Methods for non-invasively monitoring organ health in xenotransplants

Abstract

The present disclosure relates to methods for detecting, predicting, diagnosing, and / or monitoring the status of xenografts in transplant recipients, as well as methods for monitoring, administering, and adjusting immunosuppressive therapy. The present disclosure describes highly sensitive, non-invasive methods and kits for detecting, predicting, diagnosing, and / or monitoring the health status of transplants, as well as for detecting, predicting, and / or monitoring the transplant rejection status in recipients of organ, cell, or tissue transplants from donors, where the donor and recipient belong to different species. Such methods and kits are based on the detection and quantification of donor-specific nucleic acids, such as DNA or RNA, in body fluids, including, but not limited to, blood, serum, plasma, or urine, after transplantation.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 322,144, filed March 21, 2022, which is incorporated by reference herein in its entirety.

[0002] (Reference to Electronic Sequence Listing) The contents of the electronic sequence listing (506612003040SEQLIST.xml, size: 21075 bytes, and creation date: March 21, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION Provided herein are methods and kits for detecting, predicting, diagnosing, and / or monitoring the status of organ, cell, or tissue xenografts, and associated therapeutic methods. [Background technology]

[0004] Xenotransplantation or xenotransplantation, the transplantation of cells, tissues, or organs from one species to another, is a promising solution to the severe shortage of human organs, cells, and tissues available for transplantation. However, xenotransplantation presents challenges due, at least in part, to the possibility of cross-species infection transmission, immune rejection by the transplant recipient, and genetic, anatomical, and physiological differences between the donor and the recipient. Immune rejection of xenografts may be mediated through both adaptive and innate immunity in the transplant recipient. In some cases, the recipient's immune response to the xenotransplant may result in hyperacute rejection, acute antibody-mediated rejection, acute cellular rejection, or mixed antibody-mediated and cellular rejection. See, e.g., Lu et al., "Xenotransplantation: Current Status in Preclinical Research." Frontiers in immunology vol.10 3060.23 Jan.2020. Therefore, it is necessary to medically suppress the recipient's immune system to prevent or inhibit immune rejection of the xenograft.

[0005] After transplantation, the status of the graft in the transplant recipient, including assessment of graft function and immune-mediated rejection of the graft, may be monitored throughout the recipient's lifetime. In cardiac transplantation, for example, monitoring for rejection may include up to 15 scheduled biopsies within the first year of transplantation to provide specimens of the myocardium for histological evaluation by a pathologist. Each biopsy procedure is invasive, stressful, inconvenient, and expensive, as well as carrying procedural risks for the patient. Furthermore, biopsy sampling is highly localized, so histological abnormalities in any non-biopsy area of ​​the heart will be missed. Biopsy grading is subjective, and discrepancies in biopsy findings between independent pathologists are common. Furthermore, while biopsy is primarily used for monitoring transplant rejection within the first year after transplantation, this invasive method has not been adequately adapted or established to guide longer-term (e.g., beyond one year after transplantation) individualized immunosuppressive therapy. Laboratory tests have been developed to assess the status of allogeneic grafts, i.e., grafts between members of a single species based on genetic variations within the same species, e.g., single nucleotide polymorphisms (SNPs) or polymorphisms based on human leukocyte antigen (HLA) genes. However, these laboratory tests may not be applicable or may not be adequately adapted to assess the status of grafts where the donor and recipient belong to different species. Thus, there is a need for improved non-invasive methods of detecting, predicting, diagnosing, and / or monitoring xenograft status in transplant recipients, as well as methods of determining the need to administer or adjust immunosuppressive therapy to xenograft transplant recipients. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lu et al. “Xenotransplantation:Current Status in Preclinical Research.” Frontiers in immunology vol.10 3060.23 Jan.2020 Summary of the Invention [Means for solving the problem]

[0007] In one aspect, provided herein is a method for detecting and monitoring transplant rejection status of a donor-derived graft in a transplant recipient, wherein the donor and the recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplantation, the sample comprising donor-derived cell-free nucleic acid and recipient-derived cell-free nucleic acid; b) determining the amount of donor-derived cell-free nucleic acid in the sample by generating sequence reads from the cell-free nucleic acid in a high-throughput sequencing assay, the generated sequence reads corresponding to donor-specific genomic sequences and recipient-specific genomic sequences, and mapping the generated sequence reads to at least the donor-specific genomic sequence, taking into account the difference in genome size between the donor and the recipient; and c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold. In some embodiments, the method further comprises adding a quantitative spike-in nucleic acid control to the sample in step a, and the sequence reads from the spike-in control are used to determine the absolute amount of donor-derived cell-free nucleic acid. In some embodiments, the determining step includes mapping the generated sequence reads to the donor-specific genome sequence and the recipient-specific genome sequence. In some embodiments, the sequencing reads are generated from a region of a selected size of the genome. In some embodiments, the size difference between the donor genome and the recipient genome is taken into account by utilizing the average coverage over a region of a selected size of the genome, where the region is large enough to have sufficient coverage and the background noise is low enough to distinguish between the donor genome and the recipient genome. In some embodiments, the region has a size of up to 1M bases, up to 10M bases, or up to 100M bases. In some embodiments, the amount of cell-free nucleic acid from the donor is a percentage of the average coverage over a region of a selected size of the genome, where the region is large enough to have sufficient coverage and the background noise is low enough to distinguish between the donor genome and the recipient genome.In some embodiments, the high throughput sequencing assay comprises a next generation sequencing assay.

[0008] In another aspect, provided herein is a method for treating transplant rejection in a recipient of a transplant from a donor, wherein the donor and recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, comprising cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient; b) determining an amount of donor-derived cell-free nucleic acid in the sample by generating sequence reads from the cell-free nucleic acid in a high throughput sequencing assay, wherein the generated sequence reads correspond to donor-specific genomic sequences and recipient-specific genomic sequences, and mapping the generated sequence reads to at least the donor-specific genomic sequence, wherein the difference in genome size between the donor and recipient is taken into account; c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold; and d) administering an immunosuppressive treatment to the transplant recipient based on the amount of donor-derived cell-free nucleic acid. In some embodiments, the method further comprises adding a quantitative spike-in nucleic acid control to the sample in step a, and the sequence reads from the spike-in control are used to determine the absolute amount of donor-derived cell-free nucleic acid. In some embodiments, the determining step comprises mapping the generated sequence reads to a donor-specific genome sequence and a recipient-specific genome sequence. In some embodiments, the sequencing reads are generated from a selected-sized region of the genome. In some embodiments, the size difference between the donor genome and the recipient genome is taken into account by utilizing the average coverage over a selected-sized region of the genome, where the region is large enough to have sufficient coverage and the background noise is low enough to distinguish between the donor genome and the recipient genome. In some embodiments, the region has a size of up to 1 M bases, up to 10 M bases, or up to 100 M bases.In some embodiments, the amount of cell-free nucleic acid from the donor is a percentage of the average coverage over a region of a selected size of the genome, where the region is large enough to have sufficient coverage and where the background noise is low enough to distinguish between the donor genome and the recipient genome. In some embodiments, the high-throughput sequencing assay comprises a next-generation sequencing assay.

[0009] In another aspect, provided herein is a method for adjusting immunosuppressive therapy in a recipient of a transplant from a donor, wherein the donor and recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, comprising cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient; b) determining an amount of donor-derived cell-free nucleic acid in the sample by generating sequence reads from the cell-free nucleic acid in a high throughput sequencing assay, wherein the generated sequence reads correspond to a donor-specific genomic sequence and a recipient-specific genomic sequence, and mapping the generated sequence reads to at least the donor-specific genomic sequence, wherein the difference in genome size between the donor and the recipient is taken into account; c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold; and d) adjusting the immunosuppressive therapy administered to the transplant recipient based on the amount of donor-derived cell-free nucleic acid. In some embodiments, the method further comprises adding a quantitative spike-in nucleic acid control to the sample in step a, and the sequence reads from the spike-in control are used to determine the absolute amount of donor-derived cell-free nucleic acid. In some embodiments, the determining step comprises mapping the generated sequence reads to a donor-specific genome sequence and a recipient-specific genome sequence. In some embodiments, the sequencing reads are generated from a selected-sized region of the genome. In some embodiments, the size difference between the donor genome and the recipient genome is taken into account by utilizing the average coverage over a selected-sized region of the genome, where the region is large enough to have sufficient coverage and the background noise is low enough to distinguish between the donor genome and the recipient genome. In some embodiments, the region has a size of up to 1 M bases, up to 10 M bases, or up to 100 M bases.In some embodiments, the amount of cell-free nucleic acid from the donor is a percentage of the average coverage over a region of a selected size of the genome, where the region is large enough to have sufficient coverage and where the background noise is low enough to distinguish between the donor genome and the recipient genome. In some embodiments, the high-throughput sequencing assay comprises a next-generation sequencing assay.

[0010] In another aspect, provided herein is a method for detecting and monitoring transplant rejection status of a donor-derived graft in a transplant recipient, the donor and the recipient being of different species, the method comprising: a) providing a sample from the transplant recipient after transplantation, the sample comprising donor-derived cell-free nucleic acid and recipient-derived cell-free nucleic acid; b) determining the amount of donor-derived cell-free nucleic acid in the sample by digital PCR assay, the amount of donor-derived cell-free nucleic acid being determined by digital PCR quantification of donor-specific nucleic acid and recipient-specific nucleic acid with respect to the amount of sample analyzed or as a ratio of donor-derived cell-free nucleic acid to total donor-specific nucleic acid and recipient-specific nucleic acid; and c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold. In some embodiments, the digital PCR assay comprises one or more singleplex digital PCR assays. In some embodiments, the digital PCR assay is a multiplex digital PCR assay consisting of two or more PCR assays in a single digital PCR reaction. In some embodiments, the first singleplex digital PCR assay has a single copy of a recipient-specific target in the haploid recipient genome, and the second singleplex digital PCR assay has a single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, the second singleplex digital PCR assay has two or more copies of a donor-specific target in the haploid donor genome. In some embodiments, the multiplex digital PCR assay has at least one or two single copies of a recipient-specific target in the haploid recipient genome, and at least one or more single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, each PCR assay has two or more copies of a donor-specific target in the haploid donor genome.

[0011] In another aspect, provided herein is a method for treating transplant rejection of a graft from a donor in a transplant recipient, wherein the donor and the recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, the sample comprising cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient; b) determining the amount of cell-free nucleic acid derived from the donor in the sample by digital PCR assay, the amount of cell-free nucleic acid derived from the donor being determined by digital PCR quantification of donor-specific nucleic acid and recipient-specific nucleic acid with respect to the amount of sample analyzed or as a ratio of donor-specific cell-free nucleic acid to total donor-specific nucleic acid and recipient-specific nucleic acid; c) detecting transplant rejection if the amount of cell-free nucleic acid derived from the donor exceeds a predetermined threshold; and d) administering immunosuppressive treatment to the transplant recipient based on the amount of cell-free nucleic acid derived from the donor. In some embodiments, the digital PCR assay comprises one or more singleplex digital PCR assays. In some embodiments, the digital PCR assay is a multiplex digital PCR assay consisting of two or more PCR assays in a single digital PCR reaction. In some embodiments, the first singleplex digital PCR assay has a single copy of a recipient-specific target in the haploid recipient genome, and the second singleplex digital PCR assay has a single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, the second singleplex digital PCR assay has two or more copies of a donor-specific target in the haploid donor genome. In some embodiments, the multiplex digital PCR assay has at least one or two single copies of a recipient-specific target in the haploid recipient genome, and at least one or more single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, each PCR assay has two or more copies of a donor-specific target in the haploid donor genome.

[0012] In another aspect, provided herein is a method for adjusting immunosuppressive therapy in a recipient of a transplant from a donor, the donor and the recipient being of different species, the method comprising: a) providing a sample from the transplant recipient after transplant, the sample comprising cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient; b) determining the amount of cell-free nucleic acid derived from the donor in the sample by digital PCR assay, the amount of cell-free nucleic acid derived from the donor being determined by digital PCR quantification of donor-specific nucleic acid and recipient-specific nucleic acid with respect to the amount of sample analyzed or as a ratio of donor-specific cell-free nucleic acid to total donor-specific nucleic acid and recipient-specific nucleic acid; c) detecting transplant rejection if the amount of cell-free nucleic acid derived from the donor exceeds a predetermined threshold; and d) adjusting the immunosuppressive therapy administered to the transplant recipient based on the amount of cell-free nucleic acid derived from the donor. In some embodiments, the digital PCR assay comprises one or more singleplex digital PCR assays. In some embodiments, the digital PCR assay is a multiplex digital PCR assay consisting of two or more PCR assays in a single digital PCR reaction. In some embodiments, the first singleplex digital PCR assay has a single copy of a recipient-specific target in the haploid recipient genome, and the second singleplex digital PCR assay has a single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, the second singleplex digital PCR assay has two or more copies of a donor-specific target in the haploid donor genome. In some embodiments, the multiplex digital PCR assay has at least one or two single copies of a recipient-specific target in the haploid recipient genome, and at least one or more single or multiple copies of a donor-specific target in the haploid donor genome. In some embodiments, each PCR assay has two or more copies of a donor-specific target in the haploid donor genome.

[0013] In some embodiments that may be combined with any of the preceding aspects or embodiments, the method further comprises testing for the presence of an infectious agent. In some embodiments, the infectious agent is selected from a virus, a bacterium, a fungus, or a parasite. In some embodiments, the infectious agent is a virus selected from a cytomegalovirus, an Epstein-Barr virus, an Anelloviridae, or a BK virus. In some embodiments that may be combined with any of the preceding aspects or embodiments, the method further comprises performing one or more gene expression profiling assays on the recipient. In some embodiments, the combined score is calculated based on the amount of donor-derived cell-free nucleic acid in the sample and the results of the gene expression profiling assays.

[0014] In another aspect, provided herein is a kit for detecting and monitoring transplant rejection status of a graft from a donor in a transplant recipient, wherein the donor and recipient belong to different species, the kit comprising one or more PCR reaction oligonucleotide primer and probe sets that hybridize to donor-specific or recipient-specific target sequences in cell-free nucleic acid from the transplant recipient for digital PCR quantification of donor-derived cell-free nucleic acid for the amount of sample analyzed or for the total cell-free nucleic acid analyzed, and instructions for data analysis to determine the amount of donor-derived cell-free nucleic acid.

[0015] In some embodiments that may be combined with any of the preceding aspects or embodiments, the cell-free nucleic acid from the donor is DNA. In some embodiments that may be combined with any of the preceding aspects or embodiments, the cell-free nucleic acid from the donor is DNA, RNA, mRNA, miRNA, double-stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, RNA hairpin, or combinations thereof.

[0016] In some embodiments that may be combined with any of the preceding aspects or embodiments, the amount of cell free nucleic acid from the donor is adjusted with a correction factor to correct for differences in genome length between the donor genome and the recipient genome. In some embodiments that may be combined with any of the preceding aspects or embodiments, the amount of cell free nucleic acid from the donor is adjusted with a correction factor to correct for differences in cell free nucleic acid fragment size between the cell free nucleic acid that is donor-derived and the cell free nucleic acid that is recipient-derived.

[0017] In some embodiments that may be combined with any of the preceding aspects or embodiments, the amount of cell-free nucleic acid from the donor is a ratio of cell-free nucleic acid from the donor to total cell-free nucleic acid or cell-free nucleic acid from the recipient. In some embodiments, the ratio is a ratio of cell-free nucleic acid mass from the donor to total cell-free nucleic acid or cell-free nucleic acid mass from the recipient. In some embodiments, the ratio is a ratio of cell-free nucleic acid genome copy equivalents from the donor to total cell-free nucleic acid genome or cell-free nucleic acid genome copy equivalents from the recipient.

[0018] In some embodiments that may be combined with any of the preceding aspects or embodiments, the amount of cell-free nucleic acid from the donor is a percentage of cell-free nucleic acid from the donor compared to the total cell-free nucleic acid. In some embodiments, the percentage is a percentage of the mass of cell-free nucleic acid from the donor compared to the mass of the total cell-free nucleic acid. In some embodiments, the percentage is a percentage of cell-free nucleic acid genome copy equivalents from the donor compared to the total cell-free nucleic acid genome copy equivalents.

[0019] In some embodiments that may be combined with any of the preceding aspects or embodiments, the graft is a solid organ, tissue, or cell graft. In some embodiments that may be combined with any of the preceding aspects or embodiments, the donor of the graft is an animal. In some embodiments, the animal is a pig.

[0020] In some embodiments that may be combined with any of the preceding aspects or embodiments, the next generation sequencing assay is amplicon-based. In some embodiments that may be combined with any of the preceding aspects or embodiments, the next generation sequencing assay is not amplicon-based.

[0021] In another aspect, provided herein is a method for detecting and monitoring transplant rejection status of a donor-derived graft in a transplant recipient, wherein the donor and the recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, the sample comprising donor-derived and recipient-derived cell-free nucleic acid; b) determining by PCR assay the amount of donor-derived cell-free nucleic acid in the sample, the amount of donor-derived cell-free nucleic acid being determined by PCR quantification of donor-specific and recipient-specific nucleic acid, either (i) as absolute copies of donor-derived cell-free nucleic acid in the sample, or (ii) as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid; and c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold. In some embodiments, the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification. In some embodiments, the PCR assay is a digital PCR assay and the PCR quantification is end-point PCR quantification. In some embodiments, the digital PCR assay comprises (a) at least one singleplex digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome, and / or (b) at least one singleplex digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome. In some embodiments, the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copy donor-specific and / or recipient-specific targets in a single digital PCR reaction, where the copy number of the donor-specific target is relative to the haploid donor genome and the copy number of the recipient-specific target is relative to the haploid recipient genome.In some embodiments, the digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay, (a) the first digital PCR assay is for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is for the haploid recipient genome, and (b) the second digital PCR assay is for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is for the haploid donor genome. In some embodiments, the second singleplex digital PCR assay is for multiple copies of the donor-specific target. In some embodiments, the multiplex digital PCR assay is for (a) at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is for the haploid recipient genome, and (b) at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is for the haploid donor genome. In some embodiments, the multiplex digital PCR assay is for at least one multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome.

[0022] In another aspect, provided herein is a method for treating transplant rejection of a graft from a donor in a transplant recipient, wherein the donor and recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, the sample comprising donor-derived cell-free nucleic acid and recipient-derived cell-free nucleic acid; b) determining by PCR assay an amount of donor-derived cell-free nucleic acid in the sample, wherein the amount of donor-derived cell-free nucleic acid is determined by PCR quantification of donor-specific and recipient-specific nucleic acid (i) as absolute copies of donor-derived cell-free nucleic acid in the sample or (ii) as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid; c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold; and d) administering an immunosuppressive treatment to the transplant recipient based on the amount of donor-derived cell-free nucleic acid. In some embodiments, the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification. In some embodiments, the PCR assay is a digital PCR assay, and the PCR quantification is end-point PCR quantification. In some embodiments, the digital PCR assay comprises (a) at least one singleplex digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome, and / or (b) at least one singleplex digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome. In some embodiments, the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copy donor-specific and / or recipient-specific targets in a single digital PCR reaction, where the copy number of the donor-specific target is relative to the haploid donor genome and the copy number of the recipient-specific target is relative to the haploid recipient genome.In some embodiments, the digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay, (a) the first digital PCR assay is for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is for the haploid recipient genome, and (b) the second digital PCR assay is for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is for the haploid donor genome. In some embodiments, the second singleplex digital PCR assay is for multiple copies of the donor-specific target. In some embodiments, the multiplex digital PCR assay is for (a) at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is for the haploid recipient genome, and (b) at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is for the haploid donor genome. In some embodiments, the multiplex digital PCR assay is for at least one multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome.

