WO2018178051A1

WO2018178051A1 - Scgb1a1 polymorphism for the prediction and therapy or prevention of primary graft dysfunction

Info

Publication number: WO2018178051A1
Application number: PCT/EP2018/057728
Authority: WO
Inventors: Pierre MORDANT; Angela HIN; Caroline KANNENGIESSER
Original assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Diderot Paris 7
Current assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Diderot Paris 7
Priority date: 2017-03-28
Filing date: 2018-03-27
Publication date: 2018-10-04
Anticipated expiration: 2019-09-28

Abstract

The present invention relates to SCGB1A1 polymorphism for the prediction and therapy or prevention of primary graft dysfunction (PGD). The outcome following lung transplantation is currently limited by the occurrence of PGD, a serious immediate postoperative complication that is a direct consequence of ischemia-reperfusion injury. The inventors showed that the presence of a AG genotype in SCGB1A1 gene (G38A polymorphism) in the donor was associated with a decreased frequency of PGD in the recipient after lung transplantation. In particular, the present invention relates to a method of identifying lungs harvested from a donor and to be grafted to a recipient, having or at risk of having or developing a primary graft dysfunction (PGD), comprising determining, in a sample obtained from said donor or from said lungs, the presence or absence of a single nucleotide polymorphism (SNP) located in SCGB1A1 gene.

Description

SCGBIAI POLYMORPHISM FOR THE PREDICTION AND THERAPY OR PREVENTION OF PRIMARY GRAFT DYSFUNCTION

FIELD OF THE INVENTION:

The invention is in the field of primary graft dysfunction prediction and therapy or prevention. In particular, the invention relates to a specific mutation (or Single Nucleotide Polymorphism, SNP) in human gene SCGBIAI that is associated with a decreased risk of primary graft dysfunction.

BACKGROUND OF THE INVENTION:

Lung transplantation is the only therapeutic option that can improve the pulmonary function of patients with end-stage respiratory failure due to various lung diseases. The outcome following lung transplantation is currently limited by the occurrence of primary graft dysfunction (PGD), a serious immediate postoperative complication that is a direct consequence of ischemia-reperfusion injury. From the clinical perspective, PGD is a complex inflammatory state associating hypoxemia, pulmonary oedema, systemic inflammatory response syndrome and radiographic appearance of diffuse pulmonary opacities without other identifiable cause. From the biological standpoint, ischemia is defined by an oxygen deficiency in the lung caused by an obstruction of the blood flow to the lung, in this case when the donor lung is collected, and reperfusion occurs when the blood flow to the organ is restored, in this case when connected to the recipient's circulation. Ischemia-reperfusion can lead to lesions of the respiratory epithelium, as the injured lung capillary endothelium leads to protein leakage from the vascular bed into the alveolar space. Even though it is triggered by ischemia- reperfusion injury, PGD represents a multifactorial injury to the transplanted lung that develops in the first 72 hours after transplantation. The natural history of PGD remains poorly delineated, but its lethal potential is well recognized. PGD is the leading cause of early morbidity and mortality after lung transplantation. Unfortunately, predicting which lung transplant recipients go on to develop PGD remains partially unknown. Recognized risk factors of PGD include donor factors (tobacco use, poor graft oxygenation before harvest), recipient factors (primary and secondary pulmonary hypertension) and factors associated with the procedure itself (high emergency procedure, longer ischemic time). However, not all risk factors of PGD have been identified to date. In this setting, the identification of additional risk factors of PGD is of tremendous importance to better select lung grafts and to identify new therapeutic target that might improve the postoperative outcome following lung transplantation. Club Cell Secretory Protein (CCSP), produced by the non-ciliated lung epithelium, is abundant in the airways. CCSP has anti-inflammatory and immunomodulatory properties besides playing a role in host defence and control of oxidative stress (6). The gene encoding CCSP is SCGBIAI.

In the particular case of a single nucleotide polymorphism (dbSNP rs3741240) of

SCGBIAI, concerning a substitution G38A (MAF / Minor Allele Count: A = 0.3451 / 40794 from ExAC), an adenine/guanine polymorphism located 38 bp downstream from the transcription initiation site within the non-coding region of exon 1 of the SCGBIAI gene, is of particular interest (13, 21, 22). Studies of the exon 1 non-coding region of SCGBIAI have identified a number of binding sites for the transcription factors involved in the regulation of CCSP protein expression (15). To this day this particular SNP has been associated with the development of several respiratory diseases including COPD, sarcoidosis, and chronic lung allograft dysfunction (CLAD), a late complication that occurred 1 to 10 years after lung transplantation (14, 15). The association between this particular SNP and earlier complications following lung transplantation has not been studied to date.

SUMMARY OF THE INVENTION:

The present invention relates to SCGBIAI gene polymorphism for the prediction and therapy or prevention of primary graft dysfunction. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

As used herein, the term « primary graft dysfunction" or "PGD" has its general meaning in the art and refers to a syndrome encompassing a spectrum of mild to severe lung injury that occurs within the first 72 hours after lung transplantation (1, 2). PGD is a serious immediate postoperative complication that is a direct consequence of ischemia-reperfusion injury. PGD is a complex inflammatory state associating hypoxemia, pulmonary oedema, systemic inflammatory response syndrome and radiographic appearance of diffuse pulmonary opacities without other identifiable cause. The natural history of PGD remains poorly delineated, but its lethal potential is well recognized. PGD has a significant impact on early morbidity and mortality after lung transplantation, resulting in prolonged length of mechanical ventilation, more frequent use of postoperative Extracorporeal Membrane Oxygenation (ECMO) support, prolonged intensive care unit (ICU) and hospital stay and increased cost. In addition, all grades of PGD are associated with an increased risk of chronic rejection.

As used herein, the term "SCGB1A1 gene" has its general meaning in the art and refers to the secretoglobin family 1A member 1 gene (NCBI Reference Sequence: NG_021331.1) that encodes CCSP protein. SCGB1A1 gene has 3 short exons and 2 introns (4.1 kb in length), located on chromosome l lql2.3-13.1 in the vicinity of other genes associated with inflammatory and immune processes (16, 20).

Example of Homo sapiens SCGB1A1 gene nucleic sequence (SEQ ID NO:l):

