US 11,987,844 B2
Use of CFDNA fragments as biomarkers in patients after organ transplantation
Valentina Favalli, Pavia (IT)
Assigned to 4BASES SA, Manno (CH)
Appl. No. 16/963,355
Filed by 4BASES SA, Manno (CH)
PCT Filed Jan. 29, 2019, PCT No. PCT/EP2019/052054
§ 371(c)(1), (2) Date Jul. 20, 2020,
PCT Pub. No. WO2019/149673, PCT Pub. Date Aug. 8, 2019.
Claims priority of application No. 18154208 (EP), filed on Jan. 30, 2018.
Prior Publication US 2021/0062264 A1, Mar. 4, 2021
Int. Cl. C12Q 1/68 (2018.01); C12P 19/34 (2006.01); C12Q 1/6883 (2018.01); G06N 20/00 (2019.01)
CPC C12Q 1/6883 (2013.01) [G06N 20/00 (2019.01); C12Q 2600/156 (2013.01)] 10 Claims
 
1. An in-vitro or ex-vivo method of monitoring the status of a transplanted organ in a subject, comprising:
a) providing cell-free DNA from a biological sample obtained from a subject who is the recipient of an organ transplant from a donor;
b) amplifying DNA regions in the cell-free DNA in a multiplex reaction utilizing 270 nucleic acid primers, wherein the nucleic acid primers are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, and wherein the nucleic acid primers are the reverse complement of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270;
c) sequencing a panel of 94 single nucleotide polymorphisms (SNPs) from the cell-free DNA amplified in step b), and
d) calculating the percentage of donor-derived cell-free DNA in the biological sample using the formula:
DFi %=(AF (SNPi)−AF (SNPirec))/(AF(SNPidon)−AF(SNPirec)) times 100 for each SNPi; and
total DF % is calculated as: DF %=mean DFi %±SD DFi;
wherein AF(SNPi) is the allele frequency of the SNP considered, calculated for the total cfDNA mixture (donor+recipient), and AF(SNPidon) and AF(SNPirec) is the allele frequency of the SNP considered in the donor and recipient cell-free DNA respectively, according to their genotype; and
wherein the status of the transplanted organ in the subject is detected by evaluating the percentage of the donor-derived cell-free DNA with respect to the recipient cell-free DNA, using the recipient genomic DNA as reference.