[0023] In another aspect, provided herein is a method for adjusting immunosuppressive therapy in a recipient of a graft from a donor, wherein the donor and recipient belong to different species, the method comprising: a) providing a sample from the transplant recipient after transplant, the sample comprising donor-derived cell-free nucleic acid and recipient-derived cell-free nucleic acid; b) determining by PCR assay an amount of donor-derived cell-free nucleic acid in the sample, wherein the amount of donor-derived cell-free nucleic acid is determined by PCR quantification of donor-specific and recipient-specific nucleic acid (i) as absolute copies of donor-derived cell-free nucleic acid in the sample or (ii) as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid; c) detecting transplant rejection if the amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold; and d) adjusting the immunosuppressive therapy administered to the transplant recipient based on the amount of donor-derived cell-free nucleic acid. In some embodiments, the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification. In some embodiments, the PCR assay is a digital PCR assay, and the PCR quantification is end-point PCR quantification. In some embodiments, the digital PCR assay comprises (a) at least one singleplex digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to a haploid donor genome, and / or (b) at least one singleplex digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to a haploid recipient genome.In some embodiments, the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copy donor-specific and / or recipient-specific targets in a single digital PCR reaction, the copy number of the donor-specific target being for the haploid donor genome and the copy number of the recipient-specific target being for the haploid recipient genome. In some embodiments, the digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay, (a) the first digital PCR assay for a single or multiple copy recipient-specific target and the copy number of the recipient-specific target being for the haploid recipient genome, and (b) the second digital PCR assay for a single or multiple copy donor-specific target and the copy number of the donor-specific target being for the haploid donor genome. In some embodiments, the second singleplex digital PCR assay is for multiple copies of the donor-specific target. In some embodiments, the multiplex digital PCR assay is for (a) at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and (b) at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the multiplex digital PCR assay is for at least one multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome.

[0024] In some embodiments that may be combined with any of the preceding aspects or embodiments, the method further comprises testing for the presence of an infectious agent. In some embodiments, the infectious agent is selected from the group consisting of a virus, a bacterium, a fungus, and a parasite. In some embodiments, the infectious agent is a virus selected from the group consisting of a cytomegalovirus, an Epstein-Barr virus, an Anelloviridae, and a BK virus. In some embodiments that may be combined with any of the preceding aspects or embodiments, the method further comprises performing one or more gene expression profiling assays on the recipient. In some embodiments, the combined score is calculated based on the amount of donor-derived cell-free nucleic acid in the sample and the results of the gene expression profiling assays.

[0025] In another aspect, provided herein is a kit for detecting and monitoring the transplant rejection status of a donor-derived graft in a transplant recipient, wherein the donor and the recipient belong to different species, the kit comprises: (a) one or more PCR reaction oligonucleotide primers and probe sets hybridizing to donor-specific or recipient-specific target sequences in the cell-free nucleic acid in a sample from the transplant recipient for PCR quantification of donor-derived cell-free nucleic acid, either as absolute copies of donor-derived cell-free nucleic acid in the sample or as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid in the sample; and (b) instructions for data analysis to determine the amount of donor-derived cell-free nucleic acid. In some embodiments, the PCR quantification is qPCR quantification. In some embodiments, the PCR quantification is digital PCR quantification.

[0026] In some embodiments that may be combined with any of the above aspects or embodiments, the donor-derived and / or recipient-derived cell-free nucleic acid is DNA. In some embodiments that may be combined with any of the above aspects or embodiments, the donor-derived and / or recipient-derived cell-free nucleic acid is DNA, RNA, mRNA, miRNA, double-stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, RNA hairpin, or combinations thereof. In some embodiments that may be combined with any of the above aspects or embodiments, the amount of donor-derived cell-free nucleic acid is adjusted with a correction factor to correct for genome length differences between the donor genome and the recipient genome. In some embodiments that may be combined with any of the above aspects or embodiments, the amount of donor-derived cell-free nucleic acid is adjusted with a correction factor to correct for cell-free nucleic acid fragment size differences between the donor-derived cell-free nucleic acid and the recipient-derived cell-free nucleic acid. In some embodiments that may be combined with any of the above aspects or embodiments, the amount of donor-derived cell-free nucleic acid is a ratio of donor-derived cell-free nucleic acid to total cell-free nucleic acid or recipient-derived cell-free nucleic acid. In some embodiments that may be combined with any of the preceding aspects or embodiments, the amount of cell-free nucleic acid from the donor is a percentage of cell-free nucleic acid from the donor compared to total cell-free nucleic acid. In some embodiments that may be combined with any of the preceding aspects or embodiments, the graft is a solid organ, tissue, or cell graft. In some embodiments that may be combined with any of the preceding aspects or embodiments, the donor of the graft is an animal. In some embodiments, the animal is a pig.

[0027] In another aspect, provided herein is a method for analyzing a biological sample from a transplant recipient that has received a solid organ transplant from a donor, wherein the donor and recipient belong to different species, the method comprising: a) isolating cell-free nucleic acid from the biological sample from the transplant recipient, wherein the cell-free nucleic acid comprises cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient; and b) isolating target regions of the cell-free nucleic acid, wherein the target regions comprise one or more target regions that comprise a nucleotide sequence that is donor-specific and / or one or more target regions that comprise a nucleotide sequence that is recipient-specific. The method includes: generating amplicons by amplifying the regions; c) generating sequence reads from the generated amplicons by sequencing the amplicons, the generated sequence reads including one or more sequence reads corresponding to a donor-specific genome sequence and / or one or more sequence reads corresponding to a recipient-specific genome sequence, and optionally mapping the generated sequence reads to at least the donor-specific genome sequence, optionally taking into account the difference in genome size between the donor and the recipient; and c) quantifying the amount of cell-free nucleic acid from the generated sequence reads in the sample. In some embodiments, the amount of cell-free nucleic acid is the amount of cell-free nucleic acid from the transplant donor in the sample. In some embodiments, the amount of cell-free nucleic acid is the amount of total cell-free nucleic acid in the sample. In some embodiments, the method further includes adding one or more quantitative spike-in nucleic acid controls to the sample and generating sequence reads corresponding to the one or more spike-in nucleic acid controls by sequencing the one or more spike-in nucleic acid controls, and the sequence reads corresponding to the spike-in controls are used to determine the absolute amount of total cell-free nucleic acid in the sample.In some embodiments, the method further comprises adding one or more quantitative spike-in nucleic acid controls to the sample and generating sequence reads corresponding to the one or more spike-in nucleic acid controls by sequencing the one or more spike-in nucleic acid controls, and the sequence reads from the spike-in controls are used to determine the absolute amount of cell-free nucleic acid from the transplant donor in the sample. In some embodiments, the one or more quantitative spike-in nucleic acid controls are added before step a). In some embodiments, the one or more quantitative spike-in nucleic acid controls are added after step a). In some embodiments, the one or more quantitative spike-in nucleic acid controls are added before and after step a). In some embodiments, the method further comprises using a correction factor to correct for the difference in genome length between the donor genome and the recipient genome to account for the difference in genome size between the donor and the recipient.

[0028] In another aspect, provided herein is a method for analyzing a biological sample from a transplant recipient who has received a solid organ transplant from a donor, wherein the donor and recipient belong to different species, the method comprising isolating cell free nucleic acid from the biological sample from the recipient, the cell free nucleic acid comprising cell free nucleic acid derived from the donor and cell free nucleic acid derived from the recipient, determining the amount of donor-derived cell free nucleic acid in the sample using a digital PCR assay, the digital PCR assay comprising: (i) at least one digital PCR assay for one or more single copy or multiple copy donor-specific targets, or (ii) one or more single copy or multiple copy donor-specific targets, by digital PCR assays comprising at least one digital PCR assay for a copy or multiple copies of a recipient-specific target and one or more single copy or multiple copies of a donor-specific target, wherein the copy number of the donor-specific target is relative to the haploid donor genome and the copy number of the recipient-specific target is relative to the haploid recipient genome, and the amount of donor-derived cell-free nucleic acid is determined by digital PCR quantification of donor-specific nucleic acid and recipient-specific nucleic acid (i) as absolute copies of donor-derived cell-free nucleic acid in the sample, or (ii) as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid. In some embodiments, the digital PCR assay comprises (a) a first digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and (b) a second digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the second digital PCR assay is for a multiple copy donor-specific target.In some embodiments, the digital PCR assay is for (a) at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and (b) at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the digital PCR assay is for multiple copies of the donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. [Brief description of the drawings]

[0029] [Figure 1A] FIG. 1 shows the accuracy and linearity of a digital PCR (dPCR) assay for determining the percentage of porcine DNA in samples containing a mixture of porcine and human DNA. The results shown in FIG. 1A were generated using a singleplex dPCR assay using two copies of a porcine-specific target ("Pig Assay A", see Table 2) and a single copy of a human-specific target ("Human Assay A", see Table 2) in control samples containing known percentages of control porcine and human genomic DNA. The x-axis shows the known percentage of porcine DNA in each sample (i.e., "Expected Pig DNA %), and the y-axis shows the percentage of porcine DNA measured using the dPCR assay ("Measured Pig DNA %). Each point in the graph represents one sample, and the fitted line on the graph and the corresponding equation show the linearity of the predicted and measured results. The results shown in FIG. 1B were generated using a multiplex dPCR assay including two single-copy porcine assays ("Pig Assay B" and "Pig Assay C", see Table 2) and one single-copy human assay ("Human Assay B", see Table 2). [Figure 1B]FIG. 1 shows the accuracy and linearity of a digital PCR (dPCR) assay for determining the percentage of porcine DNA in samples containing a mixture of porcine and human DNA. The results shown in FIG. 1A were generated using a singleplex dPCR assay using two copies of a porcine-specific target ("Pig Assay A", see Table 2) and a single copy of a human-specific target ("Human Assay A", see Table 2) in control samples containing known percentages of control porcine and human genomic DNA. The x-axis shows the known percentage of porcine DNA in each sample (i.e., "Expected Pig DNA %), and the y-axis shows the percentage of porcine DNA measured using the dPCR assay ("Measured Pig DNA %). Each point in the graph represents one sample, and the fitted line on the graph and the corresponding equation show the linearity of the predicted and measured results. The results shown in FIG. 1B were generated using a multiplex dPCR assay including two single-copy porcine assays ("Pig Assay B" and "Pig Assay C", see Table 2) and one single-copy human assay ("Human Assay B", see Table 2). [Diagram 2] FIG. 1 provides a longitudinal analysis of percent cfDNA derived from pig donors (dd-cfDNA%) and copies per ml of plasma (cp / ml) in cfDNA samples from human transplant recipients of pig heart grafts, assessed using singleplex dPCR ("Human Assay A" and "Pig Assay A", see Table 2) to detect a single copy of a human-specific target and two copies of a pig-specific target (two pig copies per haploid genome). The percent cfDNA derived from pig donors and cp / ml (y-axis) were determined in cfDNA samples obtained from human organ transplant recipients at the post-transplant days indicated on the x-axis (i.e., days 6, 13, 19, 25, 33, 46, 55, and 60 after organ transplant). [Diagram 3]

[0023] Figure 1 provides a longitudinal analysis of percent pig-derived cfDNA in cfDNA samples from human recipients of pig heart transplants, assessed using shotgun next-generation sequencing (NGS). NGS results were analyzed according to filtering models M1-M7 described in Example 1 and Table 3 herein. Percent pig donor derived cfDNA (y-axis) was determined in cfDNA samples obtained from human organ transplant recipients at the post-transplant days indicated on the x-axis (i.e., days 6, 13, 19, 25, 33, 46, 55, and 60 after organ transplant). [Figure 4] Figure 2 shows a comparison of estimated percent cfDNA from pig donors in samples from human recipients of pig heart transplants, measured by singleplex dPCR ("human assay A" and "porcine assay A", see Table 2) or shotgun NGS. Percent cfDNA from pig donors (y-axis) was determined in cfDNA samples obtained from human organ transplant recipients at the post-transplant days indicated on the x-axis (i.e., 6, 13, 19, 25, 33, 46, 55, and 60 days after organ transplant). NGS results were analyzed according to filtering model M7, as described in Example 1 and Table 3 herein. [Diagram 5] Figure 2 shows a comparison of the percent cfDNA derived from the pig donor in samples from human recipients of pig heart grafts, as measured by singleplex dPCR ("human assay A" and "pig assay A", see Table 2), multiplex dPCR ("human assay B", "pig assay B", and "pig assay C", see Table 2), and shotgun NGS. The percent cfDNA derived from the pig donor (y-axis) was determined in cfDNA samples obtained from human organ transplant recipients at the post-transplant days indicated on the x-axis (i.e., 33, 46, 49, and 60 days after organ transplant). NGS results were analyzed according to the filtering model M7 described in Example 1 and Table 3 herein. [Figure 6A]Figure 6 shows a comparison of percent cfDNA derived from the pig donor (xcfDNA%) in samples from human deceased model recipients of pig heart grafts (recipient 1 and recipient 2) as measured by multiplex dPCR ("human assay C" and "porcine assay D", see Table 2) or shotgun NGS. Percent cfDNA derived from the pig donor (y-axis) was determined in cfDNA samples obtained from human deceased model recipients at the times indicated on the x-axis (i.e., 30, 48, 72 hours after organ transplantation for recipient 1, FIG. 6A, and 0, 12, 24, 60, 66 hours for recipient 2, FIG. 6B). NGS results were analyzed according to filtering model M7 described in Example 1 and Table 3 herein. [Figure 6B] Figure 6 shows a comparison of percent cfDNA derived from the pig donor (xcfDNA%) in samples from human deceased model recipients of pig heart grafts (recipient 1 and recipient 2) as measured by multiplex dPCR ("human assay C" and "porcine assay D", see Table 2) or shotgun NGS. Percent cfDNA derived from the pig donor (y-axis) was determined in cfDNA samples obtained from human deceased model recipients at the times indicated on the x-axis (i.e., 30, 48, 72 hours after organ transplantation for recipient 1, FIG. 6A, and 0, 12, 24, 60, 66 hours for recipient 2, FIG. 6B). NGS results were analyzed according to filtering model M7 described in Example 1 and Table 3 herein. [Figure 6C] Comparison of pig genome copies per mL of samples from post-transplant human mortality models Recipient 1 (FIG. 6C) and Recipient 2 (FIG. 6D) as measured by multiplex dPCR ("Human Assay C" and "Pig Assay D", see Table 2). [Figure 6D] Comparison of pig genome copies per mL of samples from post-transplant human mortality models Recipient 1 (FIG. 6C) and Recipient 2 (FIG. 6D) as measured by multiplex dPCR ("Human Assay C" and "Pig Assay D", see Table 2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The following description is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein, but should be accorded the scope consistent with the claims.

[0031] Overview The present disclosure describes sensitive, non-invasive methods and kits for detecting, predicting, diagnosing, and / or monitoring the health status of a transplant, and for detecting, predicting, and / or monitoring the transplant rejection status in a recipient of an organ, cell, or tissue transplant from a donor, where the donor and recipient belong to different species. Such methods and kits are based on the detection and quantification of donor-specific nucleic acids, such as DNA or RNA, in body fluids, including, but not limited to, blood, serum, plasma, or urine, after transplantation.

[0032] The transplantation of organs, cells, or tissues from a donor to a recipient induces an immune response from the recipient's immune system, which may result in acute and / or chronic transplant rejection. Without wishing to be bound by theory, it is believed that transplant rejection is associated with the death of cells from the organ, tissue, or cell transplant, which results in the release of donor-derived nucleic acids, such as donor-derived cell-free DNA (dd-cfDNA), from the dead, no longer intact donor cells into the recipient's bloodstream and other body fluids. Thus, the method of the present disclosure includes the analysis of cell-free DNA (cfDNA) in a sample from a recipient of an organ, cell, or tissue transplant from a donor, where the donor and the recipient belong to different species, to diagnose the status of the transplant based on the amount of dd-cfDNA. As disclosed herein, analysis of cfDNA in samples obtained from transplant recipients can be performed using nucleic acid sequencing-based methods (e.g., high-throughput sequencing and / or next-generation sequencing), as well as PCR-based methods for quantifying nucleic acids (e.g., quantitative PCR [qPCR] and digital PCR [dPCR]). See Examples 1 and 2 herein. Advantageously, the nucleic acid sequencing-based methods of the present disclosure provide improved accuracy in estimating the amount of dd-cfDNA by taking into account genome size differences between donors and recipients. See Examples 1 and 2 herein. Similarly, the nucleic acid PCR-based methods (e.g., dPCR-based methods) of the present disclosure provide improved sensitivity and accuracy in estimating the rare amount of dd-cfDNA, for example, by detecting donor-specific targets present in multiple copies in the donor's haploid genome. Thus, as demonstrated herein, the methods of the present disclosure allow for sensitive, accurate, non-invasive monitoring of the status of organ, cell, or tissue grafts in transplant recipients, for example, by repeatedly and over time monitoring the amount of dd-cfDNA in bodily fluids (e.g., blood) from the transplant recipient. See, e.g., Examples 2 and 3 herein.

[0033] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0035] The term "sample" as used herein refers to any sample obtained from a transplant recipient, such as whole blood, plasma, serum, lymph, peripheral blood mononuclear cells, buccal swab, saliva, urine, lung lavage, or tissue from a biopsy.

[0036] As used herein, the term "graft" refers to the transplantation of any cell, tissue, or organ (including combinations thereof) from a donor to a recipient. The term "graft" with respect to a tissue or organ can refer to the entire tissue or organ (e.g., the entire liver) or a portion thereof.

[0037] The term "nucleic acid" as used herein refers to RNA or DNA that is linear, circular or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. In some cases, nucleic acid refers to any of DNA, RNA, mRNA, miRNA, double stranded DNA, single stranded DNA, single stranded DNA hairpin, DNA / RNA hybrid, RNA hairpin, and fragments and combinations thereof.

[0038] The term "cell-free nucleic acid" as used herein refers to nucleic acid that exists outside of cells. In some cases, cell-free nucleic acid is nucleic acid that exists outside of cells and exists in the body fluids (e.g., blood, plasma, serum, urine, etc.) of transplant recipients. In some embodiments, cell-free nucleic acid refers to any DNA, RNA, mRNA, miRNA, double-stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, RNA hairpin, and fragments and combinations thereof that exists outside of cells. This term also includes organ-specific or tissue-specific RNA transcripts.

[0039] The term "providing a sample" as used herein refers to, for example, providing a sample for use in the methods of the present disclosure. In some cases, providing a sample includes performing a process to obtain a sample (e.g., performing a physical method). In some cases, the sample is provided by the party or entity performing the methods of the present disclosure. In other cases, the sample is provided by an entity or party other than the party or entity performing the methods of the present disclosure. Thus, in some cases, the methods of the present disclosure include receiving a sample from another party or source (e.g., a third party that obtained the sample).

[0040] The term "generating sequence reads" as used herein refers to performing a process to obtain sequence reads, for example for use in the methods of the present disclosure. For example, generating sequence reads can include performing a sequencing method (e.g., a next generation sequencing (NGS) method or a high throughput sequencing (HTS) method). Such sequencing methods can include whole genome sequencing or targeted sequencing, and can sequence nucleic acids, including but not limited to genomic DNA, mitochondrial DNA, cDNA obtained from RNA transcripts, or RNA. In some embodiments, at least 500, at least 1000, at least 5000, at least 10000, at least 20000, at least 50000, at least 100000, or more sequence reads can be generated. Each sequence read can include at least 20 bases, at least 50 bases, at least 75 bases, at least 100 bases, at least 150 bases, or more per read. The sequence reads can be generated by the party or entity performing the methods of the present disclosure. In other cases, the sequence reads may be generated by an entity or party other than the party or entity performing the methods of the present disclosure. Thus, in some cases, the methods of the present disclosure involve receiving information or knowledge of the sequence reads or receiving the sequence reads from another party or source (e.g., a third party laboratory that directly generated or obtained the sequence reads).

[0041] As used herein, the term "target sequence" refers to a region of a nucleic acid that contains a sequence of interest. As used herein, the term "target" can include one or more target sequences.

[0042] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided with respect to certain embodiments herein, or the use of exemplary language (e.g., "etc."), are intended merely to illustrate the embodiments and do not impose limitations on the scope of the otherwise claimed embodiments. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the embodiment.