TTGGCAGGAGCTGTTCCGATTCACTGCCATCCTCATGGTACTAATAGCCACATGGCTTATCTTCCCTCGG AACAGTTTGTCTTTGCACGGTGACCAGGTAGGTAACTGACCACATGTGGCCAGAAACAGCAGGTCCCAGT CCTCTCCACATGCTAAGTCAGGCTTGAGGGTTCTGGCCTGGAACTCACAGTGTATAGAGTTTAAGGATTT GAGGAGCAGGTGTTAGAGTTTAAGAAGTTCTGAGTTCTGGCCAGGCATGGTGACTCACGCCTATAATCCC AGCACTTGCGGAGGCCAAGGCGGGAGAATCACCTGAGGTCAGAATTTGGAGACCAGCCTGGCCAACATGG TGAAATCCCATCTTTATTAATAATACGAAAATTAACAGGGCGTGGTGACGCACGCCTATAATACCAGCTA TTTGGGAGGCTGAGGCAGGAGAATCGTCTGAACTCGGGAGGTGGAGGTTGCAGTGAGCCGCTGAGATCGT GCCATTGCACTCCAGCCTACGCAACAAAAGCGAAACTCCGTTTCCAGAAAAAAAAAAAAAAAAGCTCTGA GT T CCAAGCC T T GAC T AACACAC TTTTTGCCT CAC T AT C T CAAGCAGAGGGT CAGAAGAGCAGGGGC T T T GCAGGGTCCTGAGGCAGGGGAGAGAAACACCCACCGCCACCAAGCAGGATCTCCCAAGGGCCAGATGGCA GAGCCTATCTGGGACACTTGGTGGTGGCAGTCGAGGTACCTTCCAGAATCAGAGCAAACCTCCAACCTCG CACCTGAAAGGCCAGTGCCCCCGGTGCCCCCAGACTCCCACTGTACCTGTGTGCTTATCTGTCCCATGCA TGTCGGACCATGGCCAGGTCCCCTCCTGAGCCTGGGCCCCCTTCATGTGGCTTCCTCCTCCTGGCACATC CCCCGCTTCACCCCAACCACCAGCATCACCCATCCACCAGAGGCCAGCTCAAATGCCAGCCCTCCACGTG CCCTCTCCCCTCTGCCCAGCTACCTTTCTTGGGGCAGCCCCCTTTCTTTTTTTTTTTTTTTTTTTCCTTT T TTTTGAGACGGAGTTTCGCTCTTGTTGCCCAGGCTGGAGTGCAGTGACGCGATCTCGGCTCACCGCAA CCTCTGCCTCCCAGGTTCAAGCGATTCTTCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCAAGCAC CACCACAAGCCCAGCCAATTTTGTATTTTTAGTACAGACAGGGTTTCTCCATGTTGTTCAGGCTGGTCTC GAACTCCCAACCTCAGGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACC ACGCCCAGCCTGGGGGCAGCCCCCTTTCAACTCCCGGAGCACCATTTCCTTCTCTGACTTCCTCCATCTC CCCACAGAGCATTCCCATCAGTTGCACCCATCTCTGTCTCTCCTCCTAGCTGTTAAGTTCCTCAAAGACC CAGGAGCCAGCTATCCTGTCCCCAGGACCTCGCACCATGAAGGTGGAAAGTAGAGAGCAGAGTCTGAGGC TATGGGTGATGTGACTCCTGCCTAAGACCCCTGCAGCCATCACAGCTGCCCCGGCCAGACCAGCCACTGC CCACAGAGGGATCCACTAGTGGCGGCACTGGATGGGCTTTAGGTGCCCCCAGTGGTCCAGCTCATCTTCC TTGCCCATTTCAGCTCCATTGGCCACAGCTTTCTTTTGTTTGTTTGTTTGTTTATTTATTTATTTATTTT TGAGATGGAGTTTCGCTCTTGTTGTCCAGGCTGGAGTGCAATGGTGTGATCTCGGCTCACTGCAACCTCC ACCTCCTGGGCTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCACCTGCCACCA CGCCCGGCTAATTTTTGTATTTTTAGTAGAGAGGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTC TCGACGACCTCAGTTGATCTGCCCTCCTCAGCCTCCCAAAGTGCTGGGGTTACAGGCAGGCTACACCTTT CAACAGTCCCTAAGAGGGGAGGGATGCAGACACCTGAAATCCTCAAGATGGGCTAACCTCAAGAGCCCAG AT GATGGGACACAGAAGGAAAC T T AAAAGAAAGAAGGCAC T T C T GAGATGC T T T AT GCAAAT ACAAGCAA ATGCATATGTGGATTCTTTTTGCTTTTTTTTGAGACGGAGTCATGCTCTGTCACCAGGCTGGAGGTGCAG TGGCATGATCTCGGCTCACTGCAACCTCTGCCTCCTGGGCTCAAGTGATTCTCGTGCCTCAGCCTCCCGA GTAGCTGGGATTACAGGCAAGCGCCACCATACCCAGCTTTTTGTATTTTTTTAGTAGAGACGGGGTTTCA CTATGTTGGCCAGGATGGTCTCAATCTGACCTTGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGAT TACAGGCGTGAGCCACCGCGCCCAGCCTTCTCTTTGTTCTTTTTTGCTCTATTCATGCCTTGTTTTTCCC CCTTTGCAGTCTGCCTTGGAGATCATTTCACCATCAGCTCAGAAGATCTTCTTTCTCTGTTTTCAGCCAC ACAGCGTTTCATTGTAAGGACACACCATAACGTACTTGACAAGTCTTTGTTGGTTGCTAGTTTAGAACGT GTGCTCTTACCGGTGTTAAGGCGTCTCGGAGTGGGACGGTTCCGGGGTCACCACAGCCTTCAACTGTCAC ATGGCACAAAGGCCTCATGCATGGATTCATTCCAGGGATTCCTGTTTAGCAAGCGTTGCGTACCTTGGCT TACAGCGGCCTCAGCCCTAAAGGAGGTCAGTCAAGTAAAGGAGTCATACAAGAAAATAAGAATTGCAAAG TTAAAATATTGTATAATGGGCCCTGTAAGCAGAGGGGGAAGAGAGGCCCATGGGGAGAGTCACCACCCAT CAGAACCACCTGTCGGGGAAGGCTTCACTCCCTATTTTCACCCCCAACCCTATTTTTCCCAAGTCTTCTC TGTGTCAGGAAATAACACCCCATTCCCCCAGCTGCCCAAGTCAGGGTCAGTTCATCAGGAAAGGGATATC AGACCATGTCACTCCCTGATTAAAACCTTCAAGAGCTTCCCACTCTGAGTGTAAAGGCCTTAGAACGAGG CCTTGTGTGCTCTGGCCCCTGAATGCCCCCTTTGCAGGCACAGCAACCCTTTCGAAGCCCTCAGGCACAA TGGGGTCCTTCTTTTTTTTTTTTTTTTCTGAGACGGAGTTTTGCTCTTGTTGCCCAGGCTGGAGTGCAAT GGCGTGATCTCAGCTCACCGCAATCTATGACTCCCAGATTCAAGCAATTCTCCTGCCTCAGCCTCCCAAG TAGCTGGGATTACAGGTGCCCACCACCACATCCAGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCA TCATGTCAGCCAGGCTGGTCTCAAACTCCTGACCTTAGGTGATCCACCTGCCTCAGCCTCCCAAAGTCCT GGGATTACAGGCATGAGCCACCCTGTCTGGCCTTTTATTTGTTTATTTATTTTTATTTATTTATTTATTT TTGAGGAGTTCTTGAAGTGCCACGCAATGCCTCTCCATGTCTATCTACCCCAGTTCCCGACTCCTAGCAC CACATCTGGCACATCATAGGTGGTCAGCATTTATTGGATGAATGAATGGATCAATATTTGATTGAGTCTT GAAGTACGAATAGAATTTGGCGAAGTGGACAAGGCAGGAAATGTGTATTCAGGCATGAGGCTTAGCTTGT GCATAGGTAGAAAGGCATGAAAAAACATGGAACATTCTCGAATCCACAAATAGTAAGAGAGGGCTAAGTC GTTGTGTGGGGGAGAGGAGAAATGAAGTTCAAATGGTAGGCAAAGGTCAGTGGGCAAGGGCCTTGTAAAT TGCACTTGAATTTTATTCTTTAGGCAAAAGGAAGAGTTTAAGGGTTTCAAGAAGAAGGACACGACCAGTG GG T AGGAAGAT CATTC GATGGCCTTT CAGAGGAT GGCT GAGGAGAGC AGCAGGGAGGCAGGGAGG TCGAGTCAGGGGATTTTGACCAGGCCAGGTTAGGTGCTATGAGGCCCGGGCTTTGGAGAAGAACCAGGAA ATGGCAACTGGAGGCAAGAAAGGGGAGAAGGCCAAAGCCTGGGAACCATCAACACTTAAGAACAGTAAAG AAT AAACATC T AAAGAGAACAAGAAGGAAT GGCCAGAAGAAGGAAAACAGGAAAGAAC T GAGGCGT GGGA GCGCAAGAGAGAGAATGGTTCGAGAAGGAAGCAGTCACAGGTGTCAAGGCCATTGGGAGGTCAAGTGAGA TAAGGACTGAAGGTCCCACCAATTGGGCCATCAGGAGCACAGGACTTGAGAGCACTTCTGGGTGGGAACA GACCCAGCCTGGGGCAGGGCTCAGGTTTCTGATGGGAATTCTGGAAGGGTCCCTTTTATTCAACATGCTT CAATCCAGGGGCCCCCGAAGTCTGACCACAGCAATGCTCCAAACCATGTGTCTTTCCTGGCTTAAGGTTC AGTCGCCCTCCTCAGAGGGGAGCCTATGAAAGAGCCCAGTGGAGTGTCAGGGTCCTGAGTCCTAGTCCTA GTCCTGTCCCTGCCACTTGTGAGGGAACTTGGGCCTCAGTTTCTCCAGGTGGGCTCCACAATTGCTTCTC TTGATCTGGACTGCCCCAGTGCCCAGGTTCAGTGAGTGACACAGGCAGCTGGGTTTCCACATCCTCTGAC TTGGGTTCCCTTCACTGCCTCCAGGCAGGGCTCGGCCCTCCACCCCCAAGTGGCCCATTGTGTGAGCTCA GTTTCAGTGGGGACAGAAACTGGGTTGAGAAAAGGGAATATTTACCTATCCCACCAAGCCAATGCCAAGT AAATAGTGCAGTATCTTATGTAGAGCCCTTGCCCTGCCCTTCCCCATCTGGGTGCTGCTGCCTAGAGCAT ATAAAAGGCACCTTGCTGGGCATGTCTCATACTAGCCCACCAGACTCAGAGACGGAACCAGAGACGGGCC AGAGCATCCCCCTCCTCCACCATGAAACTCGCTGTCACCCTCACCCTGGTCACACTGGCTCTCTGCTGCA GCTCCGGTGAGTGCTCAGAGACCCTTCCCTCCCTCCTGGACTTAGGAACTCTCAGGACCCCCCAGTTCTG CTCAGAAGAAGGAGTGAGCTGCCCATTCCTGCTCTGGAGCTGCTGGGAGGACCTGGGCATGCTGAGTCTC AGAAAACTGGGTCTGGTGAGCAAGCTCATCTTGGAAACTTGGAGAGAGCCCCAGGCTGTAAGGAAGCCTA AAAAGGGTCCCATCTTCTATATCCAACAACCCTCAGAATCCCAGGGAATGGAATAGCCTGGAGGGAGGAG TGGAGAATACCCCATAAAGATGAGTACATCCAGCATAGGAATAATGAGGCCCTCATCCCAGATCTGGACA GACTCCAAGATGCTGAGACCTTGGTGCAGCCTCCAAGTCTGGGGTCTCCACTCCATGCTGGCAGCTGAAG TCACTGGAAGGGGAGCTGCAGGGACTCGTGACCCCAAAAGAAACCCAACCCAAGACAAGGTCTCTCATTC