracting nucleic acids from the urine sample of the kidney transplant recipient, wherein the extracted nucleic acids comprise one or more RNA molecules associated with a risk of kidney transplant rejection;
- (b) preparing the composition of nucleic acids from the extracted nucleic acids from step (a) by isolating RNA molecules and removing contaminating molecules;
- optionally wherein preparing the composition comprises performing reverse transcription of the RNA molecules to synthesize cDNA;
- (c) measuring an amount of RNA molecules associated with a risk of kidney transplant rejection in the composition of nucleic acids, and generating one or more transplant rejection scores from the measured amount of one or more RNA molecules, wherein the one or more transplant rejection scores provide a quantitative value of kidney transplant rejection risk or the presence of kidney transplant rejection.
A method of preparing a composition of protein derived from a urine sample of a kidney transplant recipient useful for determination of kidney transplant rejection or risk of kidney transplant rejection comprising:
- (a) extracting protein from the urine sample of the kidney transplant recipient, wherein the extracted proteins are associated with a risk of kidney transplant rejection;
- (b) preparing the composition of protein from the protein extracted in step (a) by removing contaminating molecules;
- (c) measuring an amount of proteins in the composition, and generating one or more transplant rejection scores from the measured amount, wherein the one or more transplant rejection scores provide a quantitative value of kidney transplant rejection risk or the presence of kidney transplant rejection.
Dependencies are inferred from the claim text.