[0043] I. Methods of the Disclosure Certain aspects of the present disclosure relate to methods for detecting, predicting, diagnosing, and / or monitoring transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient, methods for treating transplant rejection of an organ, tissue, or cell graft from a donor in a transplant recipient, and methods for adjusting immunosuppressive therapy in a transplant recipient of an organ, tissue, or cell graft from a donor. Other aspects of the present disclosure relate to methods of analyzing biological samples from transplant recipients (e.g., using sequencing or PCR-based methods) to quantify cell-free nucleic acid, such as donor-derived, recipient-derived, and / or total cell-free nucleic acid. In some embodiments, an organ, tissue, or cell graft according to the present disclosure is a xenogeneic graft (i.e., a xenogeneic or heterologous graft), where the organ, tissue, or cell transplant donor is of a different species than the transplant recipient. Thus, in some embodiments, the methods of the disclosure include detecting, predicting, diagnosing, and / or monitoring transplant rejection status of a xenogeneic, xenogeneic, or heterologous organ, tissue, or cell graft in a transplant recipient, treating transplant rejection of a xenogeneic, xenogeneic, or heterologous organ, tissue, or cell graft in a transplant recipient, and / or adjusting immunosuppressive therapy in a xenogeneic or xenogeneic organ, tissue, or cell graft transplant recipient.

[0044] In some embodiments, the disclosed method includes providing a sample from a recipient of an organ, tissue, or cell graft from a donor after transplantation, the sample from the transplant recipient comprising donor-derived cell-free nucleic acid (e.g., cfDNA and / or cfRNA) and recipient-derived cell-free nucleic acid (e.g., cfDNA and / or cfRNA), determining an amount of donor-derived cell-free nucleic acid in the cell-free nucleic acid from the sample, and detecting transplant rejection based on the determined amount of donor-derived cell-free nucleic acid. In some embodiments, the disclosed method includes detecting transplant rejection if the determined amount of donor-derived cell-free nucleic acid exceeds a predetermined threshold. In some embodiments, the disclosed method includes administering an immunosuppressive treatment to the transplant recipient based at least in part on the detection of transplant rejection (e.g., based on the determined amount of donor-derived cell-free nucleic acid). In some embodiments, the disclosed method includes adjusting the transplant recipient's immunosuppressive treatment based at least in part on the detection of transplant rejection (e.g., based on the determined amount of donor-derived cell-free nucleic acid).

[0045] In some embodiments, the amount of cell-free nucleic acid from the donor is determined using a sequencing-based method, such as high-throughput sequencing (HTS) and / or next-generation sequencing (NGS), as described in more detail below. In some embodiments, the amount of cell-free nucleic acid from the donor is determined using a nucleic acid PCR-based quantitative method, such as quantitative PCR (qPCR) or digital PCR (dPCR), as described in more detail below.

[0046] A. Quantification of Cell-Free Nucleic Acid Certain aspects of the present disclosure relate to methods for determining the amount of donor-derived cell-free nucleic acid in cell-free nucleic acid from a sample from a transplant recipient using sequencing-based methods or nucleic acid PCR-based quantification methods, as described in more detail below.

[0047] Other aspects of the present disclosure relate to methods for quantifying cell-free nucleic acid (e.g., recipient-derived, donor-derived, and / or total cell-free nucleic acid) in a biological sample from a transplant recipient.

[0048] Sequencing-based methods In some embodiments, the methods of the disclosure include determining the amount of cell-free nucleic acid (e.g., recipient-derived, donor-derived, and / or total cell-free nucleic acid) in cell-free nucleic acid from a sample from a transplant recipient using a nucleic acid sequencing-based method.

[0049] Various methods and protocols for nucleic acid sequencing and analysis that are well known in the art can be used in the methods of the present disclosure.For example, DNA sequencing can be achieved using high-throughput sequencing (HTS) technology, whole genome sequencing, shotgun sequencing, whole exome sequencing, targeted sequencing, Sanger sequencing, massively parallel sequencing technology, or next-generation sequencing (NGS). Examples of next generation and high throughput sequencing include, for example, massively parallel signature sequencing, polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing using HiSeq, MiSeq and other Illumina platforms, SOLiD sequencing, ion semiconductor sequencing (IonTorrent), nanopore sequencing (see, for example, the website: nanoporetech.com / applications / short-fragment-mode), DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, MassARRAY®, and Digital Analysis of Selected Regions (DANSR™).For example, see Stein RA (2008). "Next-Generation Sequencing Update". Genetic Engineering & Biotechnology News 28(15), Quail et al., (2012). "A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers". BMC Genomics 13(1):341, Liu et al., (2012). "Comparison of Next-Generation Sequencing Systems". Journal of Biomedicine and Biotechnology 2012:1-11, Qualitative and quantitative genotyping using single base primer extension coupled with matrix-assisted laser desorption / ionization time-of -flight mass spectrometry (MassARRAY®). Methods Mol Biol. 2009;578:307-43, Chu et al., A novel approach toward the challenge of accurately quantifying fetal DNA in maternal plasma. Prenat Diagn 2010;30:1226-9, and Suzuki et al., Characterization of circulating DNA in healthy human plasma. Clinica chimica acta; international journal of clinical chemistry 2008;387:55-8.

[0050] In some embodiments, the method of the present disclosure includes generating sequence reads corresponding to one or more donor-specific and / or recipient-specific genomic sequences in cell-free nucleic acid from a sample obtained from a transplant recipient. In some embodiments, the sequence reads are generated using any suitable sequencing method known in the art, such as, for example, high-throughput sequencing (HTS) techniques or next-generation sequencing (NGS), as described above. In some embodiments, the sequence reads are generated using NGS shotgun sequencing, for example, using MiSeq or NextSeq sequencing platforms from Illumina. In some embodiments, the sequence reads are generated using paired-end sequencing. In some embodiments, the sequence reads are generated using amplicon-based NGS, where the target region of the genome is amplified, for example, using PCR, prior to sequencing. In some embodiments, the sequence reads are generated using non-amplicon-based NGS, for example, using hybrid capture NGS. In some embodiments, the sequence reads are grouped into selected sized regions of equal size for the donor and recipient genomes. In some embodiments, the method includes mapping the sequence reads to the donor-specific and / or recipient-specific genomic sequences. In some embodiments, mapping comprises aligning the sequence reads to a reference sequence. Alignment or mapping may be performed using any suitable method known in the art, such as using a Burrows-Wheeler Aligner (BWA). In some embodiments, sequence read alignment is performed with a minimum alignment score cutoff of about 90%, a tolerance gap percentage of about 5%, and / or no split alignment. In some embodiments, the reference sequence comprises a reference genome corresponding to the donor and / or a reference genome corresponding to the recipient. In some embodiments, the reference sequence is a combined reference sequence comprising a reference genome corresponding to the donor and a reference genome corresponding to the recipient.In some embodiments, the method comprises excluding one or more sequence reads having multiple matches to the reference sequence from the aligned or mapped sequence reads. In some embodiments, the method comprises excluding one or more sequence reads having missing mates, translocated sequence reads, and sequence reads having improper orientation from the aligned or mapped sequence reads. In some embodiments, the method comprises excluding one or more sequence reads having an alignment score of less than 95% or less than 98% from the aligned or mapped sequence reads. In some embodiments, the method comprises excluding one or more partially aligned sequence reads or soft-clipped sequence reads from the aligned or mapped sequence reads. In some embodiments, the method comprises excluding one or more partially aligned sequence reads or hard- and soft-clipped sequence reads from the aligned or mapped sequence reads, or the alignment in MAPQ is less than 10.

[0051] In some embodiments, the amount of donor-derived cell-free nucleic acid in the cell-free nucleic acid from a sample from a transplant recipient is determined based on the percentage of sequence reads that align to the donor genome as a proportion of sequence reads that align to the donor genome and the recipient genome.

[0052] In some embodiments, the methods of the disclosure include determining the amount of donor-derived cell-free nucleic acid in a sample, taking into account the genome size difference between the recipient and the donor.

[0053] In some embodiments, the genome size difference between the recipient and the donor is considered based on the sequencing coverage of the donor and recipient genomes in the generated sequence reads. In some embodiments, the genome size difference between the recipient and the donor is considered based on the average coverage over one or more regions of a selected size of the donor and recipient genomes in the generated sequence reads. In some of such embodiments, the amount of cell-free nucleic acid from the donor is determined as a percentage of the average coverage over one or more regions of a selected size of the donor and recipient genomes. In some embodiments, the one or more regions of a selected size are large enough to have sufficient coverage. In some embodiments, the background noise is low enough to distinguish between the donor genome and the recipient genome. In some cases ... d / (m d +m r ) × 100, where m d is the median of the average coverage across one or more regions of a selected size in the donor genome, and m r is the median of the average coverage over one or more regions of a selected size of the recipient genome. In some embodiments, the selected size regions of the donor genome and the recipient genome include any of up to 1 megabase (Mb), about 1 Mb to about 10 Mb, up to 10 Mb, about 10 Mb to about 100 Mb, or up to 100 Mb. In some embodiments, the selected size regions of the donor genome and the recipient genome include up to 1 M bases, up to 10 M bases, or up to 100 M bases.

[0054] In some embodiments, the genome size difference between the recipient and the donor is taken into account using a correction factor. For example, for a donor genome that is smaller than the recipient genome, the correction factor can be obtained by multiplying the proportion of sequence reads that map to donor-specific genome sequences by the ratio of the donor genome size to the recipient genome size.

[0055] The size of cell-free nucleic acid fragments from donor and recipient may be different, see for example Example 2 and Table 4 herein, which shows that a difference was observed in the average fragment size of cell-free DNA fragments from pig donors compared to cell-free DNA fragments from human transplant recipients. Such a difference in fragment size between cell-free nucleic acid from donor and cell-free nucleic acid from recipient may result in overestimation or underestimation of the amount of cell-free nucleic acid from donor. Thus, in some embodiments, the method includes considering the possibility of overestimation or underestimation of the amount of cell-free nucleic acid from donor due to the difference in fragment size of cell-free nucleic acid from donor and recipient, for example, using a correction factor. For example, the correction factor may be determined by fragment size comparison study in artificial samples and in silico samples. In addition, the difference in fragment size between cell-free nucleic acid from donor and cell-free nucleic acid from recipient may also result in overestimation or underestimation of the sequencing coverage of donor genome and recipient genome in generated sequence reads. For example, greater coverage may be obtained with smaller nucleic acid fragments compared to larger nucleic acid fragments.Thus, in some embodiments, the method includes, for example, using a correction factor to consider the possibility of overestimating or underestimating the sequencing coverage of donor genome and recipient genome due to the fragment size difference between cell-free nucleic acid from donor and cell-free nucleic acid from recipient.For example, the correction factor can be determined by multiplying the coverage rate (e.g., the average coverage rate over the region of selected size of donor and recipient genome) by the ratio of the average donor nucleic acid fragment size to the average recipient nucleic acid fragment size.

[0056] In some embodiments, absolute quantification of the amount of donor-derived cell-free nucleic acid in the cell-free nucleic acid from the sample from the transplant recipient can be achieved by including a known concentration or amount of a control nucleic acid (e.g., a quantitative spike-in nucleic acid control) in the sample from the transplant recipient or in the cell-free nucleic acid from the transplant recipient prior to generating sequence reads. Thus, the absolute amount of donor-derived cell-free nucleic acid is determined based on the sequence reads corresponding to the control nucleic acid and the donor-derived cell-free nucleic acid.

[0057] If there are multiple cell-free nucleic acid samples from a transplant recipient to be sequenced, for example, where multiple samples are taken from a transplant recipient over time or where samples are taken from different transplant recipients, each sample can be sequenced individually or the multiple samples can be sequenced together using multiplex sequencing.

[0058] Nucleic Acid PCR-Based Quantitation-Based Methods In some embodiments, the methods of the disclosure include determining the amount of cell-free nucleic acid (such as recipient-derived, donor-derived, and / or total cell-free nucleic acid) in cell-free nucleic acid from a sample obtained from the transplant recipient using nucleic acid PCR quantification.

[0059] A variety of methods and protocols for nucleic acid quantification are well known in the art, such as quantitative nucleic acid amplification methods including quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), and real-time dPCR.

[0060] qPCR or real-time quantitative PCR refers to a method that uses PCR to simultaneously amplify and quantify a target nucleic acid. As is known in the art, quantification of accumulated amplified nucleic acid in a qPCR reaction is performed in real time after each amplification cycle. Absolute quantification may be performed using a standard curve, and quantification may be performed using detectably labeled probes, primers, or dyes, such as intercalating dyes that bind to double-stranded DNA products, e.g., YO-PRO-I, SYBR green, etc. (see, e.g., Ishiguro et al. Anal., 229:207-213 (1995); Tseng et al Anal. Biochem., 245:207-212 (1997); Morrison et al. Biotechniques, 24:954-962 (1998)), primers with hairpin structures with fluorescent molecules held in close proximity to a fluorescent quencher until forced apart by primer extension (see, e.g., Whitecombe et al. Nature Biotechnology, 17:804-807 (1999); "AMPLIFLUORO"). primers), or sequence-specific probes that typically contain a fluorescent molecule in close proximity to a fluorescent quencher until the oligonucleotide moiety to which they are attached specifically binds to an amplification product (see, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 (TAQMAN); Nazarenko et al. Nucleic Acids Research, 25:2516-2521 (1997) (Scorpion probes); Tyagi et al. Nature Biotechnology, 16:49-53 (1998) (Molecular Beacons)). Other suitable dyes, primers, or probes that may be used include dual hybridization probes, eclipse probes, LUX PCR primers, and QZyme PCR primers. Such dyes, primers, or probes may be used in conjunction with qPCR as described herein, or they may be used to measure the total amount of reaction product upon completion of the reaction.In some embodiments, the detectably labeled probe, primer, or dye is fluorescently labeled, for example, using one or more of FAM, HEX, ATTO550, ROX, Cy5, Cy5.5, or any other suitable dyes.

[0061] Digital PCR or dPCR refers to a method that allows absolute quantification of target nucleic acid. As known in the art, dPCR is performed by dividing a PCR reaction into a number of compartments, each of which contains one, a few, or no target sequence. After the PCR reaction, i.e., at the end of the PCR reaction, the total number of positive amplifications and compartments is used to quantify the target sequence using Poisson statistics. The positive compartments can be identified, for example, using detectably labeled probes, primers, or dyes, as described above for qPCR. In some embodiments, the detectably labeled probes, primers, or dyes are fluorescently labeled, for example, using one or more of FAM, HEX, ATTO550, ROX, Cy5, Cy5.5, or any other suitable dyes. For descriptions of dPCR methods, see, e.g., Hindson et al. (2011) Anal. Chem. 83(22):8604-8610, Pohl and Shih (2004) Expert Rev. Mol. Diagn. 4(1):41-47, Pekin et al. (2011) Lab Chip 11(13):2156-2166, Pinheiro et al. (2012) Anal. Chem. 84(2):1003-1011, Day et al. (2013) Methods 59(1):101-107, and Quan et al., Sensors (Basel). 2018 Apr; 18(4):1271. Multiple compartments can be generated using any suitable method known in the art, such as by dividing the PCR reaction into microwells, chambers, or droplets. In cases where multiple compartments are generated by dividing the PCR reaction into droplets, the dPCR method may be referred to as droplet digital PCR, available, for example, from BioRad (ddPCR™) or Stilla (e.g., Crystal Digital PCR™).

[0062] In some embodiments, the methods of the disclosure include determining the amount of donor-derived cell-free nucleic acid in a sample using a digital PCR assay.

[0063] In some embodiments, the dPCR assay comprises one or more singleplex dPCR assays. In some of such embodiments, the singleplex dPCR assay may comprise one or more recipient-specific singleplex dPCR assays and / or one or more donor-specific singleplex dPCR assays. In some embodiments, the recipient-specific singleplex PCR assay and the donor-specific singleplex PCR assay each have a single copy of the target per haploid genome. In some embodiments, some or all of the recipient-specific singleplex dPCR assay and / or the donor-specific singleplex dPCR assay have multiple copies (e.g., 2 or more copies) of the target per haploid genome, which generates more target-positive sections for the same amount of sample input (e.g., 2-fold or multiple-fold more target-positive sections for the same amount of sample input), increasing the sensitivity and accuracy of assay detection and quantification. This may be particularly useful when the cell-free nucleic acid from the donor is a rare abundance in the total cell-free nucleic acid from the sample. In some embodiments, the multiple copy target PCR assay may target multiple members of overlapping gene families such as actin, tubulin, rRNA gene families, or multiple members of repetitive sequences such as Alu sequences, or multiple copies of mitochondrial DNA per diploid cell. In some embodiments, the dPCR assay comprises at least one singleplex digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome, and / or at least one singleplex digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome.In some embodiments, the digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay, (a) the first digital PCR assay is for a single or multiple copy recipient-specific target, and the copy number of the recipient-specific target is for the haploid recipient genome, and (b) the second digital PCR assay is for a single or multiple copy donor-specific target, and the copy number of the donor-specific target is for the haploid donor genome. In some embodiments, the second singleplex digital PCR assay is for multiple copies of the donor-specific target. Quantification of cell-free nucleic acid by different dPCR assays targeting different copy number targets can be normalized to genome copy quantification for analysis of relative quantification of donor-derived genome to total donor and recipient genome, or absolute quantification of donor-derived genome copies per unit of sample, e.g., copies / ml of plasma sample.

[0064] In some embodiments, the dPCR assay is a multiplex dPCR assay that includes two or more PCR assays in a single dPCR reaction. In some of such embodiments, the multiplex dPCR assay may include at least one donor-specific PCR assay. In some embodiments, the multiplex dPCR assay may include at least one donor-specific PCR assay and at least one recipient-specific PCR assay for quantifying cell-free nucleic acid from the donor relative to the recipient or total cell-free nucleic acid. In some embodiments, the multiplex dPCR assay may include a recipient-specific PCR assay and a donor-specific PCR assay, each targeting at least one single copy target per haploid genome of the species. In some embodiments, the multiplex dPCR assay may include a recipient-specific PCR assay and a donor-specific PCR assay, each targeting at least one multiple copy target per haploid genome to improve target detection and quantification sensitivity with limited sample input. In some embodiments, a multiplex dPCR assay can include at least one donor-specific PCR assay with at least one single- or multiple-copy target (e.g., 2 or more copies of a target) corresponding to a haploid donor genome (i.e., each donor-specific PCR assay detects at least one single- or multiple-copy target corresponding to a donor), where the copy number of the target is with respect to the haploid genome of the donor. In some embodiments, a multiplex dPCR assay can include one or more, e.g., 1-5, donor-specific PCR assays with at least one single- or multiple-copy target (e.g., at least 1, 2, or more copies of a target) corresponding to a donor genome (i.e., each donor-specific PCR assay detects at least one single- or multiple-copy target corresponding to a donor), where the copy number of the target is with respect to the haploid genome of the donor.In some embodiments, the multiplex dPCR assay may include at least one recipient-specific PCR assay with at least one single-copy or multiple-copy target corresponding to a haploid recipient genome (i.e., each recipient-specific PCR assay detects at least one single-copy or multiple-copy target corresponding to a recipient), and the copy number of the target is with respect to the haploid genome of the recipient. In some embodiments, the multiplex dPCR assay may include one or more, e.g., 1-5, recipient-specific PCR assays with at least one single-copy or multiple-copy target corresponding to a recipient genome (i.e., each recipient-specific PCR assay detects at least one single-copy or multiple-copy target corresponding to a recipient), and the copy number of the target is with respect to the haploid genome of the recipient. In some embodiments, the digital PCR assay includes at least one multiplex digital PCR assay for two or more single-copy or multiple-copy donor-specific and / or recipient-specific targets in a single digital PCR reaction, and the copy number of the donor-specific target is with respect to the haploid donor genome and the copy number of the recipient-specific target is with respect to the haploid recipient genome. In some embodiments, the multiplex digital PCR assay is for (a) at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and (b) at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the multiplex digital PCR assay is for at least one multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome.

[0065] In some embodiments, the dPCR assay comprises at least one digital PCR assay for one or more single or multiple copy donor-specific targets. In some embodiments, the dPCR assay comprises at least one digital PCR assay for one or more single or multiple copy recipient-specific targets and one or more single or multiple copy donor-specific targets. In some embodiments, the dPCR assay comprises a first digital PCR assay for a single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and a second digital PCR assay for a single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the second digital PCR assay is for a multiple copy donor-specific target.