TGGGCACAGAGGAATATTCCAGAAAGAGAGCTTCCCTTTGGGGAACTGCCAACCCAGAGTGAAGTTTTCT AAACATTTCCGTCCTCTGCAAAAGGGATTAGGAGTCTCTGAGTAGTTGCTGCTGTCACTAAAAGGAAAAG AAC TGT GGGGGGAAGAGGGGCAGAAAGAGAGACGGAGAGAGGGGGAGAAAGGAAGGAAAGAAGGAT CACA GCTCTCTCCAAGATCCCCCGTCTTTGGGGAACTGGGTTATCTAACTCTGTTTTTCACTCTGCGTCAGCCT CTTCCATTCTCATCTGAAAATGCTGTTGTTATTTTTTAATAAACAAACTCCAATTAATTCACTTGGAAAG CTTCACAACACCCATGGAGATAAGTTTTATGACCCTGGGGAGTTAGAAAACCCAAACCAAGAAGCAGTAG GAACAACTATTTTGCAGAGAGGTTTATTTGTTTTTCAGAGAAAATGACATCATTTTGGACTGAAATGTGT ATTAATTAGAAGATCTCAGTGCTGTCTGCGTACAGAGGTGGGTGGCTGAGCAAGATAGGACTGCAACATG TTAAGGGGTGGGTCAGAGATGCATTTGTCTATTTGTGTGCACATGCATACAATATTTAACACTTACTTCA CACAATGTGCCAATCACTGTCACCCTTTCACATAATATCTCTTTTCATTCTTTTTTTTTTTTTTTTTTGA GACAGAGTCTCGCTCTGTTGCCAGGCTGGAGTGCAGTGGCGCGATCTCAGCTCACTGCAACCTCCGCCTC CCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGGTGGGATTACAGGCACGCACCACTGCACCC AGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGATGGTCTCCATCTCTTGAC CTCGTGATCCACCCACCTAGGCCTCCCAAAGTGCTGGGATTAGGCGTGAGCCACCATGCCTGGCCTCTCT TTTTATTCTTACAACAACCCTATGAAGTAGGGATATTGGGCCAGGCACGGTGGCTCACGCCTGTAATCCC AGCAATTTGGGAGGCCGAGGTGGGTAGATCACTTGAGGTCAGGAGTTCAGGACCAACCTGGCCAACATGG TGAAACCTTGTCTCTATTAAAAATACAAAAATTAGCCAGGCATGGTGGCGCATGCCTGTAGTCCCAGCTA CTTGGGAGGCCGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTGCAGTCAGCCGAGATGGCATC ACTGCACTCCAGCCTGGGCAATAGAGCGAGACTCCGTCTGAAAACAAATAAATAAATAAAAATAAAATTT AAATTAAATTAAAATTTAAAAAATAAAAAATAAAATGAAGTAGGGATATTGTTCCCATTTTACAGATGAG AAAACTGAGCTACAGAAACACAGAGTGACTTGCCTGGTGACACAGTAAGTTACACATCAAGGACCTAAGT TCTGGAGAGGGTCTGACTTGGAGTGGCAATTTCTAGTGAGGCCCTAGAGTCAGAGGAGGGAAGGCAAATT TGTTCAGAAGGCAGAGAATTCAAGGAAAAGGGATTTGAGACTCACTGGGAAGATGGAGGCAAGCAGTGGG TAGAAAATGGTGACTTTCCCCCATGTTCCTGGTTGTAAGGACCTGAGAAGAAAACAGAGTCTGGAAGCTC TGTGTTGAAGGGAATGAAGTGGTACAAGTGGCTGCTCTGTCCATGAGCTGAGTGTGCCACAGGGCCCGCT GTGCACATGTGCACACCTCTTCCCGGCCAGGTTCGGGGGCCCATGTTTGGCTGGTACAATCTCAATGGCT TCTTTTCTTTTCTTTTCTTTTCTTCTTTTTCTTTTCTCTTGCTTGCTTGCTTGCTTGCTTGCTTGCTTGC TTTTTGAGACAGAATCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGACGAGATCTCAGCTCACTGCAACT TCTGCTTCCTGGATTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGTAGCTAGGATTACGGGTGCCCAGAA CCACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGA ACTCCTGACCTCGTGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGTGC CTGGCTTACAATCGCTTTTTCCTGCCAGAGCCTGAATTTGTCACATGCCCCCAGTGAAGCATGGCTCAGG GCATCTCTAACCACTGATGAGAGGCAAGTTTCTGGTGGGAAATAAAACCCTCAGTGGCCTCTTCCCAGCC TCCACACTGCATTAAAAAATCAGGCCAGCAGCTTCTATGATCAATACTCTGCCTTGATCTCCAACAGAAA GAAAAACGGCACCTTGCTCACCTCAACCCAAGAAGTCTAAGGAAGACTCGGGCAATCCACAAATCTTACA CTCTAGTCCATCGATGAAAAGGCTGCTATCTCTCGCTGATGGGCCTGGCTGTTTGCATCTGGGCAGACCC AGCCAGAGGGCTAGCCAGCTTGGAAAGGGGCCTGGAGACATGTGCCTTCTCTCCTCTGTGTTGCAGCTTC TGCAGAGATCTGCCCGAGCTTTCAGCGTGTCATCGAAACCCTCCTCATGGACACACCCTCCAGTTATGAG GCTGCCATGGAACTTTTCAGCCCTGATCAAGACATGAGGGAGGCAGGGGCTCAGCTGAAGAAGCTGGTGG ACACCCTCCCCCAAAAGCCCAGAGAAAGCATCATTAAGCTCATGGTAACCAGCACCTTTCACGTCACACT GGTTAGAAGTGGCTTCCCCAAGTGGGGCTGCAGGATTGCCCCAGTTTTCAGACCTGTTTCTAATCCAGAG AGGAGAGTCACAGTGCCACTGTCCCCAGGCAGGCAGCACAGTGATCTTTCTAGACATCTCCTCTTCCTTT TT TTT TTT TTT TTTGAGACAGAGTCTCGCTCTGTCGCCCAGACTAGGGTGCAATAGCACGATCTTG GCTTACTGCAACCTCCACCTCCCAGGTTCAAGCGATTCTCCGGCCTCAGCCTCTTGAGTAGCTGGGATTA CAGGCACCCACCATCATGCCGAGCTAATTTCTGTATTTTTGTAGAGATGGGGTTTCACCGTGTTTGCCAG GCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGCATTACAGGCG TGAGCCACCACGCCCAGCCTCCCCTTAC TAT TTTGTAAGAGGCTT TGAGAAACAATCCAAGCCC TACT ACCTTAGTTCCTCCTAGAGTTGACTGCACCTCTCGGTTAATGTTGAAGTTTCTGTGGCTCGTCATCTCTG CCTAACTATGCAATTCATTCACTGTTGTATTGGGTTTTTCTGTTTCTTTGTCTATTTGTTTTAGGAAAAA ATAGCCCAAAGCTCACTGTGTAATTAGCATTTAGAAGCTGAAGATCCCCAACTGCTCCAGCCTCTGCCGC TGCCATGCTTTGAGTCCACGCCCACCAGCCTTGCTCTCTTCAATAAACCACAAGCATCTCACTTTTGTGC CCCCTGCGTGGTTTTGGTCTCCTTGTTATGGTGGGGGGTCTGCCCCTGTCAGGGTAAGGTGAGCCAGGAC AAAAGGAAAGCAGCCGAGCTCTGCCCTTGGATCCCAGCAGAAATCTCTGTGCTCTTCCAAAGCTCTTTTC CTCTAGGTTTCTGCCTCCAACAGAGCTCAAGCCCCCTCCCCTTATATCAGACCATCTACTCAACATCACA GACTCTACTCAACATCACAGACTCCACTTGTCCAACAGAACCAGTGATCCTGCCCCAGTCTGTTCCTATC TCAGTGAGTTCTGGCTCCATCCTCCCCGCTGGGCTGCACCAGCCTGAGGAGCATCCGCGATCCTTCCTTC TCTAGCCAACCCAGGCTACCTCTAAAGGGCCTCTGGAATCCTTCACTCCAAGTCCACAGGCAACACCCCC ATCCCTGCCTGTACAGCCCCCGGGGCCTCCTCTCTGGCTCCCAGCCAGACTCCAGGGCCCCCACAATCCC TTCTCCAACAACCCAAAGGAATTCTTCTCAAGTGCAAAGTCCCTCCCCATCCTTCAAAGCTTTCTAGGGC TCTTATTGTAAAATCCTAAATCCTGACGATAGATAGTGGCCCCTCACCAGTCTTTCCTGTGCTCCTCCTT CCCACGGCTCCTTCATTCTGCTCAGTCCCCACACACACCCCCACCTTCCCTGCCCTCAGTTTTTAAAAAG AGCCAAGCCCCTTTCAACCTTAAGACCTTTGTACCAGCCACACAGCCCTGGCTGCTCAAAAAGTGTGGCT GATGGCCAGGCACGGTGGCTCACGCCGGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGAGGATCACCTG AGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAATATACAAAAAATTA GCCAGGTATGGTGGTGCGTGCCTGTAATCCCAGCTACTCGGGTGGCTGAGGCAGGAGAATCACTTAACCT GGGAGGCAGAGGTTGCAGTGAGCTGAGATCGTGACACTGCACTCCAGCCTGGACAACAAGAGTGAAACTC CATCTCAAAAAAAAAAAAAATTGTGGCTGATTGGTTTTTAAAATTGCTCTAGAGCAAAATGTGCAAAGAA TAGAACTCATCCTAAAGATGCATCCATGTGGATATAAAGACCCCCAAATCCCCGGCCAGTTTCATGGCAG CCAGCCCCCTGCTGGGCCACATTATCCACAGCACCTAAATAGAAGCGGAGGATTGGCTGGGCTTGGTGGC TCATGCCTGTAATTCCAGCACTTCGGGAGGCTGAGGTAGGAAGATGGCTTGGGCCCAGGAGTTCAAGACC AGCCTGGCAACATAGTGAGACTCCATCTCTACAAAAATAAAAATAAATTAGCCGAGTTTGGTAACGCATG CCTGTAGAGCTGGCTACTCTGGAGGCTGAGGCAGGAGGATCTCTTGAGTCCAGGAGTTCCAGGCTGCAGT GAGCTATGATCATGCCACTGCACTCCAGCCTGGGTGACAAAGGTGAGACCCTGTCTCAAAAGGAAAGAAA GAAAAAGGTGGAGGACTGCAGAGGGGCTGCAGGAATCGGAAGGGCGTCGGCAGAGGGCACGGAAGGAGGC GGCGGGTTAGAGACGGAGCTCTCCAGTTCGAGCCCCTCCATGGAGCAACGTGGTCCGGTGGGCCTTGTGC CCCGCGGCCACTAGGGGGCAGCATCGTCCACAGGGTCCTGGAGCGGACGCGCTGTCGGGGCTGAGGCACC CAGAGCTCCGGCTCTGAGGATCTGGTGTCATCCGTATAATTTTTTAAATCTCGGCCGTGCGCGGTGGCTG ACTCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACCTGAGGTCGGGAGTTCGAGACCAG CCTGTCCAACATGGTGAAACCCCATCTCTACTAAAAATAAAAAATTAGCTGGGTGTGGTGGCGCGCACCT GTAGTCCCAGCTATTTGGGAAGCTGAGGCAGGAGAATCGCTT

