Non-invasive method for monitoring transplanted organ status in organ-transplant recipients
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Publication Number
US-11479819-B2
Publication Date
2022-10-25
Expiration Date
Abstract
The present invention pertains to a non-invasive method for monitoring transplanted organ status in organ-transplant recipients by determining the ratio of donor derived marker sequences to the marker sequences of the transplant recipient from circulating cell free DNA of the transplant recipients using digital droplet PCR. The invention also determined a normalized threshold value of the total circulating cell free nucleic acids healthy as well as in post-transplantation patients for assessing and monitoring transplanted organ status in organ-transplant recipients.
Core Innovation
The invention relates to a method of assessing the status of a transplanted organ in transplant recipients using circulating cell-free nucleic acids. The method identifies one or more recipient-derived marker sequences having homozygous genotype by screening listed markers using each recited primer pair, and identifies donor-derived marker sequences having heterozygous genotype by analyzing the circulating cell-free nucleic acids of the recipient using each primer pair by digital droplet polymerase chain reaction (DDPCR).
The method calculates a ratio of the donor-derived marker sequences to the recipient-derived marker sequences expressed in percent. The organ status of the transplant recipients is assessed by comparing the calculated percent with a cut off value. The markers include a defined set consisting of rs2493132, rs6667487, rs553921764, rs4233335, rs10900556, rs1449265, rs7603052, rs1317808, rs2229813, rs4851521, rs7653603, rs6791557, rs7652776, rs9131, rs352007, rs1818782, rs4701997, rs251022, rs714459, rs1801020, rs2842949, rs657941, rs805294, rs2523860, rs2766535, rs1554497, rs35024671, rs7794745, rs15775, rs2002792, rs2575694, rs756627, rs1800392, rs11794980, rs10738924, rs1197943, rs4405241, rs2704522, rs1512705, rs10734083, rs1073525, rs668393, rs1522662, rs481235, rs2277312, rs2043055, rs10841697, rs657197, rs9554250, rs7328030, rs1626923, rs978511, rs741761, rs10162971, rs12449089, rs7193058, rs11866418, rs4323, rs2159132, rs150384, rs3909244, rs12459052, rs67233828, rs3918251, rs2327088, rs1051266, rs2742630, rs1801198, rs6609533, rs2298065, and S1.No. 71, with each marker identified by a pair of primers comprising specified nucleic acid sequences as SEQ ID NOs.
In dependent refinements, the approach constrains the recipient-specific number of identified markers and incorporates an average marker amount reported as absolute copy numbers (copies/μl). Additional refinements specify that the cut off value used for assessment depends on the transplant type. Further refinements include selecting biological sample types for analysis and specifying permitted organ transplant categories. Another refinement determines assessment frequency as a time-dependent function based on the transplanted organ and the percent over time ratio of donor-derived marker sequences to recipient-derived marker sequences.
Claims Coverage
The document includes one independent claim describing a complete workflow to assess transplanted organ status using recipient-derived homozygous markers and donor-derived heterozygous markers from circulating cell-free nucleic acids quantified by DDPCR. Dependent claims further constrain marker handling, cut off selection, sample type, transplant types, and assessment timing.
Ddpcr ratio of donor-derived to recipient-derived marker sequences
Screening listed markers to identify recipient-derived marker sequences with homozygous genotype, identifying donor-derived marker sequences with heterozygous genotype by analyzing circulating cell-free nucleic acids using DDPCR, calculating a ratio of donor-derived to recipient-derived marker sequences expressed in percent, and assessing organ status by comparing the calculated percent with a cut off value.
Defined marker set with specified primer pairs
Using a defined set of markers consisting of rs2493132, rs6667487, rs553921764, rs4233335, rs10900556, rs1449265, rs7603052, rs1317808, rs2229813, rs4851521, rs7653603, rs6791557, rs7652776, rs9131, rs352007, rs1818782, rs4701997, rs251022, rs714459, rs1801020, rs2842949, rs657941, rs805294, rs2523860, rs2766535, rs1554497, rs35024671, rs7794745, rs15775, rs2002792, rs2575694, rs756627, rs1800392, rs11794980, rs10738924, rs1197943, rs4405241, rs2704522, rs1512705, rs10734083, rs1073525, rs668393, rs1522662, rs481235, rs2277312, rs2043055, rs10841697, rs657197, rs9554250, rs7328030, rs1626923, rs978511, rs741761, rs10162971, rs12449089, rs7193058, rs11866418, rs4323, rs2159132, rs150384, rs3909244, rs12459052, rs67233828, rs3918251, rs2327088, rs1051266, rs2742630, rs1801198, rs6609533, rs2298065, and S1.No. 71, wherein each marker is identified by a pair of primers comprising nucleic acid sequences as SEQ ID NOs.
Marker count range with average marker amount reporting
Constraining the identified markers to be ranged between 3 and 9 per recipient and calculating and reporting an average marker amount, measured as absolute copy numbers (copies/μl), as a percentage of donor-derived ccfDNA markers relative to recipient-derived markers.
Transplant-type dependent cut off value range
Using a cut off value for comparison that depends on transplant type and falls within 0.1% to 15%.
Biological sample selection from specified matrices
Selecting the biological sample from whole blood, plasma, serum, or urine.
Permitted transplanted organ types
Selecting the organ transplant used from kidney, heart, liver, pancreas, lungs, intestine, bone marrow, or thymus transplants, or a combination of more than one organ transplant.
Time-dependent assessment frequency based on percent over time ratio
Determining assessment frequency for transplant organ status as a time-dependent function based on the transplanted organ and the percent over time ratio of donor-derived marker sequences to recipient-derived marker sequences.
Overall, claim coverage centers on using DDPCR to quantify donor-derived heterozygous marker sequences and recipient-derived homozygous marker sequences from circulating cell-free nucleic acids, calculating a donor/recipient marker percent ratio, and comparing that ratio to a cut off value. Dependent claim refinements add constraints on marker count, include average copy-number reporting, define transplant-type dependent cut off ranges, restrict biological sample matrices and organ transplant categories, and optionally make assessment frequency time-dependent on the percent ratio over time.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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