[0066] In some embodiments, the digital PCR assay is for at least one single or multiple copy recipient-specific target, where the copy number of the recipient-specific target is relative to the haploid recipient genome, and at least one single or multiple copy donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome. In some embodiments, the digital PCR assay is for multiple copies of the donor-specific target, where the copy number of the donor-specific target is relative to the haploid donor genome.

[0067] In some embodiments, the single copy target corresponding to the recipient is present as a single copy in the haploid genome of the donor and is not present in the genome of the donor. In some embodiments, the single copy target corresponding to the donor is present as a single copy in the haploid genome of the donor and is not present in the genome of the recipient. In some embodiments, the multiple copy target corresponding to the donor (e.g., two or more copies of the target) is present in multiple copies (e.g., two or more copies) in the haploid genome of the donor and is not present in the genome of the recipient. In some embodiments, the target corresponding to the donor and / or the target corresponding to the recipient is in an intron region of the donor genome or the recipient genome. In some embodiments, the target corresponding to the donor and / or the target corresponding to the recipient is in the mitochondrial DNA of the donor genome or the recipient genome. In some embodiments, the multiple copy target corresponding to the donor (e.g., two or more copies) comprises a sequence in a duplicated gene group in the genome of the donor. Non-limiting examples of duplicated genes include rRNA, Hox, histone, and tubulin gene families.

[0068] In some embodiments, each recipient-specific PCR assay includes a primer pair that detects a recipient-specific target (e.g., a single or multiple copy target corresponding to the recipient), e.g., by direct amplification of the recipient-specific target in a PCR reaction. In some embodiments, each donor-specific PCR assay includes a primer pair that detects a donor-specific target (e.g., a single or multiple copy target corresponding to the donor), e.g., by direct amplification of the donor-specific target in a PCR reaction. In some embodiments, each primer in a primer pair is about 5 to about 50, about 10 to about 50, about 15 to about 30, about 15 to about 25, about 18 to about 30, or about 20 to about 30 bases in length, including any value within each of the recited ranges. In some embodiments, each primer in a primer pair has a melting temperature (T m In some embodiments, each primer in the primer pair comprises a melting temperature (T m ).

[0069] As described above, detection of donor-specific or recipient-specific positive compartments in a dPCR assay can be performed using any suitable detection reagent, such as a DNA-binding fluorescent dye (e.g., SYBR Green or EvaGreen) or a detectably labeled probe (e.g., a fluorescently labeled probe). When donor-specific or recipient-specific positive compartments are detected using a detectably labeled probe, the recipient-specific or donor-specific PCR assay comprises a detectably labeled probe suitable for separately detecting the recipient-specific or donor-specific target. In some of such embodiments, each recipient-specific PCR assay comprises a primer pair that detects a recipient-specific target (e.g., a single copy or multiple copies of a target corresponding to the recipient) and a detectably labeled probe that hybridizes to the recipient-specific target, and / or each donor-specific PCR assay comprises a primer pair that detects a donor-specific target (e.g., a single copy or multiple copies of a target corresponding to the donor) and a detectably labeled probe that hybridizes to the donor-specific target. In some embodiments, the detectably labeled probe comprises an oligonucleotide configured to hybridize to a recipient-specific target or a donor-specific target, a fluorescent label, and a quencher. In some embodiments, the fluorescent label is at one end of the oligonucleotide and the quencher is at the other end of the oligonucleotide. In some embodiments, the fluorescent label is any label or dye known in the art, including but not limited to FAM, HEX, ATTO550, ROX, Cy5, and Cy5.5. In some embodiments, the quencher is any quencher known in the art, e.g., BHQ, IABkFQ, TAMRA. In some embodiments, the oligonucleotide comprises a sequence of about 5 to about 50, about 10 to about 50, about 15 to about 30, about 15 to about 25, or about 20 to about 30 bases in length, including any value within each of the recited ranges. In some embodiments, the probe has a melting temperature (T mIn some embodiments, the probe has a melting temperature (T) of about 65° C. to about 70° C., including any value within this range. m ).

[0070] In some embodiments, a dPCR assay includes one or more singleplex PCR assays (e.g., a recipient-specific PCR assay and / or a donor-specific PCR assay), each singleplex PCR assay includes the use of a detection reagent having a unique dye or detectable label (e.g., a unique fluorescent dye or label). For example, in a dPCR assay that includes one or more singleplex PCR assays (e.g., a recipient-specific PCR assay and / or a donor-specific PCR assay), the recipient-specific PCR assay includes a detection reagent having a first fluorescent label and the donor-specific PCR assay includes a detection reagent having a second fluorescent label that is different from the first fluorescent label. Thus, in one embodiment, the dPCR assay comprises a recipient-specific PCR assay comprising a primer pair that detects a recipient-specific target (e.g., a single copy or multiple copy target corresponding to the recipient) and a detectably labeled probe that hybridizes to the recipient-specific target, where the detectably labeled probe is labeled with a first fluorescent label, and a donor-specific PCR assay comprising a primer pair that detects a donor-specific target (e.g., a single copy or multiple copy target corresponding to the donor) and a detectably labeled probe that hybridizes to the donor-specific target, where the detectably labeled probe is labeled with a second fluorescent label that is different from the first fluorescent label.

[0071] In some embodiments, the dPCR assay is a multiplex dPCR assay comprising two or more PCR assays in a single dPCR reaction, e.g., at least one donor-specific PCR assay, for quantification of donor-derived cell-free nucleic acid as absolute copies of donor-derived cell-free nucleic acid in a sample, e.g., absolute donor (genomic) copies per mL of sample, e.g., copies per mL of plasma, or donor (genomic) copies per amount of DNA, e.g., copies per ng of DNA. In some embodiments, the dPCR assay is a multiplex dPCR assay comprising two or more PCR assays, e.g., at least one donor-specific PCR assay and one or more recipient-specific PCR assays, in a single dPCR reaction for recipient-derived cell-free nucleic acid quantification or relative donor-derived cell-free nucleic acid quantification for total donor- and recipient-derived cell-free nucleic acid in a sample, where a detection agent comprising a unique dye or detection label (e.g., a unique fluorescent dye or label) is used for each PCR assay in the multiplex dPCR reaction, or at least two PCR assays in the multiplex dPCR reaction comprise detection reagents comprising the same dye or detectable label (e.g., the same fluorescent dye or label). Thus, in one embodiment, in a multiplex dPCR assay comprising at least one recipient-specific PCR assay and at least one donor-specific PCR assay in a single dPCR reaction, each PCR assay in the dPCR reaction may comprise a detection reagent with a unique fluorescent dye or label. For example, a multiplex dPCR assay may include at least one recipient-specific PCR assay using a single or multiple copy target corresponding to the recipient and at least one donor-specific PCR assay using a single or multiple copy (e.g., two or more copies) of a target corresponding to the donor, where each PCR assay in the dPCR reaction (i.e., each recipient-specific PCR assay and each donor-specific PCR assay) includes a detection reagent having a unique fluorescent dye or label.In another embodiment, in a multiplex dPCR assay comprising at least one recipient-specific PCR assay and at least one donor-specific PCR assay in a single dPCR reaction, at least two recipient-specific or donor-specific PCR assays in the dPCR reaction comprise detection reagents comprising the same dye or detectable label (e.g., the same fluorescent dye or label). For example, a multiplex dPCR assay may comprise at least one recipient-specific PCR assay using a single copy or multiple copies of a target corresponding to a recipient, and two or more donor-specific PCR assays using single copy or multiple copies (e.g., two or more copies) of a target corresponding to a donor, where at least two donor-specific PCR assays in the dPCR reaction comprise detection reagents with the same fluorescent dye or label.

[0072] The amount of cell-free nucleic acid from the donor present in a cell-free nucleic acid sample from a transplant recipient (e.g., determined according to the methods of the present disclosure) can be expressed in various ways. In some embodiments, the amount of cell-free nucleic acid from the donor is determined as a percentage of the total cell-free nucleic acid in the sample. In some embodiments, the amount of cell-free nucleic acid from the donor is determined as an absolute copy of cell-free nucleic acid from the donor in the sample. In some embodiments, the amount of cell-free nucleic acid from the donor is determined as a ratio of the total cell-free nucleic acid from the donor to the total donor- and recipient-derived cell-free nucleic acid in the sample. In some embodiments, the amount of cell-free nucleic acid from the donor is determined as a ratio of the cell-free nucleic acid from the donor to the cell-free nucleic acid from the recipient in the sample. In some embodiments, the amount of cell-free nucleic acid from the donor is determined as a ratio or percentage compared to one or more reference nucleic acid molecules in the sample. For example, the amount of cell-free nucleic acid from the donor can be determined to be 10% of the total nucleic acid (or total DNA or RNA) molecules in the cell-free nucleic acid sample. Alternatively, the amount of cell-free nucleic acid from the donor may be a 1:10 ratio compared to the total nucleic acid (or DNA or RNA) molecules in the cell-free DNA sample. In some embodiments, the amount of cell-free nucleic acid from the donor may be determined as a concentration. For example, the amount of cell-free nucleic acid from the donor in the cell-free nucleic acid sample may be determined to be 1 μg per mL of sample, 100 donor (genomic) copies per mL of sample, 10 donor (genomic) copies per ng of DNA, etc. The values ​​described herein are merely exemplary to illustrate various ways to express the amount of cell-free nucleic acid from the donor. The percentage of cell-free nucleic acid from the donor in a cell-free nucleic acid sample from a transplant recipient may be extremely low. It is noted that the amount of cell-free nucleic acid from the recipient in a cell-free nucleic acid sample may also be expressed in the manner described for cell-free nucleic acid from the donor. Further methods of expressing the amount of a given source, type, or sequence of nucleic acid in a cell-free nucleic acid sample will be readily apparent to those skilled in the art.

[0073] In some embodiments, the amount of cell-free nucleic acid from the donor is determined with respect to the amount of sample analyzed by dPCR assay. In some embodiments, the amount of cell-free nucleic acid from the donor can be determined as the number of copies of donor genome in a sample from a transplant recipient (e.g., the number of copies of donor genome per mL of sample, or per amount of cell-free nucleic acid in a sample). In one embodiment, the amount of cell-free nucleic acid from the donor can be determined as the number of copies of donor genome per volume of sample from the recipient, such as the number of copies of donor genome per volume (e.g., mL) of plasma from the recipient. In another embodiment, the amount of cell-free nucleic acid from the donor can be determined as the number of copies of donor genome per amount of total cell-free nucleic acid in a sample from the recipient, such as the number of copies of donor genome per ng of cell-free nucleic acid in a sample from the recipient. In one example, the copy number of the donor-specific target sequence molecule determined by dPCR assay (e.g., as the copy number of donor DNA molecules per μL) is adjusted to the copy number of the donor-specific haploid genome in the dPCR assay (e.g., in a donor-specific PCR assay for a target with two copies corresponding to a donor haploid genome, the copy number of the donor target sequence determined by dPCR assay is divided by 2 copies / haploid genome). The copy number of the recipient target sequence molecule determined by dPCR assay can be adjusted as described above for the donor-specific target sequence. The copy number of the donor genome in the sample from the transplant recipient (e.g., the copy number of the donor genome per mL of sample or per amount of cell-free nucleic acid in the sample) is then determined based on the volume used in the dPCR assay, the volume of cell-free nucleic acid from the sample (e.g., the volume of cell-free nucleic acid extracted from the sample from the transplant recipient), and the volume of the sample from the transplant recipient (e.g., the volume of the sample from the transplant recipient from which the cell-free nucleic acid was extracted). If two or more PCR assays in a dPCR reaction (e.g., in a multiplex dPCR assay) contain detection reagents with the same fluorescent dye or label, the method further includes normalizing for the number of PCR assays that contain detection reagents with the same fluorescent dye or label.In one example of a multiplex dPCR assay using two donor-specific single copy target PCR assays in a dPCR reaction containing detection reagents with the same fluorescent dye or label, the copy number of the donor-specific target sequence determined by the dPCR assay (e.g., as the sum of the copy number of the donor-specific sequence per μL) is adjusted (divided) by the number of donor-specific PCR assays in the dPCR reaction containing detection reagents with the same fluorescent dye or label (e.g., in a dPCR assay using two donor-specific PCR assays containing detection reagents with the same fluorescent dye or label, the copy number of the donor genome per μL is obtained by dividing the measured total copies / μL by 2), and for the copy number of each donor-specific target in the PCR assay (e.g., in a donor-specific PCR assay for a two-copy target corresponding to a donor, the copy number of the donor genome is obtained by dividing the measured copies / μL by 2). The copy number of the recipient genome determined by the dPCR assay (e.g., as the copy number of the recipient genome per μL) can also be adjusted as described above for the copy number of the donor genome. The number of copies of the donor genome in the sample from the transplant recipient (e.g., the number of copies of the donor genome per mL of sample or per amount of cell free nucleic acid in the sample) is then determined based on the volume used in the dPCR assay, the volume of cell free nucleic acid from the sample (e.g., the volume of cell free nucleic acid extracted from the sample from the transplant recipient), and the volume of the sample from the transplant recipient (e.g., the volume of the sample from the transplant recipient from which the cell free nucleic acid was extracted).In some embodiments, the amount of cell-free nucleic acid from the donor is determined using one or more or all of the following steps: (1) dividing the donor target copies / μl from the dPCR instrument by the number of donor target copies per haploid genome (and also by the number of donor target assays in the fluorescence channel if two or more donor target assays are combined in a fluorescence channel) to obtain donor target copies / μl in the reaction; (2) calculating the total donor genome copies in the reaction by multiplying the donor target copies / μl in the reaction by the volume of the reaction; (3) dividing the total donor genome copies in the reaction by the volume of nucleic acid sample used in the reaction to obtain total donor genome copies per volume of nucleic acid sample; (4) multiplying the total donor genome copies per nucleic acid volume of sample by the total volume of nucleic acid sample obtained from nucleic acid extraction to obtain total donor genome copies obtained from nucleic acid extraction; and (5) calculating the total donor genome copies per volume of plasma from the recipient by dividing the total donor genome copies obtained from nucleic acid extraction by the volume of plasma used for nucleic acid extraction.

[0074] In some embodiments, the amount of cell-free nucleic acid from the donor is determined as a ratio of the cell-free nucleic acid from the donor to the total donor-specific and recipient-specific nucleic acid based on the quantification of both donor-specific and recipient-specific nucleic acids in the sample by dPCR assay. In some embodiments, the copy number of the donor genome determined by dPCR assay (e.g., as the copy number of the donor genome per μL of reaction) is adjusted for the copy number of each donor-specific target in the dPCR assay (e.g., for a target with 2 copies corresponding to the donor, the copy number of the donor genome determined by dPCR assay is divided by 2). The copy number of the recipient genome determined by dPCR assay (e.g., as the copy number of the recipient genome per μL of reaction) can be adjusted as described above for the copy number of the donor genome. Thus, the ratio of the donor genome copy to the total number of copies of the recipient genome and the donor genome is determined. In some cases, the ratio or percentage of the donor genome copy to the total number of copies of the recipient genome and the donor genome is determined.

[0075] B. Organ, Tissue, or Cell Transplant Rejection Conditions For example, following organ, tissue, or cell transplantation, the recipient may experience immunoquiescence, which is a state that may be characterized by the absence or low levels of immune activity, or the absence of rejection-associated clinical symptoms, such as biopsy-confirmed rejection, or a significant change in organ function as indicated, for example, by elevated serum creatinine levels, decreased estimated glomerular filtration rate, abnormal echocardiogram results, or any other clinical concern indicating the clinical need for a biopsy.

[0076] Alternatively, the recipient may experience active rejection, which may be due to T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR or AMR), or a combination of the two (i.e., ("mixed" rejection). ABMR refers to antibody-mediated rejection of organ, tissue, or cell grafts, including but not limited to acute active antibody-mediated rejection, chronic active antibody-mediated rejection, and chronic stable antibody-mediated rejection. TCMR refers to cellular or T cell-mediated rejection of organ, tissue, or cell grafts, including but not limited to TCMR IA, IB, IIA, IIB, borderline TCMR, and chronic TCMR.

[0077] Thus, in some embodiments, the status of an organ, tissue, or cell graft determined by any of the methods provided herein can be the absence of rejection, e.g., characterized by immunoquiescence. In other embodiments, the status of an organ, tissue, or cell graft determined by any of the methods provided herein can be the presence of rejection, such as active rejection, T cell mediated rejection, antibody mediated rejection, or mixed rejection. In further embodiments, the status of an organ, tissue, or cell graft determined by any of the methods provided herein can be a predicted risk or likelihood of rejection, such as active rejection, T cell mediated rejection, antibody mediated rejection, or mixed rejection.

[0078] Detection of transplant rejection In some embodiments, the methods provided herein include detecting, predicting, diagnosing, and / or monitoring a transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient. In some embodiments, the methods include detecting transplant rejection when the amount of donor-derived cell-free nucleic acid in cell-free nucleic acid in a sample obtained from the transplant recipient (e.g., determined according to a method of the present disclosure) exceeds a predetermined threshold.

[0079] A predetermined threshold generally refers to any predetermined level or range of levels that indicates the presence or absence of a condition, or the presence or absence of a risk or likelihood of a condition, such as rejection or non-rejection. A predetermined threshold according to the present disclosure may take a variety of forms. It may be a single cut-off value, such as a median or mean value. As another example, a predetermined threshold may be determined from a baseline value before the presence of a condition, or the risk or likelihood of the presence of a condition, or after a course of treatment. Such a baseline may indicate a normal or other condition in the transplant recipient that does not correlate with the risk or condition being tested. For example, a baseline value may be a level of donor-derived cell-free nucleic acid, such as donor-derived cell-free DNA, in a sample from the transplant recipient prior to transplant, which may be zero or negligible, but may also indicate a baseline error in the system. In some embodiments, the predetermined threshold may be a baseline value for the transplant recipient being tested. The predetermined threshold may vary widely as it relates to distinguishing significant changes in the amount of donor-derived cell-free nucleic acid (e.g., DNA) in the transplant recipient. Those skilled in the art will recognize appropriate parameters and means for determining significant changes over time in the amount of donor-derived cell-free nucleic acid (e.g., DNA) in a transplant recipient. When appropriate analytical parameters are selected, determining the change in the amount of donor-derived cell-free nucleic acid (e.g., DNA) in a transplant recipient over a period of time can provide information about the status of the graft.

[0080] In some embodiments, an increase in the amount of donor-derived cell-free nucleic acid, e.g., DNA, in a transplant recipient over time indicates transplant rejection or a risk or likelihood of transplant rejection, the need to administer or adjust immunosuppressive therapy, immunosuppressive treatment nephrotoxicity, infection, and / or the need for further investigation of the graft status. Without wishing to be bound by theory, it is believed that if the amount of donor-derived cell-free nucleic acid, e.g., DNA, is increasing in a transplant recipient over time, the cells of the graft are increasingly undergoing apoptosis and / or necrosis over time, which is indicative of transplant rejection or a risk or likelihood of transplant rejection.

[0081] In some embodiments, a decrease in the amount of donor-derived cell-free nucleic acid, e.g., DNA, in the transplant recipient over time indicates transplant tolerance, a need to adjust (e.g., decrease) immunosuppressive therapy, and / or a need for further investigation of the graft status. Without wishing to be bound by theory, it is believed that if the amount of donor-derived cell-free nucleic acid, e.g., DNA, is decreasing in the transplant recipient over time, the cells of the graft are undergoing increasingly less apoptosis and / or necrosis over time, which is indicative of transplant tolerance, excessive immunosuppression, or adequate immunosuppression.

[0082] In some embodiments, the amount of donor-derived acellular nucleic acid, e.g., DNA, in the transplant recipient does not change over time, indicating a stable transplant rejection state and / or an opportunity to adjust the immunosuppressive therapy. Without wishing to be bound by theory, it is believed that if the amount of donor-derived acellular nucleic acid, e.g., DNA, in the transplant recipient does not change over time, the cells of the transplant are experiencing a steady-state level of apoptosis over time, which indicates a stable transplant rejection state. A stable transplant rejection state may provide information on the state of the transplant during the analyzed time window, but may not provide information on whether the transplant is progressing toward rejection or tolerance. For example, the transplant may be undergoing active rejection in the transplant recipient, but a stable transplant rejection state indicates that the rejection rate has not changed (i.e., the transplant rejection rate has not increased or decreased) during the time it was analyzed. Similarly, although the graft may be undergoing active tolerance in the transplant recipient, a stable transplant rejection state indicates that the rate of tolerance has not changed during the time period analyzed (i.e., the rate of transplant tolerance has not increased or decreased).