As used herein, the term "CCSP" has its general meaning in the art and refers to Club Cell Secretory Protein secreted by Club cells (previously named Clara cells) in lung. This protein is known by many names including: secretoglobin family 1A member 1 (SCGBIAI, current gene symbol), club cell protein (CC16 or CC10), human protein 1 (PI), urine protein 1, uteroglobin, and blastokinin. The Human protein CCSP (mass: 15.8 kDa) secreted by Club cells is a homodimer without sugar residues; each unit is composed of 70 amino acid residues bound with covalent bonds. Its structure includes a hydrophobic pocket for binding lipophobic ligands. It is resistant to the action of proteases, low temperature, and pH changes. CCSP abundant in the airways has anti- inflammatory and immunomodulatory properties besides playing a role in host defense and control of oxidative stress. Uniprot reference is PI 1684.

Example of Homo sapiens Club Cell Secretory Protein amino acid sequence (SEQ ID

NO:2):

MKLAVTLTLVTLALCCSSASAEICPSFQRVIETLLMDTPSSYEAAMELFSPDQDMREAGA QLKKLVDTLPQKPRESI IKLMEKIAQSSLCN

As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. The term "treatment" encompasses the prophylactic treatment. As used herein, the term "prevent" refers to the reduction in the risk of acquiring or developing a given condition.

The term "preventing" as used herein refers to minimizing, reducing or suppressing the risk of developing a disease state or parameters relating to the disease state or progression or other abnormal or deleterious conditions.

A "sample" in the context of the present invention is a biological sample isolated from a subject or an organ ex vivo and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue. In a preferred embodiment, the sample to be tested is blood or lung biopsy. As used herein "blood" includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.

The term "organ" as used herein refers a group of several tissue types that perform a given function. Exemplary organs include, but are not limited to heart, kidney, liver, pancreas, and lung.

As used herein the term "lungs" has its general meaning in the art and refers to saccular thoracic organs that constitute the basic respiratory organ of air-breathing vertebrates. "Risk" in the context of the present invention, relates to the probability that an event (i.e PGD) will occur over a specific time period. The term "risk of having or developing a primary graft dysfunction" as used herein refers to the predisposition of lungs to suffer or develop a primary graft dysfunction.

As used herein, the term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes.

The term "mutation" or "allelic variant" means a sequence variation of a gene. Allelic variants can be found in the exons, introns, untranslated regions of the gene, or in the sequences that control expression of the gene. Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype occurs in a sufficiently large population.

As used herein, the term "single nucleotide polymorphism", or "SNP", refers to single nucleotide position in a genomic sequence for which two or more alternative alleles are present at appreciable frequency (e.g., at least 1%) in a population.

The term "graft" as used herein refers to the tissue and/or organ derived from a donor for transplantation into a recipient.

The term "donor" as used herein refers to the subject that provides the organ and/or tissue transplant or graft to be transplanted into the recipient and/or host. The term "potential donor" refers to a subject which is likely to provide the organ and/or the tissue transplant or graft to be grafted. In others words, a potential donor may provide or not the organ and/or the tissue transplant or graft to be grafted.

The term "recipient" or "host" as used herein refers to any subject that receives an organ and/or tissue transplant or graft.

The term "ex vivo" means that which takes place outside an organism. An "ex vivo organ" refers to an organ which has been removed from an organism. "Ex vivo lungs" refers to lungs which have been removed from an organism. "Ex vivo" lungs can be stored on ice (topical cooling), perfused through the pulmonary artery and/or the left atrium using cellular or acellular solution, and/or ventilated through the trachea, in hypothermia or in normothermia.

Prediction method:

For the first time, the inventors studied the link between the presence of a SCGB1A1 polymorphism (such as G38A) in the donor or the recipient, and ultimately the occurrence of PGD. Indeed, the inventors put forth the hypothesis that the concentration of CCSP in the lung or the blood of the donor or the recipient might play a role in the answer to the epithelial aggression related to the phenomena of ischemia-reperfusion injury in pulmonary transplantation, in other words as mediators or indicators on the potential occurrence of severe PGD. For the first time, they showed that the presence of a AG genotype in SCGB1A1 gene (G38A polymorphism) in the donor was associated with a decreased frequency of PGD in the recipient after lung transplantation.

A first aspect of the present invention relates to a method of identifying lungs harvested from a donor and to be grafted to a recipient, having or at risk of having or developing a primary graft dysfunction (PGD), comprising determining, in a sample obtained from said donor or from said lungs, the presence or absence of a single nucleotide polymorphism (SNP) located in SCGB1A1 gene.

In a particular embodiment, the SNP is selected from the group consisting of SCGB1A1 dbSNP rs3741240 G>A and: - the presence of the allele (A) of SCGB1A 1 dbSNP rs3741240 G>A indicates a decreased risk of having or developing primary graft dysfunction.

In a preferred embodiment, the sample is a blood sample or a lung biopsy.

According to the invention, the determination of the presence or absence of said SNP may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridation, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said variant may be detected in a DNA sample, preferably after amplification. For instance, the isolated DNA may be subjected to amplification by polymerase chain reaction (PCR), using specific oligonucleotide primers that are specific for the SNP or that enable amplification of a region flanking the SNP. According to a first alternative, conditions for primer annealing may be chosen to ensure specific amplification; so that the appearance of an amplification product be a diagnostic of the presence of the SNP according to the invention. Otherwise, DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. Actually numerous strategies for genotype analysis are available (Cooper et al, 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype of the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase- PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real- time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalentlyjoin the two oligonucleotides only if they are perfectly hybridized to one of the allele.

Therefore, short DNA sequences, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the one of the allele of the polymorphism.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides.

Kit of the invention

A second object of the invention is a kit suitable for identifying whether lungs have or are at risk of having or developing primary graft dysfunction, comprising:

- at least a means for detecting the SNP selected from the group consisting of SCGB1A1 dbSNP rs3741240 G>A and

- instructions for use.

In one embodiment, the kit according to the invention comprises: - at least one primer and/or at least one probe for amplification of a sequence comprising a SNP consisting of SCGBIAI dbSNP rs3741240 G>A,

- instructions for use.

In one embodiment of the invention, the primer or probe may be labelled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.

Examples of primer sequences for PCR amplification of the SCGBIAI gene are: SEQ ID N°3: 5' - TCCCTTCACTGCCTCCAG - 3' (forward : 18nt)

SEQ ID N°4: 5' - CTCCTCCCTCCAGGCTATTC - 3' (reverse : 20nt)

Therapeutic methods and uses

As previously mentioned, the inventors showed that the presence of a AG genotype in

SCGBIAI gene (G38A polymorphism) was associated with a decreased frequency of PGD. Thus, in the case of the GG genotype identification associated with high risk of PGD, it would be decided to administer the donor or the recipient or to deliver to the ex vivo lungs an appropriate treatment(s) for the prevention or treatment of PGD.

In one embodiment, the present invention relates to a method of grafting lungs comprising the following steps:

detecting the presence or the absence of the allele (A) of SCGBIAI dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs; grafting said lungs on the recipient when the presence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of CSSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising delivering to ex vivo lungs a therapeutically effective amount of CSSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for preparing ex vivo lungs comprising delivering to said ex vivo lungs a therapeutically effective amount of CSSP protein or functional equivalent thereof. In a particular embodiment, said ex vivo lungs are maintained into a device comprising an organ container filled with a preservation solution.

detecting the presence or the absence of the allele (A) of SCGBIAI dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs; administering to said donor a therapeutically effective amount of CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected;

grafting said lungs on the recipient.

detecting the presence or the absence of the allele (A) of SCGBIAI dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs;

grafting said lungs on the recipient;

administering to said recipient a therapeutically effective amount of CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected.

delivering to said ex vivo lungs a therapeutically effective amount of CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected;

grafting said lungs on the recipient.

As used herein, a "functional equivalent of CCSP" is a polypeptide in which a given amino acid residue has been changed without altering the overall conformation and function of the CCSP protein. The term "functionally equivalent" thus includes any equivalent of CCSP obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions (replacement of an amino acid with one having similar properties such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like) or additions such that the protein analogue retains the function of CCSP. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence. The term "functional equivalent" includes fragments, mutants, and muteins of CCSP.

In some embodiments, the functional equivalent of CCSP is at least 70% homologous to the amino acids sequence SEQ ID NO:2.

In some embodiments, the functional equivalent of CCSP is at least 80% homologous to the amino acids sequence SEQ ID NO:2. In some embodiments, the functional equivalent of CCSP is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acids sequence SEQ ID NO:2.

In a preferred embodiment, the functional equivalent of CCSP is at least 90% homologous to the amino acids sequence SEQ ID NO:2 as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).

As used herein, the terms "homology" or "homologous," used in reference to polypeptides, refers to amino acid sequence similarity between two polypeptides. When an amino acid position in both of the polypeptides is occupied by identical amino acids, they are homologous at that position.

The amino acid changes may be achieved by changing codons in the DNA sequence, according to Table 1.

Table 1

Amino acids Codons

Alanine Ala A GCA, GCC, GCG, GCU

Cysteine Cys C UGC, UGU

Aspartic Acid Asp D GAC, GAU

Glutamic acid Glu E GAA, GAG

Phenylalanine Phe F UUC, UUU

Glycine Gly G GGA, GGC, GGG, GGU

Histidine His H CAC, CAU

Isoleucine lie I AUA, AUC, AUU

Lysine Lys K AAA, AAG

Leucine Leu L UUA, UUG, CUA, CUC, CUG, CUU

Methionine Met M AUG

Asparagine Asn N AAC, AAU

Proline Pro P CCA, CCC, CCG, CCU

Glutamine Gin Q CAA, CAG

Arginine Arg R AGA, AGG, CGA, CGC, CGG, CGU

Serine Ser S AGC, AGU, UCA, UCC, UCG, UCU

Threonine Thr T ACA, ACC, ACG, ACU

Valine Val V GUA, GUC, GUG, GUU

Tryptophan Trp w UGG

Tyrosine Tyr Y UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of protein function. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the polypeptide sequences of the invention, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity.

In making the changes in the amino sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: iso leucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (- 3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.

Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. In order to produce sufficient amounts of polypeptides or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. In particular, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

The polypeptides of the invention and fragments thereof can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N-linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine).

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising delivering to ex vivo lungs a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for preparing ex vivo lungs comprising delivering to said ex vivo lungs a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof. In a particular embodiment, said ex vivo lungs are maintained into a device comprising an organ container filled with a preservation solution.

detecting the presence or the absence of the allele (A) of SCGBIA1 dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs; administering to said donor a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1AI dbSNP rs3741240 G>A polymorphism in said sample is detected;

grafting said lungs on the recipient.

detecting the presence or the absence of the allele (A) of SCGB1A1 dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs;

grafting said lungs on the recipient;

- administering to said recipient a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1AI dbSNP rs3741240 G>A polymorphism in said sample is detected.

- detecting the presence or the absence of the allele (A) of SCGBIA1 dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs;

delivering to said ex vivo lungs a therapeutically effective amount of a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1A1 dbSNP rs3741240 G>A polymorphism in said sample is detected;

- grafting said lungs on the recipient.

The vector used according to the present invention comprises a nucleic acid sequence encoding for CCSP protein (SEQ ID NOT) or a nucleic acid sequence encoding for a functional equivalent of CCSP protein.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. The vectors may further comprise one or several origins of replication and/or selectable markers. The promoter region may be homologous or heterologous with respect to the coding sequence, and provide for ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate host cell, including for in vivo use. Examples of promoters include bacterial promoters (T7, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV- IE, etc.), mammalian gene promoters (albumin, PGK, etc), and the like.

Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for treating or preventing primary graft dysfunction comprising delivering to ex vivo lungs a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

In one embodiment, the present invention relates to a method for preparing ex vivo lungs comprising delivering to said ex vivo lungs a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof. In a particular embodiment, said ex vivo lungs are maintained into a device comprising an organ container filled with a preservation solution.

- detecting the presence or the absence of the allele (A) of SCGB1A1 dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs;

administering to said donor a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected;

grafting said lungs on the recipient.

grafting said lungs on the recipient;

administering to said recipient a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected.

delivering to said ex vivo lungs a therapeutically effective amount of cells which have been transduced or transfected with an amino acid sequence encoding for CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGBIAI dbSNP rs3741240 G>A polymorphism in said sample is detected;

grafting said lungs on the recipient.