[0083] C. Administering or adjusting immunosuppressive therapy In some embodiments, the methods provided herein include treating transplant rejection of an organ, tissue, or cell graft from a donor in a transplant recipient and / or adjusting immunosuppressive therapy in a transplant recipient of an organ, tissue, or cell graft from a donor.

[0084] Immunosuppressive therapy generally refers to the administration of immunosuppressants or other therapeutic agents that suppress immune responses in transplant recipients. Medical practice of immunosuppression in transplant recipients has evolved to include a regimen of preventive pharmacological agents that typically begins with induction therapy to deplete lymphocytes, followed by maintenance drugs intended to inhibit lymphocyte activation or replication, such as corticosteroids, calcineurin inhibitors (such as tacrolimus), and further inhibitors of lymphocyte replication (such as mycophenolate mofetil). After transplantation, the dosage of immunosuppressants can be reduced over time to reduce the incidence and severity of side effects, such as increased risk of infection, while still avoiding immune rejection of the graft.

[0085] In some embodiments, immunosuppressive therapy may be administered or may be recommended in response to identification of rejection of an organ, tissue, or cell graft, including T-cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft. In other embodiments, the dosage or frequency of immunosuppressive therapy may be increased or may be recommended to be increased in response to identification of rejection, or risk or likelihood of rejection, including T-cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft. For example, in response to identification of TCMR, bolus steroid therapy may be initiated (or may be recommended to be initiated), or maintenance immunosuppressive therapy may be increased (or may be recommended to be increased) in terms of dose and / or frequency. In another example, in response to identification of ABMR, plasma exchange or intravenous immunoglobulin (IVIg) may be initiated (or may be recommended to be initiated).

[0086] In other embodiments, the dosage or frequency of immunosuppressive therapy may be maintained, or may be recommended to be maintained, in response to the absence of organ, tissue, or cell transplant rejection, e.g., the identification of immunoquiescence. In other embodiments, the dosage or frequency of immunosuppressive therapy may be reduced, or may be recommended to be reduced, in response to the absence of organ, tissue, or cell transplant rejection, e.g., the identification of immunoquiescence. In other embodiments, the dosage or frequency of immunosuppressive therapy may be stopped or discontinued, or may be recommended to be stopped or discontinued, in response to the absence of organ, tissue, or cell transplant rejection, e.g., the identification of immunoquiescence.

[0087] In other embodiments, the methods of the present disclosure may include modifying the immunosuppressive therapy with respect to the drugs administered or recommended to be administered. In some embodiments, the immunosuppressive therapy may be replaced (or recommended to be replaced) with another immunosuppressive therapy in response to identification of rejection of an organ, tissue, or cell transplant, including, for example, T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR or AMR), or mixed rejection. In other embodiments, the immunosuppressive therapy may be administered (or recommended to be administered) in combination with one or more additional immunosuppressive therapies in response to identification of rejection of an organ, tissue, or cell transplant, including, for example, T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR or AMR), or mixed rejection.

[0088] Immunosuppressive therapies, and modifications to immunosuppressive therapies (e.g., substituting one therapy for another or combining therapies), may be selected based on a variety of factors, such as the potential for the immunosuppressive therapy to ameliorate or reduce organ, tissue, or cell graft rejection, ameliorate or reduce the symptoms of organ, tissue, or cell graft rejection, reduce any direct or indirect pathological consequences of organ, tissue, or cell graft rejection, reduce the rate of organ, tissue, or cell graft rejection, ameliorate or ameliorate the condition of organ, tissue, or cell graft rejection, improve the prognosis of an organ, tissue, or cell graft, or slow the progression of organ, tissue, or cell graft rejection.

[0089] It should be noted that the amount of donor-derived cell-free nucleic acid, e.g., DNA, in a transplant recipient may not be the only factor considered when determining the need or lack of administering or adjusting immunosuppressive therapy. For example, for transplant recipients that show both graft rejection and increased severity of infection, it may not be advisable to increase or even maintain the current immunosuppressive therapy. Therefore, it should be noted that the immunosuppressive therapy administered to a transplant recipient may be increased, decreased, or maintained regardless of the determined amount of donor-derived cell-free nucleic acid, e.g., DNA, in the transplant recipient, depending on the presence or absence of other controlling or contributing clinical factors. Thus, additional factors that may be considered when selecting, administering, modifying, or adjusting immunosuppressive therapy include prevention, control, improvement, or reduction of undesirable effects of immunosuppressive therapy, such as adverse events, undesirable side effects, toxicity, undesirable drug-drug interactions, undesirable symptoms, etc.

[0090] Exemplary immunosuppressive therapies that may be used in accordance with the methods of the present disclosure include, for example, aspirin, azathioprine, B7RP-1-fc, brequinar sodium, Campath-1H, celecoxib, chloroquine, coumadin, cyclophosphamide, cyclosporine A, DHEA, deoxyspergualin, dexamethasone, diclofenac, dolobid, etodolac, everolimus, FK778, feldene, fenoprofen, flurbiprofen, heparin, hydralazine, hydroxychloroquine, CTLA-4 or LFA3 immunoglobulin, ibuprofen, indomethacin, ISAtx-247, ketoprofen ... including trolac, leflunomide, meclofenamate, mefenamic acid, mepacrine, 6-mercaptopurine, meloxicam, methotrexate, mizoribine, mycophenolic acid derivatives, naproxen, oxaprozin, plaquenil, NOX-100, prednisone, methylprednisolone, rapamycin (sirolimus), sulindac, tacrolimus (FK506), thymoglobulin, tolmetin, tresperimus, UO126, calcineurin inhibitors, mTOR inhibitors, ACE inhibitors, anticoagulants, antimalarials, beta blockers, corticosteroids, cardiovascular drugs, non-steroidal anti-inflammatory drugs anti-inflammatory drugs (NSAIDs), and steroids, as well as antibodies, including, for example, alpha lymphocyte antibodies, adalimumab, anti-CD3, anti-CD25, anti-CD52, anti-IL2R, anti-TAC antibodies, basiliximab, daclizumab, etanercept, hu5C8, infliximab, OKT4, and natalizumab, and any combination thereof.

[0091] Administering or coordinating transplant-related therapy The methods of the present disclosure can also be used to inform the need to administer or adjust other transplant-related therapies. In general, the amount of donor-derived cell-free nucleic acid, e.g., DNA, in a transplant recipient that exceeds a certain threshold can be informative in determining the need to administer or adjust other transplant-related therapies.

[0092] Other transplant-related therapies include treatments or therapies other than transplant or immunosuppressive therapy administered to the transplant recipient to promote graft survival or treat transplant-related conditions (e.g., cytokine release syndrome, neurotoxicity). Examples of other transplant-related therapies include, but are not limited to, administration of antibodies, antigen-targeting ligands, non-immunosuppressive drugs, and other agents that stabilize or destabilize graft components that are important for transplant activity or that directly activate or inhibit one or more transplant activities. These activities may include the ability to induce, modify, or attenuate immune responses, recognize and replicate specific antigens, and / or induce repair of damaged tissue.

[0093] Coordination of surveillance of transplant recipients The method of the present disclosure can also be used to inform the need to adjust the monitoring of the recipient of the transplant.In general, the amount of donor-derived cell-free nucleic acid, e.g., DNA, in the transplant recipient that exceeds a certain threshold can be useful for determining the need to adjust the monitoring of the recipient of the transplant.In some embodiments, determining the status of the transplant as described above is useful for determining the need to adjust the monitoring of the recipient of the transplant.

[0094] Depending on the status of the graft, monitoring of the recipient may be adjusted accordingly. For example, monitoring may be adjusted by increasing or decreasing the frequency of monitoring, as appropriate. Monitoring may be adjusted by changing the means of monitoring, for example, by changing the metrics or assays used to monitor the recipient.

[0095] D.Report In some embodiments, the methods described herein may include generating and / or providing a report.

[0096] In some embodiments, the report includes an assessment or diagnosis of the rejection status of an organ, tissue, or cell graft in a transplant recipient, e.g., as determined according to the methods described herein. In some embodiments, the report indicates the presence of rejection of an organ, tissue, or cell graft, including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft. In some embodiments, the report indicates the risk or likelihood of rejection of an organ, tissue, or cell graft, including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft. In some embodiments, the report indicates the absence of rejection of an organ, tissue, or cell graft, e.g., immunoquiescence. In some embodiments, the report includes a recommendation to administer immunosuppressive therapy to the transplant recipient, based at least in part on detection of rejection of the organ, tissue, or cell graft by the methods provided herein, including, e.g., T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of the organ, tissue, or cell graft, e.g., as described in more detail above. In some embodiments, the report includes a recommendation to adjust or modify the immunosuppressive therapy administered to the transplant recipient, e.g., to increase, decrease, or maintain the dose or frequency, or to discontinue or modify the immunosuppressive therapy, based at least in part on detection of the presence or absence of rejection of the organ, tissue, or cell graft by the methods provided herein, e.g., as described in more detail above.

[0097] In some embodiments, the report indicates the presence of rejection of an organ, tissue, or cell graft in the recipient (e.g., including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft), e.g., as described in more detail above), and includes a recommendation to administer immunosuppressive therapy to the transplant recipient. In some embodiments, the report indicates the presence of rejection of an organ, tissue, or cell graft (e.g., including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of an organ, tissue, or cell graft), e.g., as described in more detail above), and includes a recommendation to adjust, e.g., increase the dose or frequency of, or modify, the immunosuppressive therapy administered to the transplant recipient. In some embodiments, the report indicates a risk or likelihood of rejection of the organ, tissue, or cell graft in the recipient (e.g., including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of the organ, tissue, or cell graft), e.g., as described in more detail above, and includes a recommendation to administer immunosuppressive therapy to the transplant recipient. In some embodiments, the report indicates a risk or likelihood of rejection of the organ, tissue, or cell graft (e.g., including T cell mediated rejection (TCMR), antibody mediated rejection (ABMR or AMR), or mixed rejection of the organ, tissue, or cell graft), e.g., as described in more detail above, and includes a recommendation to adjust, e.g., increase the dose or frequency of, or modify, the immunosuppressive therapy administered to the transplant recipient. In some embodiments, the report indicates the absence of rejection of the organ, tissue, or cell transplant, as described in more detail above, and includes a recommendation to adjust or modify the immunosuppressive therapy being administered to the transplant recipient, e.g., to maintain or reduce the dosage or frequency, or to discontinue or modify the immunosuppressive therapy.

[0098] Reports according to the present disclosure may be in any suitable form, such as digital, electronic, web-based, or paper form, In some embodiments, reports may be provided to one or more parties, such as the transplant recipient, a caregiver, a physician, a hospital, a clinic, a third party payor, an insurance company, a government agency, and any combination thereof.

[0099] In some embodiments, the report may include information regarding a prognosis or potential or proposed treatment options. The report may include information regarding the likely effectiveness of a treatment option, the acceptability of a treatment option, or the appropriateness of applying a treatment option to the transplant recipient. For example, the report may include information or recommendations regarding administration of drugs, such as immunosuppressive therapy, and recommended dosages or treatment regimens, and / or combinations of other drugs.

[0100] In some embodiments, the report may be generated and / or provided to the party within less than one day, or within any of about one day, about two days, about three days, about four days, about five days, about six days, about seven days, about one week, about two weeks, about three weeks, about four weeks, or about one month, of obtaining or receiving a sample from a transplant recipient or determining the status of the organ, cell, or tissue transplant (e.g., whether or not the organ, cell, or tissue transplant has been rejected).

[0101] II. Cell-free nucleic acids The methods provided herein include analysis of cell-free nucleic acids (e.g., cell-free DNA, RNA, mRNA, miRNA, double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA / RNA hybrids, RNA hairpins, and combinations thereof) from a transplant recipient to detect and monitor the transplant rejection status of an organ, tissue, or cell graft in the transplant recipient.

[0102] Cell-free nucleic acid generally refers to nucleic acid present or circulating outside of cells, such as, for example, nucleic acid present in the body fluids (e.g., blood, plasma, serum, urine, etc.) of a transplant recipient. Cell-free nucleic acid may originate from various locations within a cell. For example, cell-free nucleic acid, such as cell-free DNA, may originate from, for example, nuclear DNA and / or mitochondrial DNA. Without wishing to be bound by theory, it is believed that cell-free nucleic acid is released from cells through cellular apoptosis or necrosis (i.e., cell death). Thus, without wishing to be bound by theory, it is believed that during transplant rejection, apoptosis or necrosis of cells from an organ, tissue, or cell transplant results in donor-derived cell-free nucleic acid being released into the body fluids of the transplant recipient. Thus, a transplant recipient undergoing transplant rejection may have a cell-free nucleic acid population in its body fluids that includes both its own endogenous cell-free nucleic acid (recipient-derived cell-free nucleic acid) as well as cell-free nucleic acid originating from the donor (donor-derived cell-free nucleic acid). Thus, as disclosed herein, assessment of levels of donor-derived cell-free nucleic acid in a transplant recipient according to the methods of the present disclosure can be used to detect, predict, diagnose, and / or monitor the status of the organ, tissue, or cell graft.

[0103] In some embodiments, the disclosed method includes determining the amount of donor-derived cell-free nucleic acid in a sample containing cell-free nucleic acid from a transplant recipient, for example, according to any of the detection / quantification methods provided herein. As described in more detail below, the sample from the transplant recipient can be or be derived from any bodily fluid, including whole blood, plasma, serum, lymph, urine, oral swab, bone marrow, saliva, sweat, lung lavage, tears, ear fluid, sputum, bone marrow suspension, semen, vaginal flow, cerebrospinal fluid, brain fluid, ascites, milk, respiratory tract secretions, intestinal fluid, or urogenital tract fluid. In some embodiments, the sample is urine or is derived from urine. In some embodiments, the sample is or is derived from whole blood or a fraction thereof (e.g., serum or plasma). In some embodiments, the sample is plasma or is derived from plasma. In some embodiments, the cell-free nucleic acid present in the sample can be entirely recipient-derived or can include a mixture of recipient-derived and donor-derived nucleic acid.

[0104] In some embodiments, the cell-free nucleic acid from the donor is cell-free DNA from the donor, and / or the cell-free nucleic acid from the recipient is cell-free DNA from the recipient. In some embodiments, the cell-free nucleic acid from the donor is cell-free RNA from the donor, and / or the cell-free nucleic acid from the recipient is cell-free RNA from the recipient. In some embodiments, the cell-free RNA comprises tissue-specific RNA transcripts. In some embodiments, the cell-free RNA from the transplant recipient may be analyzed for gene expression levels to detect, predict, diagnose, and / or monitor the status of the organ, tissue, or cell graft.

[0105] A. Extraction Cell-free nucleic acid (e.g., cell-free DNA or cell-free RNA) from a sample from a transplant recipient can be extracted prior to analysis by the methods of the present disclosure. Methods for the extraction of cell-free nucleic acid, such as cell-free DNA or cell-free RNA, are known in the art, see, for example, Current Protocols in Molecular Biology, latest edition. Exemplary, non-limiting methods that may be used include the Triton-Heat-Phenol (THP) method (Xue et al., (2009) Clin. Chim. Acta 404, 100-104), the phenol-chloroform isoamyl alcohol isolation (PCI) protocol (Yuan et al., (2012) Yonsei Med. J. 53:132; Schmid et al., (2005) Clin. Chem. 51, 1560-1561; and Hufnagl et al., (2013) J. Nucleic Acids Investig. 4, 1-3), and the salting-out method (Miller et al., (1988) Nucleic Acids Res. 16:1215; Jorgez et al., (1989) Nucleic Acids Res. 20:132; al., (2006) Genet. Med. 8, 615-619).Other exemplary, non-limiting methods for extracting cell-free nucleic acids are known in the art and are described, for example, in Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock. Clin. Chem. 2008, v. 54, p. 1000-1007; Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction. JCO 2005, v. 23, p. 5205-5210; Circulating Nucleic Acids in Blood of Healthy Male and Female Donors. Clin. Chem. 2005, v. 51, p. 1317-1319; Use of Magnetic Beads for Plasma Cell-free DNA Extraction: Toward Automation of Plasma DNA Analysis for Molecular See Diagnostics.Clin.Chem.2003,v.49,p.1953-1955, Chiu RWK, Poon LLM, Lau TK, Leung TN, Wong EMC, Lo YMD.Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma.Clin Chem 2001;47:1607-1613, and Swinkles et al.Effects of Blood-Processing Protocols on Cell-free DNA Quantification in Plasma.Clinical Chemistry,2003,vol.49,no.3,525-526. Any method known in the art for extracting cell-free nucleic acid may be used in the methods of the present disclosure.In some embodiments, extraction of cell-free nucleic acids, such as cell-free DNA or cell-free RNA, from a sample can be performed using commercially available kits, such as the Qiagen QIAamp Circulating Nucleic Acid Kit, BioChain cfPure Cell-Free DNA Extraction Kit, ThermoFisher Scientific MagMax Cell-Free DNA Isolation Kit, Roche MagNa Pure 24 System, Zymo Quick-cfDNA Serum & Plasma Kit, or Macherey-Nagel NucleoSnap cfDNA Kit, NucleoSpin Gel and PCR Clean-Up Kit, or NucleoSpin Plasma XS Kit. In some embodiments, extraction of cell-free nucleic acids, such as cell-free DNA or cell-free RNA, from a sample is performed using the Qiagen QIAamp Circulating Nucleic Acid Kit.

[0106] In some embodiments, cell-free nucleic acids, such as cell-free DNA or cell-free RNA, can be further assessed for quality, quantity (e.g., concentration or absolute amount), and / or fragment size, for example, prior to analysis by the methods of the present disclosure. Quantification, quality analysis, and / or fragment size analysis of cell-free nucleic acids can be performed using any method known in the art, such as fluorescent quantitative dsDNA assays (e.g., using the Quant-iT PicoGreen dsDNA Assay Kit from ThermoFisher), using a fluorometer, such as using Qubit assays, real-time PCR, quantitative PCR, or digital droplet PCR (see, e.g., Rago et al. (2007) Cancer Res. 67, 9364-9370; Takai et al. (2015) Sci. Rep. 5:18425; Rostami et al., (2020) Cell Rep. 31:107830; He et al. (2019) Sci. Rep. 9:5599), using a bioanalyzer (e.g., using the High Sensitivity DNA Microchip Kit (Agilent Technologies) and an Agilent 2100 Bioanalyzer). In some embodiments, the cell-free nucleic acid can also be assessed for the presence or absence of modifications such as methylation. Any suitable method for analyzing modifications of nucleic acids known in the art can be used, such as, for example, bisulfite-based methylation analysis using a kit such as the EpiTect Plus DNA Bisulfite Kit or the PyroMark PCR Kit from Qiagen.

[0107] In some embodiments, at least about 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1000 ng, 2000 ng, 3000 ng, 4000 ng, 5000 ng, or more of cell-free nucleic acid (e.g., cell-free DNA or RNA) may be obtained from a sample from a transplant recipient, e.g., after extraction. In some embodiments, about 50 ng to about 5000 ng, about 100 ng to about 4000 ng, or about 200 ng to about 4000 ng (including any value within each of the recited ranges) of cell-free nucleic acid (e.g., cell-free DNA or RNA) may be obtained from a sample from a transplant recipient, e.g., after extraction. In some embodiments, cell-free nucleic acid (e.g., cell-free DNA or RNA) for use in the methods of the disclosure may have a concentration, for example, after extraction, of at least about any of 0.01 ng / μL, 0.1 ng / μL, 0.5 ng / μL, 0.75 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, 15 ng / μL, 20 ng / μL, 25 ng / μL, 30 ng / μL, 35 ng / μL, 40 ng / μL, 45 ng / μL, 50 ng / μL, 55 ng / μL, 60 ng / μL, 65 ng / μL, 70 ng / μL, or more. In some embodiments, cell-free nucleic acid (e.g., cell-free DNA or RNA) for use in the methods of the present disclosure may have a concentration, e.g., after extraction, of about 1 ng / μL to about 100 ng / μL, about 1 ng / μL to about 70 ng / μL, or about 4 ng / μL to about 70 ng / μL, including any value within each of the recited ranges.