The cells used according to the invention are transduced or transfected cells with CCSP amino acid sequence or CCSP functional equivalent amino acid sequence or a vector comprising a nucleic acid encoding for CCSP or a CCSP functional equivalent.

As used herein, the term "transduction" refers to the delivery of a gene using a retroviral vector particle by means of infection, in particular, introduction of a gene carried by the retroviral vector into a cell via lentivirus or vector infection and provirus integration.

The term "transfection" as used herein refers to the use of methods, such as chemical methods, to introduce exogenous nucleic acids, such as the synthetic, modified RNAs described herein, into a cell, preferably a eukaryotic cell. As used herein, the term transfection does not encompass viral-based methods of introducing exogenous nucleic acids into a cell. Methods of transfection include physical treatments (electroporation, nanoparticles, magnetofection), and chemical-based transfection methods. Chemical-based transfection methods include, but are not limited to, cyclodextrin, polymers, liposomes, and nanoparticles.

In one embodiment, the transduced or transfected cells used according to the invention are mesenchymal cells.

As used herein, the term "mesenchymal cells" has its general meaning in the art and refers to cells from mesenchyme tissue.

In one embodiment, the transduced or transfected cells used according to the invention are mesenchymal stem cells.

As used herein, the terms "mesenchymal stem cells" or "MSCs" has its general meaning in the art and refers to multipotent stem cells that can differentiate into a variety of cell types such as cartilage chondrocytes, osteoblasts and fat cells.

The transduced or transfected cells used according to the invention are obtained for instance from bone marrow, umbilical cord, peripheral blood, fallopian tube or cells banks.

In one embodiment, the transduced or transfected cells used according to the invention derive from the donor. In one embodiment, the transduced or transfected cells used according to the invention derive from the recipient. In one embodiment, the transduced or transfected cells used according to the invention derive from a person other than the subject who received the cell therapy.

In one embodiment, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administered to the donor, before the lungs transplantation.

In one embodiment, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administered to the recipient, after the lungs transplantation.

In one embodiment, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are delivered to the ex vivo lungs, before the lungs transplantation. In a preferred embodiment, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administered to the donor or delivered to the ex vivo lungs, before the lungs transplantation.

In a particular embodiment, the ex vivo lungs are perfused with a preservation solution comprising the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

In a particular embodiment, the ex vivo lungs are preserved into a device comprising an organ container filled with a preservation solution comprising CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

As used herein, the terms "preservation solution" or "organ preservation solution" refer to an aqueous solution having a pH between 6.5 and 7.5, including salts, preferably chloride, sulfate, sodium, calcium, magnesium and potassium; sugars, preferably mannitol, raffinose, sucrose, glucose, fructose, lactobionate (which is a water resistant), or gluconate; antioxidants, for instance glutathione; active agents, for instance xanthine oxidase inhibitors such as allopurinol, lactates, amino acids such as histidine, glutamic acid (or glutamate), tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran.

In one embodiment of the invention, the organ preservation solution is selected from:

- the solution from the University of Wisconsin (UW or ViaSpan®), which has an osmolality of 320 mOsmol / kg and a pH of 7.4, of the following formulation for one liter in water: potassium lactobionate: 100 mM, KOH: 100 mM, NaOH: 27 mM, KH2P04: 25 mM, MgS04: 5 mM, Raffinose: 30 mM, Adenosine: 5 mM, Glutathione: 3 mM, Allopurinol : 1 mM, Hydroxyethyl starch: 50 g / L,

- IGL-1®, having an osmolality of 320 mOsm / kg and a pH of 7.4, of the following formulation, per liter in water: NaCL:125 mM, KH2P04: 25 mM, MgS04: 5 mM, Raffinose: 30 mM, potassium lactobionate: 100 mM, Glutathione: 3 mM, Allopurinol: 1 mM, Adenosine: 5 mM, Polyethylene glycol (molecular weight: 35 kDa): 1 g / L, - Celsior®, having an osmolality of 320 mOsm / kg and a pH of 7.3, of the following formulation per liter in water: Glutathione: 3 mM, Mannitol: 60 mM, lactobionic acid: 80 mM, Glutamic acid: 20 mM, NaOH: 100 mM, calcium chloride dehydrate: 0.25 mM, MgS04: 1.2 mM, KC1: 15 mM, magnesium chloride hexahydrate: 13 mM, Histidine 30 mM,

- BMPS Belzer® or Belzer solution infusion machine or KPS1, especially comprising

100 mEq / L of sodium, 25 mEq / L potassium, pH 7.4 at ambient temperature, and having an osmolarity of 300 mOsm / L,

- Custodiol® HTK solution having the following formulation per liter in water, the pH of 15 being 7.20 at room temperature, and the osmolality was 310 mOsm / kg: NaCl: 18.0 mM, C1: 15.0 mM, KH2P04: 9 mM, 2-ketoglutarate hydrogenated potassium: 1.0 mM, hexahydrate magnesium chloride: 4.0 mM; histidine, HCl, H20: 18.0 mM, histidine: 198.0 mM, Tryptophan : 2.0 mM, Mannitol: 30.0 mM, calcium chloride dihydrate : 0.015 mM

- Soltran®, having an osmolality of 486 mOsm / kg and a pH of 7.1 and the following formulation per liter in water: Sodium: 84 mM, Potassium: 80 mM, Magnesium: 41 mM, Sulfate: 41 mM, Mannitol 33.8 g / 1, Citrate: 54 mM, Glucose: 194 mM,

- Perfadex®, having an osmolarity of 295 mOsmol / L and the following formulation in water: 50 g / L of Dextran 40 (molecular weight: 40,000), Na + 138 mM, K + 6 mM, Mg2 +: 0.8 mM, CI - 142 mM, S042 0.8 mM, (+ H2P04- HP042-): 0.8 mM, glucose 5 mM,

- Ringer lactate®, of the following formulation, in water, the pH being between 6.0 and 7.5 at ambient temperature, and having an osmolarity of 276.8 mOsmol / L: Na + 130 mM, K

+ 5.4 mM, Ca2 +: 1.8 mM, C1-: 11 1 mM, Lactate: 27.7 mM,

- Plegisol®, of the following formulation, in water: KCI: 1.193 g / 1, MgC12, H20: 3.253 g / L, NaCl : 6.43 g / L, CaC12: 0.176 g / 1,

- Solution Hospital Edouard Henriot, of the following formulation in water, the pH being equal to 7.4 at ambient temperature, and having an osmolarity of 320 mOsmol / L: OH: 25 mM, NaOH: 125mm, KH2P04: 25 mM, MgC12 : 5 mM, MgS04: 5 mM, Raffmose: 30 mM, lactobionate: 100 mM, Glutathione: 3 mM, Allopurinol: 1 mM, Adenosine: 5 mM, Hydroxyethyl starch 50g / L,

- And Steen® solution comprising human serum albumin, dextran and extracellular electrolyte with a low concentration of potassium.

All these organ preservation solutions are commercial products.

In a particular embodiment, the preservation solution according to the invention is the solution from the University of Wisconsin (UW or Viaspan®). Pharmaceutical compositions

In some embodiments, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administered to the subject or delivered to the ex vivo lungs with a therapeutically effective amount.

By a "therapeutically effective amount" is meant a sufficient amount of CCSP protein or functional variant thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof to treat or prevent primary graft dysfuntion at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage may be varied over a wide range from 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg kg to 7 mg/kg of body weight per day.

As used herein, the term "administering" or "delivering" refers to administration of the compounds, vectors or cells as needed to achieve the desired effect. The compositions according to the invention are formulated for parenteral, transdermal, oral, rectal, subcutaneous, sublingual, topical, intrapulmonary or intranasal administration.

Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In a particular embodiment, the pharmaceutical compositions are formulated for parenteral administration. The pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

In a particular embodiment, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administrated by intratracheal or intrabronchial route.

In a particular embodiment, the pharmaceutical compositions are formulated for ex vivo lungs delivering. More particularly, the pharmaceutical compositions are formulated for intravascular route, through the perfusate (for instance the perfusate is the preservation solution) or for intratracheal or intrabronchial route.

The CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof may be combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In some embodiments, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof may be administered in combination with conventional treatment usually used for treating or preventing primary graft dysfunction, such as the parenteral corticosteroids administered to the donor before aortic cross clamp and pneumoplegia.