[0108] B. Amplification Cell-free nucleic acid (e.g., cell-free DNA or cell-free RNA) isolated from a sample obtained from a transplant recipient can be amplified for downstream techniques and analyses, for example, according to the methods of the present disclosure.

[0109] Methods for amplifying nucleic acids are well known in the art. Amplification generally refers to any device, method, or technology that can generate copies of nucleic acids. Amplification of cell-free nucleic acids (e.g., cell-free DNA or RNA) can include, for example, isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP), and linear amplification (see U.S. Pat. No. 6,132,997), polymerase chain reaction (PCR) techniques such as rolling circle amplification, and the like. Cell-free nucleic acids (e.g., cell-free DNA or RNA) can be amplified for use in downstream analysis, for example, by qPCR, dPCR, or sequencing. If the cell-free nucleic acid is cell-free RNA, the method can include, for example, using reverse transcriptase to generate DNA from cell-free RNA. Fluidigm Access Array™ system, RainDance Technologies RainDrop system, or other technologies for multiplex amplification can be used for multiplex or highly parallel simplex nucleic acid amplification. Amplification may include the use of a high-fidelity polymerase, such as, for example, FastStart High Fidelity (Roche), Expand High Fidelity (Roche), Phusion Flash II (ThermoFisher Scientific), Phusion Hot Start II (ThermoFisher Scientific), KAPA HiFi (Kapa BioSystems), or KAPA2G (Kapa Biosystems).

[0110] Amplification may include an initial PCR cycle that adds a unique sequence to each individual molecule, referred to as molecular indexing. The amplified nucleic acid may also be subjected to additional processes such as indexing (also referred to as barcoding or tagging). Methods for indexing nucleic acids are well known in the art and are described herein. Indexing allows the use of multiplex sequencing platforms that are compatible with various sequencing systems, such as Illumina HiSeq, MiSeq, and ThermoFisher Scientific Ion PGM and Ion Proton. Multiplex sequencing allows the sequencing of nucleic acids from multiple samples in a single reaction through the use of indexing to specifically identify the sample source of the sequenced nucleic acid.

[0111] III. Samples In some embodiments, the methods provided herein include determining the amount of donor-derived cell-free nucleic acid in one or more samples from the transplant recipient, e.g., one or more biological samples, such as one or more samples containing cell-free nucleic acid.

[0112] Generally, the sample according to the methods provided herein can be (or can be derived from) any bodily fluid including a sample of whole blood, plasma, serum, lymph, peripheral blood mononuclear cells urine, oral swab, bone marrow, saliva, sweat, lung lavage, tears, ear fluid, sputum, fluid from bone marrow suspension, semen, vaginal fluid, cerebrospinal fluid, brain fluid, ascites, milk, respiratory tract secretions, intestinal fluid, or urogenital tract fluid. In some embodiments, the sample is urine or derived from urine. In some embodiments, the sample is or is derived from whole blood or a fraction thereof (e.g., serum or plasma). In some embodiments, the sample is or is derived from plasma. In some embodiments, the sample is or is derived from serum. In some embodiments, when the sample is (or is derived from) blood, serum, or plasma, the blood, serum, or plasma is derived from the venous or arterial blood of the transplant recipient. In some embodiments, the blood, serum, or plasma is derived from the venous blood of the transplant recipient.

[0113] In some embodiments, a sample from a transplant recipient according to the present disclosure comprises cell-free nucleic acid, hi some embodiments, the cell-free nucleic acid present in the sample can be entirely cell-free nucleic acid from the recipient, or the cell-free nucleic acid present in the sample can comprise a mixture of cell-free nucleic acid from the recipient and cell-free nucleic acid from the donor.

[0114] In some embodiments, the methods provided herein include providing a sample from a recipient. In some embodiments, the methods provided herein further include obtaining a sample from the recipient, e.g., in a manner that prevents degradation and / or contamination of the sample and / or analytes, and in a specially prepared container that prevents degradation and / or contamination of the sample and / or analytes. Once the sample is obtained, the sample can be used directly, frozen, or otherwise stored under conditions that maintain the integrity of the sample (e.g., of the nucleic acids in the sample, such as cell-free nucleic acids in the sample) and prevent degradation and / or contamination of the sample. The amount of sample taken at a particular time can vary and can depend on additional factors, such as any need to repeat analysis of the sample. In some embodiments, up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 mL of sample is obtained. In some embodiments, 0.1-1, 1-50, 2-40, 3-30, or 4-20 mL of sample is obtained. In some embodiments, greater than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL of sample may be obtained.

[0115] Samples may be obtained from the transplant recipient one or more times. When multiple samples are obtained from the transplant recipient, the frequency of sampling may vary. For example, samples may be obtained about once a day, about once every other day, about once every 3 days, about once a week, about once every 2 weeks, about once every 3 weeks, about once a month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, about once a year, or about once every 2 years, or more, after the first sampling event. In some embodiments, any of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 or more samples may be obtained from the recipient. In some embodiments, any of 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, or 400-500 or more samples may be obtained from a recipient.

[0116] In some embodiments, one or more samples may be obtained from the transplant recipient over a time interval for use in determining, i.e., detecting, predicting, diagnosing, and / or monitoring the status of an organ, cell, or tissue transplant in the recipient according to the methods of the present disclosure. The time interval at which samples are taken from the transplant recipient after the transplant event may vary. Exemplary intervals for sampling are described, for example, in U.S. Application No. 14 / 658,061, which is incorporated herein by reference in its entirety. For example, samples may be taken from the transplant recipient at various times and over various periods for use in determining, i.e., detecting, predicting, diagnosing, and / or monitoring the status of an organ, cell, or tissue transplant in the recipient according to the methods of the present disclosure. In some embodiments, one or more samples may be taken from the transplant recipient before the recipient receives the organ, cell, or tissue transplant. In some embodiments, the one or more samples are taken at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 2 years, after the transplant event or after the first sample is taken from the recipient (i.e., after the first sampling event). , about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, or more, or any combination of any of them. In some embodiments, the sample may be taken from the transplant recipient within about 3 months, about 6 months, about 9 months, or less than one year after the transplant event. In some embodiments, the sample may be taken from the transplant recipient at various times before the one-year anniversary of the transplant event, at the one-year anniversary of the transplant event, or at various times after the one-year anniversary of the transplant event.For example, at the first anniversary of transplantation, samples may be taken from the transplant recipient starting at the 12th month (i.e., the first anniversary of the transplantation event) and continuously over this period. In some embodiments, the time period for obtaining samples from the transplant recipient is within the first few days after the donor-to-recipient transplant has taken place. This may be done to monitor induction therapy. In some embodiments, the time period for obtaining samples from the transplant recipient includes the period that typically occurs during the first 12 months after the donor-to-recipient transplant has taken place during the tapering of the immunosuppressive regimen administered to the recipient. In some embodiments, the time period for obtaining samples from the transplant recipient includes during the initial long-term immunosuppressive maintenance phase, which may begin about 12-14 months, or earlier or later, after the donor-to-recipient transplant has taken place. In some embodiments, the time period for obtaining samples from the transplant recipient includes the entire long-term maintenance of the immunosuppressive regimen, which may be any time greater than 12 months, or earlier or later, after the donor-to-recipient transplant has taken place.

[0117] In some embodiments, one or more samples are obtained from the transplant recipient twice weekly for the first three weeks after transplant. In some embodiments, one or more samples are obtained daily for the first one or two weeks after transplant. In some embodiments, one or more samples are obtained weekly for the first three months after transplant. In some embodiments, one or more samples are obtained monthly for the first year after transplant. In some embodiments, one or more samples are obtained four times per year after the first year after transplant.

[0118] In some embodiments, samples are obtained from the transplant recipient for 1-3 consecutive months beginning on the first anniversary of the transplant event (i.e., 12 months after the transplant event), providing a total of 4-6 samples for analysis taken over a 3-month time interval, with samples collected approximately every 2 weeks. In some embodiments, the transplant recipient has samples taken once a week for 1-3 consecutive months beginning on the first anniversary of the transplant event (i.e., 12 months after the transplant event), providing a total of 12 samples for analysis taken over a 3-month time interval. The total period over which samples are obtained from the transplant recipient, as well as the frequency with which such samples are obtained, can vary and depend on various factors, such as clinical progression. For example, the transplant recipient can have samples obtained throughout their lifetime. The appropriate timing and frequency of sampling can be determined by one of skill in the art for a given transplant recipient.

[0119] In some embodiments, the methods provided herein include providing one or more samples from a recipient, eg, for use in accordance with the methods of the present disclosure.

[0120] IV. Donors and Recipients The methods of the disclosure include detecting, predicting, diagnosing, and / or monitoring the transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient, treating transplant rejection of an organ, tissue, or cell graft from a donor in a transplant recipient, and / or adjusting immunosuppressive therapy in a transplant recipient of an organ, tissue, or cell graft from a donor.

[0121] In some embodiments, an organ, tissue, or cell graft according to the present disclosure is a xenogeneic graft (i.e., a xenogeneic or heterologous graft), where the organ, tissue, or cell graft donor is of a different species than the transplant recipient. Thus, in some embodiments, the methods of the present disclosure include detecting, predicting, diagnosing, and / or monitoring the status of a xenogeneic, xenogeneic, or heterologous organ, tissue, or cell graft, treating transplant rejection of a xenogeneic, xenogeneic, or heterologous organ, tissue, or cell graft, and / or adjusting immunosuppressive therapy in a transplant recipient of a xenogeneic, xenogeneic, or heterologous organ, tissue, or cell graft, where the organ, tissue, or cell graft donor is of a different species than the transplant recipient.

[0122] In some embodiments, the transplant recipient and the transplant donor are animals. In some embodiments, the transplant recipient and / or the transplant donor are vertebrates. In some embodiments, the transplant recipient and / or the transplant donor are mammals. In some embodiments, the transplant recipient and / or the transplant donor are non-human mammals. In some embodiments, the transplant recipient is a human and the transplant donor is a non-human animal, such as a non-human vertebrate or a non-human mammal. In some embodiments, the transplant recipient is a non-human animal, such as a non-human vertebrate or a non-human mammal, and the transplant donor is a humanized non-human organ. In some embodiments, the non-human animal is a non-human primate, cow, horse, pig, sheep, goat, dog, cat, rodent, bird, reptile, or other non-human animal. In some embodiments, the non-human mammal is a non-human primate, cow, horse, pig, sheep, goat, dog, cat, rodent, or other non-human mammal. In some embodiments, the rodent is a mouse, rat, squirrel, prairie dog, porcupine, beaver, guinea pig, hamster, or another rodent. In some embodiments, the non-human primate is a monkey or ape. In some embodiments, the non-human primate is a chimpanzee, bonobo, gorilla, orangutan, rhesus monkey, cynomolgus monkey, pig-tailed macaque, African green monkey, marmoset, capuchin monkey, spider monkey, vervet monkey, baboon, squirrel monkey, night monkey, or another non-human primate. In some embodiments, the transplant recipient is a human and the transplant donor is a pig. In some embodiments, the pig is of the species Sus scrofa, Sus scrofa domesticus, Phacochoerus aethiopicus, Potamochoerus porcus porcus, Potamochoerus porcus, Babirousa babyrussa. In some embodiments, the pig is a Hanford pig, a mini pig or a micro pig, a Yucatan pig, a Yucatan micro pig, a Sinclair pig, a Gottingen pig, a Duroc pig, a Yorkshire pig, a Landrace pig, or a combination, hybrid, or crossbreed thereof.

[0123] In some embodiments, the donor and / or recipient are genetically modified or contain one or more cells, organs, or tissues that have been genetically modified. In some embodiments, the organ, tissue, or cell graft is genetically modified. Such genetic modification may include, for example, gene inactivation or complete gene knockout, and / or genetic integration or expression of a gene that was not carried by the original genome of the recipient or donor, or non-recipient or non-donor, transplant donor.

[0124] In some embodiments, the graft is from an adult (e.g., an adult human or non-human animal). In some embodiments, the graft is from a fetus, an embryo, an embryonic stem cell, an induced pluripotent stem cell, a child, or a teenager, a young adult, or an adult. In some embodiments, the graft is from a man or a woman.

[0125] V. Organ, Cell, and Tissue Grafts The methods of the disclosure include detecting, predicting, diagnosing, and / or monitoring the transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient.

[0126] In some embodiments, the graft according to the present disclosure is a graft of one or more organs. In some embodiments, the organ is a solid or hollow organ. In some embodiments, the graft is a whole organ or a portion of an organ. In some embodiments, the organ can be any of the following: kidney, pancreas, liver, heart, lung, intestine, bladder, adrenal gland, appendix, brain, ear, esophagus, eye, gallbladder, bladder, small or large intestine, mouth, muscle, nose, parathyroid gland, pineal gland, pituitary gland, splenic parathyroid gland, stomach, thymus, thyroid gland, trachea, uterus, or appendix. In some cases, the organ is a glandular organ. For example, the organ can be an organ of the digestive system or the endocrine system. In some cases, the organ can be both an endocrine gland and a digestive organ. In some cases, the organ can be derived from endoderm, ectoderm, primitive endoderm, or mesoderm. In some embodiments, the graft is a vascularized composite graft. In some embodiments, the organ graft is an intact organ, a fragment of an intact organ, a disrupted organ, or cells from an organ.

[0127] In some embodiments, the transplant is a kidney transplant. A kidney transplant may also be referred to as a renal transplant. In some embodiments, the kidney transplant is from a deceased or living donor. In some embodiments, the kidney is transplanted together with the pancreas. In some embodiments, the kidney transplant is from a pig donor.

[0128] In some embodiments, the graft is a heart graft. A heart graft is also referred to as a cardiac transplant. In some embodiments, the heart graft is from a pig donor.

[0129] In some embodiments, the graft according to the present disclosure is a graft of one or more cells. In some embodiments, the one or more cells are taken directly from the donor for administration to the recipient, the one or more cells are taken from the donor and genetically engineered before administration to the recipient, the one or more cells are taken from the donor and cultured before administration to the recipient, the one or more cells are taken from the donor and subjected to a manufacturing process before administration to the recipient, or a combination thereof. The one or more cells may also be stored (e.g., "off-the-shelf" cells) before administration to the recipient. In some embodiments, the one or more cells are blood cells, stem cells, cardiomyocytes, neurons, lymphocytes, natural killer (NK) cells, NK T cells, T regulatory (T-reg) cells, neurons, macrophages, dendritic cells, pancreatic islet cells, or any combination thereof. In some embodiments, blood cells may include hematopoietic stem cells (i.e., HSCs), T cells, B cells, chimeric antigen receptor (CAR) T cells, NK cells, NK T cells, tumor-infiltrating lymphocytes (TILs), and any combination thereof. In certain embodiments, the one or more cells are CAR T cells, universal CAR T cells (i.e., the CAR binds to an antibody that binds a specific antigen), split CAR T cells (i.e., a dimerizer activates CAR T cell function), activatable CAR T cells, suppressible CAR T cells, polyphasic CAR T cells (i.e., the CAR must bind multiple specific antigens and / or agents that induce T cell activation), tumor infiltrating lymphocytes, regulatory T cells, genetically modified T cells, T cells with genetically modified or synthetic T cell receptors (TCR), virus-specific T cells (e.g., EBV, HPV, BKV, CMV, etc.), antigen-specific T cells, neoantigen-specific T cells, or any cells isolated from a donor. In some embodiments, the one or more cells are administered as bone marrow cells, cord blood cells, or purified cells.In some embodiments, the one or more cells are bone marrow cells. In some embodiments, the one or more cells are cord blood cells. In some embodiments, the graft comprises stem cells. In some embodiments, the stem cells are administered as bone marrow stem cells, cord blood stem cells, or purified stem cells. In some embodiments, the stem cells are from a donor. In some embodiments, the stem cells are administered as a hematopoietic cell transplant. In some embodiments, the blood cells comprise one or more of white blood cells (e.g., monocytes, lymphocytes, neutrophils, eosinophils, basophils, macrophages, etc.), red blood cells (erythrocytes), and platelets. In some embodiments, the blood cells are administered as a transfusion (e.g., whole blood transfusion), a transfusion of individual components of blood, or a transfusion of purified cells such as white blood cells (e.g., monocytes, lymphocytes, neutrophils, eosinophils, basophils, or macrophages), red blood cells (erythrocytes), or platelets. In some embodiments, the one or more cells are from an organ (e.g., an organ described herein or any other organ known in the art). In some embodiments, the one or more cells are pancreatic cells, liver cells, cardiac cells, kidney cells, nervous system cells, and the like.

[0130] In some embodiments of any of the methods of the present disclosure, the graft comprises stem cells. In some embodiments, the stem cells are embryonic stem cells, tissue-specific stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, skeletal muscle stem cells, myogenic stem cells, cardiac stem cells, neural stem cells, epidermal stem cells, or intestinal stem cells. In some embodiments, the stem cells are hematopoietic stem cells, embryonic stem cells, adult stem cells, multipotent stem cells, pluripotent stem cells, neural stem cells, cardiac stem cells, umbilical cord blood-derived cells, or induced pluripotent stem cells.

[0131] In some embodiments, the one or more cells are gallbladder cells, cardiomyocytes, glomerular cells (e.g., parietal cells, podocytes), renal proximal tubule brush border cells, Henle's loop thin segment cells, thick ascending limb cells, renal distal tubule cells, renal collecting duct cells, interstitial renal cells, enterocytes, goblet cells, enterocytes, caveolated tuft cells, enteroendocrine cells, ganglion neurons, parenchymal cells, non-parenchymal cells, hepatocytes, sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, muscle cells, pancreatic beta cells, endothelial cells, or exocrine cells, or any combination thereof.

[0132] In some embodiments, the one or more cells are genetically engineered and / or subjected to a manufacturing process and / or cultured prior to administration to a recipient. In some embodiments, the one or more cell grafts comprise multiple cell types. In some embodiments, the grafts comprise one or more cells that are genetically distinguishable from one another. In some embodiments, the one or more cell grafts comprise two or more independent administrations. For example, one cell type may be administered in a first administration and a second cell type may be administered in a second administration.

[0133] In some embodiments, the graft according to the present disclosure is a tissue graft. Exemplary tissues include, but are not limited to, connective tissue, epithelial tissue, muscle tissue, nerve tissue, lymphatic tissue, blood or blood components, adipose tissue, cartilage, dense fibrous tissue, skeletal muscle, cardiac muscle, or smooth muscle. Muscle tissue may include muscle fibers or muscle cells. In some embodiments, the tissue is a cornea, tendon, heart valve, vein or artery, skin, bone, birth tissue, or parts, fragments, and / or combinations thereof. In some embodiments, the birth tissue is a placenta tissue, amniotic tissue, chorionic tissue, amniotic fluid tissue, umbilical cord tissue, umbilical vein, or Wharton's jelly. In some cases, the tissue is a bone or tendon (both referred to as musculoskeletal graft tissue).

[0134] VI. Additional Analysis The methods of the disclosure may be performed in addition to, or in conjunction with, other analyses of samples from the transplant recipient and / or transplant donor.

[0135] In some embodiments, the presence or level of an infectious agent in the transplant recipient is tested. Infectious agents that can be tested include, for example, viruses, bacteria such as Pseudomonas aeruginosa, Enterobacteriaceae, Nocardia, Streptococcus pneumonia, Staphyloccous aureus, and Legionella, fungi such as Candida, Aspergillus, Cryptococcus, Pneumocystis carinii, or parasites such as Toxoplasma gondii. In some embodiments, the presence or level of a viral infectious agent in the transplant recipient is tested. Viral biomarkers can be analyzed in nucleic acid obtained from a sample from the transplant recipient to determine the presence or level of the virus in the transplant recipient. Viruses that can be tested include, for example, cytomegalovirus, Epstein-Barr virus, Anelloviridae, and BK virus. The results of the test for the presence or level of the virus can be used to classify the immune status of the transplant recipient and / or to determine the status of infection in the transplant recipient. In some embodiments, immunosuppressive therapy may be decreased, or at least not increased, in transplant recipients classified as having a high risk of clinically significant infection. In some embodiments, immunosuppressive therapy may be increased, or at least not decreased, in transplant recipients classified as having a low risk of clinically significant infection. It should be noted that transplant recipients may increase, decrease, or maintain their currently administered immunosuppressive therapy regardless of the results of testing for the presence or levels of virus and / or classification as to risk of clinically significant infection, as other clinical factors may inform the decision to adjust immunosuppressive therapy.