In some embodiments, the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof may be administered in combination with conventional treatment usually used before, during or after the organ transplantation, such as the immune suppression administered to the recipient during and after transplantation

The invention will be further illustrated by the following examples, including patients, methods, results, tables, and figures. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Survival curves in lung transplant recipients according to Primary Graft Dysfunction (PGD). Median follow-up was 1687 days (IQR 1110-1897) for the study group, 1755 days (IQR 1368-1924) for the group 1 - no PGD, and 1114 days (IQR 847-1658) for the group 2 - PGD. Log-rank (Mantel-Cox) test p-value = 0.23.

Figure 2. (A) Serum CCSP concentrations in donors and recipients (two-tailed Wilcoxon matched-pairs signed rank test, p<0.001). (B) Serum CCSP concentrations in recipients according to underlying diagnosis (two-tailed Mann Whitney test, p<0.001). (C) Serum CCSP concentrations in donors and recipients according to the occurrence of a severe PGD (two-tailed Mann Whitney test when paired, p=0.93 for donors, p=0.69 for recipients). Serum CCSP concentration remained significantly different between donors and recipients when deciphered according to PGD group (group 1-no PGD, p-value=0.0015; group 2-PGD, p-value=0.0063). Representation of Mean with SEM. Figure 3. (A). DNA sequence readout of patients and donors CCSP gene to report G38A SNP. (B). Pretransplant CCSP serum concentration according to CCSP G38 polymorphism in donors (two-tailed Mann Whitney test when paired, p). (C). Pretransplant CCSP serum concentration according to CCSP G38 polymorphism in recipients (two-tailed Mann Whitney test when paired). Representation of mean with SEM.

EXAMPLE:

Patients and methods

Study design. COLT is a French national, multicentric, prospective study initiated in 2009 with the goal to follow a cohort of LT candidates and recipients, and to identify biological risk factors of CLAD (http://clinicaltrials.gov/ identification : NCT00980967). This cohort is associated with a collection of whole blood, serum, and DNA harvested from the donor and the recipient at various time points pre- and post-transplantation. Our study included all donor and recipients involved in the COLT study, who underwent single or double LT between January 2009 and December 2014, with at least one pretransplant serum and DNA samples available from the donor and the recipient (n=480). Patients with missing clinical data, missing donor or recipient pretransplant samples, heart-lung or lobar transplant, were then excluded (n=376). All together, 104 patients completed the study criteria and account for the study group. For each recipient included in the study, demographic characteristics, lung disease diagnosis and severity, and associated comorbidities were retrieved. For each donor, demographic characteristics, tobacco use, cause of death, and lung graft assessment were collected. For each procedure, the type of graft allocation (regular vs high emergency), and the type of LT performed (single vs. double) were recorded. For each donor and recipient, peripheral blood both was collected by participating centres at the time of transplantation and centralized. The main outcome was severe PGD, defined as the occurrence of a Grade 3 PGD at anytime during the first 72 hours following lung transplantation. The study protocol has been approved by COLT scientific committee on June 17^th, 201 , and by the Greater Paris Committee for Patients ' Protection (Comite de Protection des Personnes - He de France 1) on December the 13^th' 2015.

CCSP G38A (rs3741240) polymorphism assessment. Genomic DNA was isolated from peripheral blood samples using a DNA extraction kit (Qiagen^®). Genomic DNA was normalized to a concentration of 20 ng^L for a 500 volume and stored in 96-well plates. Specific PCPv amplification of the CCSP coding gene SCGB1A1 (5'UTR of exon 1) was achieved as follows: 40 ng of genomic DNA was added to a PCR master mix containing H₂0, MgCi2 25mM, dNTP mix 5mM, lOpmol of forward and revers primers, Buffer Gold 10X and Taq Gold (5υ/μ1). Primers were designed with Primer3, further verified using BLAST and SNPCheck version:3.2.1 , Reference Genome Build version:37.1 and dbSNP Build version: 141 for alternative unwanted amplifications and primer slipping. Primers used were: 5' - TCCCTTCACTGCCTCCAG - 3' (forward: 18nt) (SEQ IDN°3) and 5 ' - CTCCTCCCTCCAGGCTATTC - 3' (reverse: 20nt) (SEQ ID N°4). PCR thermocycler run following an initial denaturation at 95 °C for seven minutes the reactions were cycled 38 times through a temperature profile of 96°C for 30 seconds, 60°C for one minute, and 72°C for one minute. A final extension was performed at 72°C for 10 minutes. The products of the reaction were visualised by Caliper^® (LabChip). PCR yielded a fragment of 624 bp comprising the G38A SNP information of the patient. After confirming PCR amplification and clear blank controls, Exostar, enzymatic PCR clean up, was performed. Exostar thermocycler run following 37°C for 20 minutes and 80°C for 15 minutes followed with a FAST Sequencing reaction: adding H20, big dye, buffer and 3,2pmol primers either forward or reverse. Product was centrifuged for 5min at 2700 rpm on Sephadex G-50 Superfine (GE Healthcare) Millipore 96- well filtration plate, to enhance genotyping quality (pre-prepared a minimum of 3h before use) and collected on a sequencing plate. Plates were read on a 3130x1 Genetic Analyzer (Applied Biosystems, Hitachi) and the output of both forward and revers sequence reactions were displayed on Chromas^® 1.4 and SeqScape^® Software allowing for G38A polymorphism assessment when compared to template sequence from 1000genomes/Primer3.

CCSP serum measurements. Serum was isolated from peripheral blood samples and stored at -80°C. CCSP serum measurements were performed using the Human Uteroglobin (CCSP) DuoSet ELISA (R&D Systems). Preparation of 96-well plate coated with 2 μ^πύ in PBS of capture rat anti-human uteroglobin (CCSP) antibody (843195) and then blocked with reagent diluent 1% BSA in PBS, pH 7.2-7.4, 0.2 μιη filtered (R&D Systems Catalog # DY995) for lh at room temperature. Serum samples were diluted at 1/100, loaded in duplicates on the 96-well plate, and incubated for 2h at room temperature. Detection biotinylated rat anti-human uteroglobin (CCSP) antibody (843196) was added for an additional 2h at room temperature. Streptavidin-HRP (890803) was then incubated for 20 minutes at room temperature in the dark. The reaction was visualized by the addition of 100 chromogenic substrate (TMB) for 10 min. The reaction was stopped with 50 μΐ, of stop solution 2 NH2SO4. Absorbance at 450 nm was measured with reductions using ELISA plate reader. As a reference for quantification, a standard curve was established by serial dilution of recombinant human uteroglobin (CCSP) to allow detection ranging from 31.20 to 2,000 pg/mL. Statistical analysis. Continuous variables with normal distribution were reported as mean and standard deviation, and compared using Student t test. Continuous variable with non-normal distribution were reported as mean, median and interquartile range (IQR) and compared using Mann Whitney test. Categorical variables were reported as count and proportion, and compared using Fisher or Chi Square tests when appropriate. As donors and recipients were considered as paired binary data, matched analyses were performed when comparing variables between donors and recipients. Continuous variables were compared using paired Student test or paired Mann Whitney test as appropriate and McNemar test was used for categorical variables. Primary outcome was severe PGD, defined as the occurrence of a grade 3 PGD at anytime during the first 72 hours following lung transplantation. Multivariate analysis was performed using Generalized Linear Models. Secondary outcome was overall survival, defined as the time interval between the date of operation and the date of death or the last follow-up visit for censored patients. Follow-up information was obtained from the hospital case records, or from a questionnaire completed by the chest physician. Median time of follow-up for the study group was 1687 days (IQR 1 110-1897). Actuarial survival curves were estimated by the Kaplan-Meier method. Statistical comparisons between survival distributions were made using the log-rank test. All data analyses were conducted with the two-sided test. A p-value < 0.05 was considered significant. Statistical analyses were conducted using Prism (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com) and R software (R Foundation for Statistical Computing, Vienna, Austria, www.r-project.org).