[0136] In some embodiments, the disclosed methods include performing gene expression analysis on a sample from the transplant recipient. In some embodiments, the gene expression analysis is performed on peripheral blood mononuclear cells (PBMCs) from the recipient. In some embodiments, the gene expression analysis is performed on a whole blood sample or a portion thereof from the recipient. In some embodiments, the gene expression analysis is performed on selected genes that provide information about the status of the cell, organ, or tissue graft in the transplant recipient. In some embodiments, the gene expression analysis is performed using any suitable method known in the art, such as RNA sequencing, microarray methods, nanostring analysis systems, and quantitative real-time PCR. In some embodiments, the gene expression analysis is performed using AlloMap testing. AlloMap testing includes performing a quantitative real-time polymerase chain reaction (qRT-PCR) assay using RNA isolated from PBMCs. The expression of a selected number of genes is analyzed, and the gene expression data is used to provide information about the status of the cell, organ, or tissue graft in the transplant recipient. AlloMap testing is known in the art. The results of gene expression analysis (e.g., obtained using the AlloMap test or any other suitable method) may be used in conjunction with the methods of the present disclosure, with or without the method of defining a single score from the combined tests, to determine the status of a cell, organ, or tissue graft in a transplant recipient and / or to inform the need to administer or adjust immunosuppressive therapy administered to the transplant recipient.

[0137] In some embodiments, the methods of the present disclosure include determining a single score that can be used to communicate the status of a cell, organ, or tissue graft in a transplant recipient.

[0138] In some embodiments, the method of the present disclosure includes determining a combination score that can be used to communicate the status of a cell, organ, or tissue graft in a transplant recipient. The combination score is generally calculated based on the results of multiple (e.g., two or more) assays used to explore the status of a cell, organ, or tissue graft in a transplant recipient. For example, the combination score can be calculated based on the determined level of donor-derived cell-free nucleic acid, e.g., DNA, in the transplant recipient and the results of a gene expression profiling assay, e.g., as described above (such as the AlloMap test). The combination score can be calculated based on a single sample taken from the transplant recipient, or can be based on samples taken from the transplant recipient over a time interval. The combination score can be used to determine the status of a cell, organ, or tissue graft in a transplant recipient and / or to administer or adjust an immunosuppressive therapy administered to the transplant recipient.

[0139] Additional biomarker analyses, gene expression assays, and other assays to determine the status of a cell, organ, or tissue graft in a transplant recipient, i.e., to detect, predict, diagnose, and / or monitor, and / or to determine the need to administer or adjust immunosuppressive therapy, may also be used in addition to or in conjunction with the methods of the present disclosure, with or without the method of defining a single score from the combined tests, as would be readily apparent to one of skill in the art.

[0140] Additional analyses may be performed to identify markers of new, metastatic, or recurrent cancer in the transplant recipient. Primers may be designed to amplify regions where genetic mutations are known to occur, to provide early detection of cancer through identification of known tumor-associated mutations. This may be advantageous, at least in part, because transplant recipients may be at increased risk of developing certain malignancies due to immunosuppression.

[0141] VII. Kits of the Present Disclosure Also provided herein are kits for use in any one of the methods described herein.

[0142] In one aspect, provided herein is a kit for detecting, predicting, diagnosing, and / or monitoring transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient, where the donor and recipient belong to different species. In some embodiments, the kit comprises reagents for use with the methods of the present disclosure. For example, in some embodiments, the kit comprises reagents for analyzing cell-free nucleic acids isolated from a transplant recipient as described herein. In some embodiments, the kit may comprise reagents for performing qPCR, dPCR, or sequencing methods (such as NGS or HTS), e.g., as described above. Such reagents may include primers (e.g., for use in qPCR or dPCR methods), probes or dyes (e.g., for use in qPCR or dPCR methods), reagents for sequencing library preparation, reagents for amplifying nucleic acids (e.g., polymerase, buffers, etc.), reagents for performing nucleic acid extraction from a sample if extraction is required, and the like. In some embodiments, the kit comprises one or more PCR reaction oligonucleotide primer and probe sets that hybridize to donor-specific or recipient-specific target sequences in cell-free nucleic acids from the transplant recipient, for example. In some embodiments, one or more PCR reaction oligonucleotide primers and probe sets of the kit are for use in qPCR or digital PCR quantification of donor-derived cell-free nucleic acid, either as absolute copies of donor-derived cell-free nucleic acid in a sample or with respect to the total cell-free nucleic acid analyzed. In some embodiments, one or more PCR reaction oligonucleotide primers and probe sets of the kit are for use in qPCR or digital PCR quantification of donor-derived cell-free nucleic acid, either as absolute copies of donor-derived cell-free nucleic acid in a sample or as a ratio of donor-derived cell-free nucleic acid to total donor-derived and recipient-derived cell-free nucleic acid in a sample. In some embodiments, one or more PCR reaction oligonucleotide primers and probe sets of the kit are for use in digital PCR quantification of donor-derived cell-free nucleic acid, either with respect to the amount of sample analyzed or with respect to the total cell-free nucleic acid analyzed.In some embodiments, the kit may contain sufficient reagents to analyze a single sample. In some embodiments, the kit may contain sufficient reagents to analyze several samples.

[0143] In some embodiments, the kit may include instructions for specifying target values ​​and control materials that may be used in conjunction with the reagents and instructions provided in the kit. In some embodiments, the kit further includes a control for use with the methods of the present disclosure. For example, in some embodiments, the kit further includes a control sample containing a known amount of nucleic acid (e.g., cell-free nucleic acid).

[0144] In some embodiments, the kit further comprises instructions for use according to the methods of the present disclosure. Instructions for carrying out any one of the methods described herein may be included. For example, in some embodiments, the kit comprises instructions for use of the kit and for data analysis to determine the amount of cell-free nucleic acid from the donor.

[0145] In some embodiments, the kit further comprises instructions and specifications for input material qualities or input preparation methods.

[0146] In some embodiments, the kit includes software instructions for analysis of sequence data (e.g., sequence reads) or PCR data (e.g., data from qPCR or dPCR), e.g., to determine the amount of donor-derived cell free nucleic acid in cell free nucleic acid obtained from a sample from a transplant recipient. In some embodiments, the kit includes instructions for accessing software, e.g., instructions for downloading and / or installing the software, that can be used to perform a statistical analysis of donor-derived cell free nucleic acid in cell free nucleic acid obtained from a sample from a transplant recipient.

[0147] VIII. Software, Systems, and Devices Certain aspects of the methods described herein for detecting, predicting, diagnosing, and / or monitoring transplant rejection status of an organ, tissue, or cell graft from a donor in a transplant recipient, where the donor and recipient belong to different species, may be performed by a computer system or device. For example, in some embodiments, the step of analyzing sequence data (e.g., sequence reads) or PCR data (e.g., data from qPCR or dPCR) to determine the amount of donor-derived cell-free nucleic acid in cell-free nucleic acid obtained from a sample from a transplant recipient may be performed using a computer system or device. Software for performing the methods described herein is also provided herein.

[0148] The above-described embodiments of the present disclosure may be implemented in a variety of ways. For example, some aspects of the embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code may be executed on any suitable processor or collection of processors, whether provided on a single computer or distributed among multiple computers. It should be understood that any component or collection of components that performs the above-described functions may be generally considered as one or more controllers that control the functions discussed above. The one or more controllers may be implemented in a number of ways, such as dedicated hardware or general-purpose hardware (e.g., one or more processors) programmed with microcode or software to perform the above-described functions.

[0149] In this regard, it should be noted that implementation of various features of the present disclosure may use at least one non-transitory computer-readable storage medium (e.g., computer memory, floppy disk, compact disk, tape, etc.) encoded with a computer program (i.e., a plurality of instructions) that, when executed on a processor, performs the functions and methods discussed above. The computer-readable storage medium may be transportable such that the program stored thereon can be loaded onto any computer resource to implement certain aspects of the present disclosure discussed herein. Furthermore, it should be noted that reference to a computer program that, when executed, performs the functions discussed above is not limited to an application program running on a host computer. Rather, the term computer program is used in a general sense herein to refer to any type of computer code (e.g., software, or microcode) that may be used to program a processor to implement certain aspects of the present disclosure. EXAMPLES

[0150] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes may be suggested to those skilled in the art in light thereof, and should be included within the spirit and scope of this application and the scope of the appended claims.

[0151] Example 1: A method for non-invasively monitoring organ graft health in xenotransplantation. This example describes a digital polymerase chain reaction (dPCR) and sequencing-based method for noninvasive monitoring of xenogeneic organ transplants using cell-free DNA (cfDNA) from transplant recipients.

[0152] In the method described below for monitoring xenotransplantation, a genetically modified pig served as an exemplary organ donor and a human served as an exemplary organ recipient. The pig was genetically modified to not express four porcine genes and to additionally express six human genes (Revivicor, a subsidiary of United Therapeutics). These modifications were taken into account and were found to represent negligible changes in donor genome size. Therefore, all calculations herein were based on the publicly available porcine genome for Sus scrofa.

[0153] In general, modification of the genome of the graft donor animal may involve gene inactivation or complete gene knockout, and / or genetic integration and expression of additional human or non-human genes not carried by the original genome of the graft donor animal. Most, if not all, genetic modifications serve the purpose of mitigating potentially aggressive immune responses elicited by the immune system of the transplant recipient.

[0154] A dPCR-based method for non-invasive monitoring of xenogeneic organ transplants General approach Single copy and multiple copy target detection assays were designed for use in both singleplex and multiplex dPCR assays, as outlined in Table 1. Precision was calculated as a percentage of measured copies to expected copies based on the input DNA measured by Qubit.

[0155] [Table 1]

[0156] Primer and probe design PCR primers and probes were designed to quantitatively detect human-specific and pig-specific genomic DNA sequences. Exemplary PCR primers and fluorophore-labeled probes are shown in Table 2 below. Genomic DNA sequences in gene intron regions are more likely to show diversity between pig and human genomes and were therefore preferably selected as at least a portion of the targeted region for PCR primer design. Primer and probe designs were designed based on, for example, an amplicon size of 60-110 base pairs, a primer melting temperature (T) of 60°C, and a fluorophore-labeled probe. m ), and probe T at 65-70°C m The primers were selected using the primer3 program (see primer3.org) with targeted assay parameters of:

[0157] Table 2. Exemplary PCR primers and fluorophore-labeled probes for detecting and quantifying human-specific and porcine-specific genomic DNA sequences. In one embodiment of the invention, as shown herein, a two-copy porcine target assay detected two copies of the PCR target per haploid genome. In another embodiment of the invention, as shown herein, an eight-copy porcine target assay detected eight copies of the same PCR target per haploid genome.

[0158] [Table 2]

[0159] To ensure the absence of cross-species reactions, PCR primers were checked for unique amplicon products in one genome, e.g., pig, and for the absence of amplicon products in the other genome, e.g., human, using an in silico PCR program. The target genome, e.g., pig genome, was computer-divided into base pair (bp) segments, e.g., 80 bp segments, and the segments were mapped back to the target genome to identify multiple mapping segments. The multiple mapping segments were aligned to non-target genomes, and any alignments were discarded. In some cases, the remaining multiple mapping segments were filtered to remove fragments, e.g., fragments with high or low GC content or fragments aligned to repeat mask regions, or to remove undesirable regions, e.g., non-exonic regions. "LOC" genes (i.e., genes of unknown function that do not have an official symbol and / or ortholog) were discarded, and overlapping segments were merged. The remaining segments were submitted to primer 3 design software and the 0th primer set output for each segment was verified by in silico PCR to confirm multiple hits in the targeted genome and the absence of hits in non-targeted genomes. Only primer 3 generated designs with a minimum separation between segments of 1 kilobase were selected for experimental evaluation.

[0160] The probes were labeled with either the same fluorescent dye for singleplex dPCR assays or different fluorescent dyes for multiplex dPCR assays (as described below).

[0161] For the multiple copy target detection assay, pig-specific overlapping gene groups were identified, and PCR primers / probes were designed to detect multiple copies of the target sequence in the pig genome, but not in the human genome. For pig-specific PCR assay primer and probe design, the genomic sequences of overlapping gene groups (e.g., rRNA, Hox, histone, and tubulin gene families) were searched for regions with high diversity between the pig genome and the human genome. Compared to the single copy target detection assay, the multiple copy target detection assay has an increased number of pig genome positive sections, which is important when pig cfDNA is present in very low copies in the sample. For example, the pig 2 copy target detection assay detects two pig target copies per haploid genome, resulting in a two-fold increase in the number of pig-specific sections compared to the single copy target detection assay.

[0162] Method 1A: A singleplex dPCR assay detecting a single copy of a human-specific target and a single copy of a pig-specific target. Various amounts of cfDNA input, for example up to 120ng per sample, are assayed separately using a human-specific dPCR assay that detects a single copy of a human-specific target, and a pig-specific dPCR assay that detects a single copy of a pig-specific target. The assay is carried out on a Stilla Naica system using 1x multiplex PCR master mix, 0.5μM forward and reverse primers, and 0.3μM FAM-labeled probe in a 25μl reaction on Crystal Droplet dPCR Sapphire Chips. The dPCR results are acquired on a Crystal Droplet dPCR Reader and analyzed with Crystal Droplet dPCR Miner software. Pig and human genome copy concentrations are normalized to the amount of input cfDNA (ng), and pig genome copy percentages are calculated relative to the total copies of pig and human genomes in the sample.

[0163] Method 1B: Singleplex dPCR assay detecting a single copy of a human-specific target and multiple copies of a pig-specific target. As described above for method 1A, separate single-plex dPCR assays were performed to detect single copy human-specific target and multiple copies of pig-specific target.To determine pig genome copy concentration, the measured pig copy number was divided by the copy number of pig-specific target per haploid genome (e.g., if 2 copy target was used in pig assay, the measured pig copy number was divided by 2).Pig and human genome copies per μl were added together, and pig genome copy percentage was calculated relative to the total copies of pig and human genome in the sample.Multiple copy pig-specific target detection assay improves quantitative accuracy and sensitivity.

[0164] Method 2A: A multiplex dPCR assay detecting one or more single copy human specific targets and one or more single and / or multiple copy pig specific targets using unique fluorescent dyes to discriminate between all the different targets or to distinguish between the two species. Individual human target-specific assays and porcine target-specific assays (e.g., as described above for methods 1A and 1B) were optionally combined into a single multiplex assay using different fluorescent dye labels (e.g., FAM, HEX, ATTO550, ROX, Cy5, Cy5.5, SUN) for the different component assay probes. Genome copy concentrations from each component were corrected for genome copy concentrations for the multi-copy target assay (if any), averaged for human and porcine genome copy concentrations, and porcine genome copy percentages were calculated relative to the total copies of the porcine and human genomes in the sample. Thus, in method 2A, more targets were used to increase the number of positive compartments for the donor, improving assay precision and sensitivity.

[0165] Method 2B: A multiplex dPCR assay detecting one or more single copy human-specific targets and at least one multiple copy pig-specific target using non-specific fluorescent dyes for targets within the same species. Multiplex dPCR assay was designed as above for method 2A, except that at least one multiple copy target assay was used in a single dPCR assay reaction, either labeled with different fluorescent dyes or labeled with the same fluorescent dye.Thus, in method 2B, assuming that different genomic targets are separate from each other and can be amplified independently in different droplets in dPCR assay, multiple pig-specific target PCR assays were detected using either multiple fluorescent channels or a single fluorescent channel, increasing the number of positive compartments for the determination of cfDNA from pig donors and improving assay accuracy and sensitivity.

[0166] Data analysis For Methods 1A-1B and 2A-2B, as described above, the outputs from the dPCR reactions (i.e., pig-specific target sequence copies per μL reaction and human-specific target sequence copies per μL reaction) were calculated in terms of genome copies per μL concentration after dividing each by the number of target sequence copies per haploid genome per assay. For relative concentrations, the proportion or percentage of pig genome copies was calculated relative to the total genome, e.g., the total number of human and pig genome copies.

[0167] The number of porcine genomes may also be determined and reported as an absolute value per unit sample input, for example, porcine genome copies per ng of cfDNA or per milliliter (mL) of plasma.

[0168] For absolute concentration, the porcine target sequence copies / μl from the dPCR instrument were first adjusted (divided) by the number of target sequence copies per target in the genome. The volume used in the dPCR reaction was then used to calculate the porcine genome equivalent cfDNA copies per ml of plasma, in addition to the volume of DNA eluted from the extraction and the volume of plasma used in the DNA extraction. Alternative methods for determining absolute concentration include, but are not limited to, obtaining the porcine (target) copies per μl (cp / μl) from the dPCR instrument, calculating the porcine genome cp / μl by dividing the porcine cp / μl by the number of target copies per haploid genome and the number of assays (if two or more assays are combined in the fluorescent channel), determining the porcine genome copies into the dPCR reaction, and determining the porcine genome cp / ml plasma. The specific steps for one method of determining the absolute number of porcine-derived sequences were as follows: 1. Obtain porcine target copies / μl from the dPCR machine and divide by the number of porcine target copies per haploid genome (if two or more porcine target assays are combined in a fluorescent channel, also divide by the number of porcine target assays in the fluorescent channel) to obtain porcine target copies / μl in the reaction; 2. Calculate the total porcine genome copies in the reaction by multiplying the porcine target copies / μl in the reaction by the volume of the reaction; 3. Dividing the total porcine genome copies in the reaction by the volume of DNA sample used in the reaction to obtain the total porcine genome copies per volume of DNA sample; 4. Multiplying the total porcine genome copies per DNA volume of the sample by the total volume of DNA sample obtained from the DNA extraction to obtain the total porcine genome copies obtained from the DNA extraction; and 5. Calculate the total pig genome copies per volume of plasma from the recipient by dividing the total pig genome copies obtained from the DNA extraction by the volume of plasma used in the DNA extraction.

[0169] Sequencing-Based Methods for Noninvasive Monitoring of Xenogeneic Organ Transplants Library preparation and sequencing 25–100 ng of cfDNA was used to prepare shotgun libraries using Illumina's DNA Prep Kit® according to the manufacturer's instructions.

[0170] The average library fragment size was determined by Tapestation® cfDNA kit and libraries were quantified by Qubit. Libraries were sequenced using paired-end reads on a MiSeq (2x65 or 2x55 cycles, 12 pM) or NextSeq (2x65 or 2x35 cycles, 1.3-1.8 pM). Pure human genomic DNA and pure porcine genomic DNA were also sequenced with samples designed between 0.2% and 0.4% of the porcine genome equivalent (GE).

[0171] Bioinformatics Using a next-generation sequencing (NGS) shotgun pipeline, we analyzed the sequencing data and quantified the amount of pig donor-derived cell-free DNA in samples obtained from human recipients of genetically engineered pig-donated heart grafts and human deceased model recipients of pig heart grafts.

[0172] Differences in genome size between the human genome and the genome of the graft donor animal (e.g., the genome of a genetically modified pig) were taken into account by either normalizing based on genome size for all reads or by using the average coverage over a defined bin size of the genome, as described in more detail below.

[0173] A combined reference FASTA file was created consisting of the 24 chromosomes of the human genome (UCSC hg19; genome.ucsc.edu / ) and the 20 chromosomes of the pig genome (build Sus scrofa 11.1). Raw sequence reads from the FASTQ files were aligned to the combined reference using the BWA aligner. Read alignment was performed with a minimum alignment score cutoff of 90%, a gap percentage setting of 5%, and no split alignment.

[0174] Various mapping filter models were applied to filter the aligned reads and determine the estimated bias and stringency (see Table 3). For each model listed in Table 3, the average coverage over a 1 megabase (Mb) region was calculated for both the human and pig genomes. The percent cfDNA from pig donors was calculated as m p / (m p+ m h ) * 100, where m p is the median average coverage over a 1 Mb region on the pig genome, and m h is the median average coverage over a 1 Mb region on the human genome. This methodology takes into account the fact that the pig genome is smaller than the human genome.

[0175] Alternatively, the percentage of cfDNA derived from the pig donor was calculated by aligning the sequence reads to a combined reference that includes the pig genome and the human genome described above, and determining the percentage of sequence reads that align to the pig genome as a percentage of the combined reference. This methodology does not take into account the size difference between the human genome and the pig genome.

[0176] To determine the accuracy of the models in Table 3, in silico samples were prepared with various proportions of the human and pig genomes using 25 million total sequencing reads.

[0177] [Table 3]

[0178] Differences were observed in the average fragment size of cfDNA from pigs versus cfDNA from humans. For example, as shown in Table 4, analysis of two samples containing cfDNA fragments from pigs and humans showed that cfDNA fragments from humans have a longer average fragment size. The average cfDNA fragment size difference between pigs and humans (e.g., as shown in Table 4) may result in overestimation of the cfDNA fraction from pig donors, for example, when smaller cfDNA fragments result in greater coverage. Therefore, a correction factor may be applied to improve accuracy by taking into account the difference in average fragment size of pig cfDNA versus human cfDNA.

[0179] [Table 4]

[0180] Example 2: Non-invasive monitoring of organ graft health in human recipients of porcine heart transplants. This example describes the non-invasive monitoring of organ graft health in human recipients of porcine heart transplants using the dPCR-based and sequencing-based methods described in Example 1 herein.

[0181] sample Blood samples from human recipients were collected into two 10 mL Streck tubes on days 6, 13, 19, 25, 33, 46, 49, 55, and 60 after they received an investigational heart (UHeart) graft from a pig genetically modified by Revivicor, a subsidiary of United Therapeutics. Cell-free DNA (cfDNA) was extracted using the Qiagen QIAamp Circulating Nucleic Acid Kit and quantified using Qubit.

[0182] As shown in Table 5, cfDNA yields varied over time after organ transplantation, from 223 ng at day 25 post-transplant to 3360 ng at day 46 post-transplant.

[0183] [Table 5]

[0184] dPCR-based analysis Control samples containing known percentages of control pig and human genomic DNA were generated to compare the performance of dPCR assays using a single copy or two copies of the pig-specific target. As shown in Table 6, the dPCR assay using two copies of the pig-specific target produced two times more positive dPCR compartments compared to the dPCR assay using a single copy of the pig-specific target.

[0185] [Table 6]

[0186] As shown in Figure 1A and Figure 1B, singleplex dPCR assays using two copies of the pig-specific target (see Figure 1A) as well as multiplex dPCR assays using two one-copy pig-specific targets (see Figure 1B) resulted in a linear relationship between the known or predicted percentage of control pig DNA and the calculated or measured percentage of pig DNA. Cell-free DNA (cfDNA) samples from human recipients of pig heart grafts were analyzed using dPCR at five time points after transplantation (i.e., 6, 13, 19, 25, 33, 46, 49, 55, and 60 days after transplantation). As shown in Figure 2, for genome copies / mL plasma samples and percentage of pig donor-derived cell-free DNA, the percentage of pig donor-derived cell-free DNA was lowest at day 6 after organ transplantation, increased over time until it peaked at day 55 after organ transplantation, and then decreased slightly at day 60 after organ transplantation.

[0187] Sequencing-based analysis cfDNA samples from human recipients of porcine heart grafts were also analyzed by shotgun next-generation sequencing (NGS) using filtering models M1-M7 as described in Example 1 herein (see Table 3). Control samples containing pure human and porcine genomic DNA were also analyzed using the same method.

[0188] As shown in Table 7, using filtering model M7, the pure human DNA control sample had a calculated percent porcine DNA of 0%, suggesting that the Limit of Blank (LOB) was very close to 0.

[0189] Filtering models M1-M7 described in Table 3 of Example 1 herein resulted in different percentages of cfDNA estimates derived from the pig donor for samples obtained from human recipients of pig heart grafts (see Table 7 and Figure 3). However, all filtering models showed a consistent trend over time, with the percentage of cfDNA derived from the pig donor being lowest at day 6 after organ transplantation, increasing over time until it peaked at day 55 after organ transplantation, and then decreasing slightly at day 60 after organ transplantation (see Table 7 and Figure 3).

[0190] [Table 7]

[0191] The calculated percent cfDNA of pig donor origin was compared in samples from human recipients of pig heart grafts using either dPCR or shotgun NGS using filtered model M7. For digital PCR assays, either singleplex assays with one two-copy pig-specific target and one single-copy human-specific target, or multiplex assays with two single-copy pig-specific targets and one single-copy human-specific target, as described in Table 1, were used.

[0192] As shown in Figure 4, Figure 5 and Table 8, the determined percentage of cfDNA from pig donors was generally different between dPCR approach and shotgun NGS, but the analyses nevertheless showed consistent trends over time. As shown in Figure 4, the percentage of cfDNA from pig donors was lowest on day 6 after organ transplantation, peaked on day 55 after organ transplantation, and then slightly decreased on day 60 after organ transplantation. The percentage of cfDNA from pig donors measured by the above singleplex dPCR assay using one 2-copy pig-specific target and one single-copy human-specific target was 2-fold lower than the percentage of cfDNA from pig donors measured by the above shotgun NGS method. The percentage of cfDNA from pig donors measured by the above multiplex dPCR assay using two single-copy pig-specific targets and one single-copy human-specific target was comparable to that measured by the shotgun NGS method (Table 8).

[0193] Table 8. Comparison of percent cell-free DNA from pig donors (xcfDNA%) in human recipients of pig heart grafts. xcfDNA% was measured by shotgun next generation sequencing ("NGS"), a singleplex dPCR assay with one two-copy pig-specific target and one single-copy human-specific target ("singleplex dPCR"), or a multiplex dPCR assay with two single-copy pig-specific targets and one single-copy human-specific target ("multiplex dPCR").

[0194] [Table 8]

[0195] Example 3: Non-invasive short-term monitoring of organ graft health in human deceased model recipients of porcine heart transplants. This example describes short-term non-invasive monitoring of organ graft health in two human deceased model recipients of porcine heart grafts that were briefly placed on artificial life support using the sequencing-based and / or multiplex dPCR-based methods described in Example 1.

[0196] sample For quantification of cell-free DNA from the porcine donors, blood samples were obtained from two human deceased model recipients (Recipient 1 and Recipient 2). For Recipient 1, blood was collected into two 10 mL Streck tubes at 30, 48, and 72 hours post-transplant. For Recipient 2, blood was collected into two 10 mL Streck tubes at 0, 12, 24, 36, 48, 60, and 66 hours post-transplant. However, the 36 and 48 hour samples were indistinguishable due to smearing of the tube labels, so they were excluded from further analysis.

[0197] dPCR-based analysis Samples for all available time points were tested in duplicate using multiplex dPCR reactions with either two single copy porcine assays and one single copy human assay ("Multiplex dPCR #1") or one multi-copy porcine assay and one single copy human assay ("Multiplex dPCR #2"). Input cfDNA amounts were 20 ng for the multiplex dPCR #1 test and 10 ng for the multiplex dPCR #2 test. All sample replicates tested using the multiplex dPCR #2 method showed nearly identical xcfDNA quantification % with low standard deviation (Table 9).

[0198] [Table 9]

[0199] For both recipients 1 and 2, the xcfDNA% measurements by the multiplex dPCR #1 and multiplex dPCR #2 assays were closer to each other than the xcfDNA% measurements by NGS (Figures 6A and 6B). Measurements of porcine genome copies per mL of plasma by both dPCR methods were also comparable to each other in recipient 1 samples (Figure 6C) and nearly identical in recipient 2 (Figure 6D and Table 9).

Claims

1. A method for detecting and / or monitoring the state of graft rejection in a transplant recipient of a graft from a donor, wherein the donor and the recipient belong to different species, and the method (a) To provide a sample from the transplant recipient after transplantation, wherein the sample includes cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient, (b) The amount of donor-derived cell-free nucleic acid in the sample determined by a PCR assay, wherein the amount of donor-derived cell-free nucleic acid is (i) as an absolute copy of the donor-derived cell-free nucleic acid in the sample, or (ii) As the ratio of donor-derived cell-free nucleic acids to total donor-derived and recipient-derived cell-free nucleic acids, The amount of donor-derived cell-free nucleic acid is determined by PCR quantification of donor-specific nucleic acids and recipient-specific nucleic acids, (c) A method comprising detecting transplant rejection when the amount of cell-free nucleic acid derived from the donor exceeds a predetermined threshold.

2. The method according to claim 1, wherein the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification.

3. The method according to claim 1, wherein the PCR assay is a digital PCR assay and the PCR quantification is endpoint PCR quantification.

4. The aforementioned digital PCR assay, (a) at least one singleplex digital PCR assay for a single copy or multiple copies of a donor-specific target, wherein the copy number of the donor-specific target relates to a haploid donor genome, and / or (b) The method according to claim 3, comprising at least one singleplex digital PCR assay for a single copy or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome.

5. The method according to claim 3, wherein the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copies of donor-specific targets and / or recipient-specific targets in a single digital PCR reaction, wherein the copy number of the donor-specific target relates to a haploid donor genome and the copy number of the recipient-specific target relates to a haploid recipient genome.

6. The digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay. (a) The first singleplex digital PCR assay is for a single copy or multiple copies of a recipient-specific target, and the copy number of the recipient-specific target is related to a haploid recipient genome, (b) The method according to claim 4, wherein the second singleplex digital PCR assay is for a single copy or multiple copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

7. The method according to claim 6, wherein the second singleplex digital PCR assay is for multiple copies of a donor-specific target.

8. The aforementioned multiplex digital PCR assay (a) at least one single copy or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome, and (b) The method according to claim 5, wherein the method is for at least one single copy or multiple copies of a donor-specific target, the copy number of the donor-specific target relating to a haploid donor genome.

9. The method according to claim 8, wherein the multiplex digital PCR assay is for at least one plurality of copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

10. An immunosuppressant for use in a method for treating transplant rejection of a graft from a donor in a transplant recipient, wherein the donor and the recipient belong to different species, and the method (a) To provide a sample from the transplant recipient after transplantation, wherein the sample includes cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient, (b) The amount of donor-derived cell-free nucleic acid in the sample determined by a PCR assay, wherein the amount of donor-derived cell-free nucleic acid is (i) as an absolute copy of the donor-derived cell-free nucleic acid in the sample, or (ii) As the ratio of donor-derived cell-free nucleic acids to total donor-derived and recipient-derived cell-free nucleic acids, The amount of donor-derived cell-free nucleic acid is determined by PCR quantification of donor-specific nucleic acids and recipient-specific nucleic acids, (c) If the amount of cell-free nucleic acid derived from the donor exceeds a predetermined threshold, a transplant rejection reaction is detected. (d) Immunosuppressants for use, comprising administering immunosuppressant therapy to the transplant recipient based on the amount of cell-free nucleic acid derived from the donor.

11. The immunosuppressant for use according to claim 10, wherein the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification.

12. The immunosuppressant for use according to claim 10, wherein the PCR assay is a digital PCR assay and the PCR quantification is endpoint PCR quantification.

13. The aforementioned digital PCR assay, (a) at least one singleplex digital PCR assay for a single copy or multiple copies of a donor-specific target, wherein the copy number of the donor-specific target relates to a haploid donor genome, and / or (b) An immunosuppressant for use according to claim 12, comprising at least one singleplex digital PCR assay for a single or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome.

14. The immunosuppressant for use according to claim 12, wherein the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copies of donor-specific targets and / or recipient-specific targets in a single digital PCR reaction, wherein the copy number of the donor-specific target is related to a haploid donor genome, and the copy number of the recipient-specific target is related to a haploid recipient genome.

15. The digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay. (a) The first singleplex digital PCR assay is for a single copy or multiple copies of a recipient-specific target, and the copy number of the recipient-specific target is related to a haploid recipient genome, (b) The immunosuppressant for use according to claim 13, wherein the second singleplex digital PCR assay is for a single copy or multiple copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

16. The immunosuppressant for use according to claim 15, wherein the second singleplex digital PCR assay is for multiple copies of a donor-specific target.

17. The aforementioned multiplex digital PCR assay (a) at least one single copy or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome, and (b) The immunosuppressant for use according to claim 14, which is for at least one single copy or multiple copies of a donor-specific target, wherein the number of copies of the donor-specific target relates to a haploid donor genome.

18. The immunosuppressant for use according to claim 17, wherein the multiplex digital PCR assay is for at least one plurality of copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

19. An immunosuppressant for use in a method for modulating immunosuppressive therapy in a recipient of a graft from a donor, wherein the donor and the recipient belong to different species, and the method (a) To provide a sample from a transplant recipient after transplantation, wherein the sample includes cell-free nucleic acid derived from the donor and cell-free nucleic acid derived from the recipient, (b) The amount of donor-derived cell-free nucleic acid in the sample determined by a PCR assay, wherein the amount of donor-derived cell-free nucleic acid is (i) as an absolute copy of the donor-derived cell-free nucleic acid in the sample, or (ii) As the ratio of donor-derived cell-free nucleic acids to total donor-derived and recipient-derived cell-free nucleic acids, The amount of donor-derived cell-free nucleic acid is determined by PCR quantification of donor-specific nucleic acids and recipient-specific nucleic acids, (c) If the amount of cell-free nucleic acid derived from the donor exceeds a predetermined threshold, a transplant rejection reaction is detected. (d) an immunosuppressant for use, comprising adjusting the immunosuppressant treatment administered to the transplant recipient based on the amount of cell-free nucleic acid derived from the donor.

20. The immunosuppressant for use according to claim 19, wherein the PCR assay is a quantitative PCR (qPCR) assay, and the PCR quantification is real-time PCR quantification.

21. The immunosuppressant for use according to claim 19, wherein the PCR assay is a digital PCR assay and the PCR quantification is endpoint PCR quantification.

22. The aforementioned digital PCR assay, (a) at least one singleplex digital PCR assay for a single copy or multiple copies of a donor-specific target, wherein the copy number of the donor-specific target relates to a haploid donor genome, and / or (b) an immunosuppressant for use according to claim 21, comprising at least one singleplex digital PCR assay for a single or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome.

23. The immunosuppressant for use according to claim 21, wherein the digital PCR assay comprises at least one multiplex digital PCR assay for two or more single or multiple copies of donor-specific targets and / or recipient-specific targets in a single digital PCR reaction, wherein the copy number of the donor-specific target is related to a haploid donor genome and the copy number of the recipient-specific target is related to a haploid recipient genome.

24. The digital PCR assay comprises a first singleplex digital PCR assay and a second singleplex digital PCR assay. (a) The first singleplex digital PCR assay is for a single copy or multiple copies of a recipient-specific target, and the copy number of the recipient-specific target is related to a haploid recipient genome, (b) The immunosuppressant for use according to claim 22, wherein the second singleplex digital PCR assay is against a single copy or multiple copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

25. The immunosuppressant for use according to claim 24, wherein the second singleplex digital PCR assay is for multiple copies of a donor-specific target.

26. The aforementioned multiplex digital PCR assay (a) at least one single copy or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome, and (b) The immunosuppressant for use according to claim 23, which is for at least one single copy or multiple copies of a donor-specific target, wherein the number of copies of the donor-specific target relates to a haploid donor genome.

27. The immunosuppressant for use according to claim 26, wherein the multiplex digital PCR assay is for at least one plurality of copies of a donor-specific target, and the number of copies of the donor-specific target is related to a haploid donor genome.

28. (a) The method further comprises testing for the presence of an infectious agent, wherein the infectious agent is selected from the group consisting of viruses, bacteria, fungi, and parasites, and / or (b) The method further comprises performing one or more gene expression profiling assays on the recipient, The method according to any one of claims 1 to 9, or an immunosuppressant for use according to any one of claims 10 to 27.

29. The infectious agent is a virus selected from the group consisting of cytomegalovirus, Epstein-Barr virus, Anelloviridae, and BK virus. The method according to claim 28 or an immunosuppressant for use.

30. The method according to claim 28 or an immunosuppressant for use, wherein the combined score is calculated based on the amount of cell-free nucleic acid derived from the donor in the sample and the results of the gene expression profiling assay.

31. A kit for detecting and monitoring the state of graft rejection in a transplant recipient, wherein the donor and the recipient belong to different species, and the kit is (a) A set of PCR reaction oligonucleotide primers and probes that hybridize to a donor-specific or recipient-specific target sequence in the donor-derived cell-free nucleic acid in the sample from the transplant recipient, for PCR quantification of donor-derived cell-free nucleic acid as an absolute copy of the donor-derived cell-free nucleic acid in the sample, or as a ratio of the donor-derived cell-free nucleic acid in the sample to the total donor-derived and recipient-derived cell-free nucleic acids, (b) A kit including instructions for data analysis to determine the amount of cell-free nucleic acid derived from the donor.

32. The method according to any one of claims 1 to 9, wherein the cell-free nucleic acid derived from the donor and / or the recipient is DNA, RNA, mRNA, miRNA, double-stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, RNA hairpin, or a combination thereof, an immunosuppressant for use according to any one of claims 10 to 27, or the kit according to claim 31.

33. (a) The amount of donor-derived cell-free nucleic acid is the ratio of the total donor-derived cell-free nucleic acid to the recipient-derived cell-free nucleic acid, or (b) The amount of donor-derived cell-free nucleic acid is the percentage of donor-derived cell-free nucleic acid compared to the total cell-free nucleic acid. The method according to any one of claims 1 to 9, an immunosuppressant for use according to any one of claims 10 to 27, or the kit according to claim 31.

34. The method according to any one of claims 1 to 9, wherein the graft is a parenchymal organ, tissue, or cell graft; the immunosuppressant for use according to any one of claims 10 to 27; or the kit according to claim 31.

35. The method according to claim 33, an immunosuppressant for use, or a kit, wherein the donor of the graft is an animal.

36. The method according to claim 34, an immunosuppressant for use, or a kit, wherein the animal is a pig.

37. A method for analyzing biological samples from a transplant recipient who has received a parenchymal organ graft from a donor, wherein the donor and the recipient belong to different species, wherein the method is (a) Isolating cell-free nucleic acids, wherein the cell-free nucleic acids include cell-free nucleic acids derived from the donor and cell-free nucleic acids derived from the recipient, from a biological sample from the recipient. (b) The amount of donor-derived cell-free nucleic acid in the sample is determined by a digital PCR assay, wherein the digital PCR assay (i) at least one digital PCR assay for one or more single or multiple copies of a donor-specific target, (ii) Determining by a digital PCR assay comprising one or more single or multiple copies of recipient-specific targets and one or more single or multiple copies of donor-specific targets, The copy number of the donor-specific target is related to the haploid donor genome, and the copy number of the recipient-specific target is related to the haploid recipient genome. The amount of cell-free nucleic acid derived from the donor is (i) as an absolute copy of the donor-derived cell-free nucleic acid in the sample, or (ii) As the ratio of donor-derived cell-free nucleic acids to total donor-derived and recipient-derived cell-free nucleic acids, A method determined by digital PCR quantification of donor-specific nucleic acids and recipient-specific nucleic acids.

38. The method according to claim 37, wherein the digital PCR assay comprises (a) a first digital PCR assay for a single or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome; and (b) a second digital PCR assay for a single or multiple copies of a donor-specific target, wherein the number of copies of the donor-specific target relates to a haploid donor genome.

39. The method according to claim 38, wherein the second digital PCR assay is for multiple copies of a donor-specific target.

40. The method according to claim 37, wherein the digital PCR assay is for (a) at least one single copy or multiple copies of a recipient-specific target, wherein the number of copies of the recipient-specific target relates to a haploid recipient genome, and (b) at least one single copy or multiple copies of a donor-specific target, wherein the number of copies of the donor-specific target relates to a haploid donor genome.

41. The method according to claim 40, wherein the digital PCR assay is for multiple copies of a donor-specific target, and the copy number of the donor-specific target is related to a haploid donor genome.

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