Results

Study group. All together, 104 recipients paired with their respective donor were included, and account for the study group. Among them, 84 patients (81%) with grade 0, 1 and 2 PGD were included in group 1 - no PGD, while 20 patients (19%) with grade 3 PGD were included in group 2 - PGD. Recipients, donors, procedures characteristics as well as specific outcomes are summarized in Table 1. As compared with group 1 - no PGD, group 2 - PGD was characterized by a higher frequency of donor tobacco use (63% vs. 33%, p=0.023), and a trend toward a higher frequency of high emergency graft allocation (21% vs. 8%, p=0.12). As compared with group 1 - no PGD, group 2 - PGD was also associated with a significantly worse outcome, as witnessed by a higher frequency of postoperative ECMO support (55% vs. 0%, p<0.001), a longer stay in ICU after transplantation (median 42.5 vs. 14 days, p<0.001), a higher mortality 90 days after the surgery (20% vs. 1%, p<0.001), a trend toward a higher mortality 1 year after the surgery (20% vs. 7%, p=0.080), and an adverse overall survival (Figure 1, p=0.196). Serum CCSP concentration. Pretransplant CCSP serum concentration was significantly higher in donors (mean 33.32 ng/mL, median 22.54, IQR 9.6-43.9) than in recipients (mean 19.85 ng/mL, median 7.03, IQR 0.89-19.2, p<0.001 , figure 2.A). In recipients, pretransplant CCSP serum concentration was significantly higher in patients with interstitial lung disease (ILD, mean 45.21 ng/mL, median 43.02, IQR 28.51-57.53) than in patients with chronic obstructive pulmonary disease (COPD, mean 8.19 ng/mL, median 6.06, IQR 1.31- 37.14) and cystic fibrosis (CF, mean 18.47 ng/mL, median 4.15 ng/mL, IQR 0.11-8.70) when bronchial diseases were considered together (p<0.001 , figure 2.B). As compared with group 1 - no PGD, group 2 - PGD was not associated with any significant difference in CCSP serum concentration in donors (mean 33.85 ng/mL and median 22.29 ng/mL vs. mean 30.81 ng/mL and median 23.24 ng/mL, respectively, p=0.93) and recipients (mean 21.52 ng/mL and median 6.58 ng/mL vs. mean 1 1.85 ng/mL and median 7.71 ng/mL, respectively, p=0.69, Figure 2.C).

CCSP polymorphism. CCSP G38A (rs3741240) polymorphism was analysed in donors and recipients (Figure 3. A). There was no significant difference in the distribution of CCSP G38A polymorphism between donors and recipients, with the A polymorphism being present in 47% of the donors and 61% of the recipient (p=0.16, Table 2). We then deciphered the pretransplant CCSP serum concentrations according to the CCSP G38A polymorphism. In donors, the presence of the CCSP G38A genotype was associated with a decrease in the serum concentration of CCSP of borderline significance (GG: 43.76±30.73 ng/mL, AG: 29.47±24.38 ng/mL, AA 29.49±21.87 ng/mL; GG vs. AG, p=0.087; GG vs. AG+AA, p=0.070, figure 3.B). In recipients, there was no significant difference in the serum concentration of CCSP according to the CCSP genotype (GG: 16.23±23.43 ng/mL, AG: 24.86±43.02 ng/mL, AA: 27.59±46.21 , p=0.73, figure 3.C). Interestingly, a very significant difference was observed in the serum concentration of CCSP between GG homozygotes donor vs. recipient (p<0.001) but not between AG heterozygotes donor vs. recipient (p=0.073) nor between AA homozygotes donors vs. recipients (p=0.33). When deciphering donors' genotypes by PGD outcomes, the presence of CCSP AG genotype was associated with a decreased frequency of PGD3 (1/18=5.5% of patients with AG genotype, 2/5=40%) of patients with AA genotype, 10/26 patients=38.4% of patients with GG genotype, p=0.022 for multiple comparisons), with a significant difference between GG and AG genotypes (p=0.033), but not between GG and AA genotypes (p=0.95, Table 2).

Prognostic analysis. In multivariate analysis including donor smoking, donor CCSP G38A polymorphism significantly impacted the risk of severe PGD (OR=0.082, 95CI 0.0041- 0.52, p=0.026, Table 3). Discussion

Main results reminder. Studying donors and recipients CCSP G38A polymorphism, pretransplant CCSP serum concentration, and their impact on primary lung graft dysfunction, we found donor CCSP G38A polymorphism to be associated (i) with a decreased concentration of CCSP in the peripheral blood prior to LT; and (ii) with a decreased risk of severe PGD following LT.

CCSP and lung transplantation. The first clinical study exploring the role of CCSP after LT focused on Bronchiolitis Obliterans Syndrome (BOS), the most frequent pattern of chronic lung allograft dysfunction (CLAD) characterized by a fibrosing process of the small airways causing irreversible airway obstruction. Following 22 LT recipients over 2 years, Nord et al. found that levels of CCSP in serum and BAL were lowered in BOS patients, suggesting that recipient serum CCSP concentration could be an early marker for BOS (16). Ten years later, Diamond et al. focused on PGD on a prospective cohort of 104 LT recipients, and determined levels of plasma CCSP at three time points: pre-transplant, 6h post-transplant, and 24h post-transplant. Interestingly, elevated CCSP levels at 6h post-transplant were associated with increased odds of PGD in univariate and multivariate analysis (17). Concomitantly, Gilpin et al. studied the kinetics of CCSP(+) Bone Marrow Cells (BMC) in the first days following LT in 30 recipients, and found a significant increase in these progenitors at 24h, with a decrease by 48h (18). All together, these data suggest that lung IR leads to increased levels of CCSP that correlate with altered alveolar epithelial permeability, while increased CCSP(+) PBMC mobilization following LT may have beneficial effects on lung oxygenation and recovery time, pointing CCSP and CCSP(+) PBMC as a key determinant of early graft function. Unfortunately, these studies did not take into account the genetic variability of donor and recipients, even though the gene encoding CCSP is subject to a frequent polymorphism that may alter its expression at baseline or its response to external injuries.

CCSP G38A polymorphism. In humans, the gene encoding CCSP has 3 short exons and 2 introns for a total length of 4.1 kb. It is located on chromosome 1 l ql2.3-13.1 in the vicinity of other genes associated with inflammatory and immune processes (19, 20). Studies of the non-coding region of the exon 1 of CCSP gene have identified a number of binding sites for transcription factors (1 ). This region is also subject to a single nucleotide polymorphism (SNP) located 38 bp downstream of the transcription initiation site, defined as dbSNP rs3741240, and characterized by an adenine/guanine substitution (21 , 22). This CCSP G38A polymorphism is found in 34% of the population and associated with 25% reduced transcription levels as compared with the G allele (11, 12). Interestingly, we report similar proportions of CCSP G38A polymorphism in donors and recipients, but associated decrease in serum CCSP levels were found in donors but not in recipients. These results suggest that in healthy individuals such as lung donors, CCSP polymorphism is associated with decreased serum CCSP levels, but that in patients with end-stage lung disease such as lung recipients, CCSP polymorphism is associated with sustained serum CCSP levels - i.e that CCSP G38A polymorphism could be associated with a resistance to cell signals associated with end-stage respiratory disease. In the literature, CCSP G38A polymorphism has been studied mostly in sarcoidosis and asthma. The association between the presence of an A allele and the development of asthma in the general population is still questioned (13, 23), while serum CCSP levels has been independently related to small airway hyperresponsiveness in asymptomatic individuals (24), COPD patients (7) and asthmatic patients (25). The fact that CCSP polymorphism has not been formally associated with the development of asthma, but serum CCSP levels has been associated with small airway hyperresponsiveness in various situations, could be explained by confounding factors. Individual exposure to cigarette smoke, air pollution, and professional toxics could indeed interact with individual genetic susceptibility to impact the serum concentration of CCSP and the development of symptoms (15).

Gene-environment interaction. This concept of gene-environment interaction could be of tremendous importance when considering lung grafts subjected to IR. In our study, the presence of A allele in the donor was associated with a decreased risk of severe PGD both in univariate and multivariate analyses. A potential mechanism could be the resistance of the A allele to p53, as recently suggested (15). Lung IR has been associated with increased levels of p5 in the airway epithelium (3), that correlates with increased apoptosis of airway epithelial cells (26). Knabc et al. recently report that p53 binds to the promoter of CCSP and causes a decrease in CCSP gene expressio n. In humans, this binding site is located at the position of the G38A polymorphism site, thus explaining the resistance of CCSP G38A to p53-mediated CCSP down regulation in vitro (15). Our hypothesis is that in lung grafts with the G allele of CCSP polymorphism, IR causes an increase in p5 associated with an increase in apoptosis and a decrease in the production of CCSP in lung epithelial cells, thus constituting a vicious circle that may lead to PGD. Conversely, in lung grafts with the A allele of CCSP G3 A polymorphism, IR-induced p53 increase has no effect on CCSP expression and CCSP levels (not shown). This p53 resistance of G38A binding site may therefore explain the decrease frequency of PGD in grafts harboring the CCSP G38A genotype.

Conclusion In this study, donor CCSP A38G gene polymorphism is associated with a decreased concentration of CCSP in the peripheral blood prior to LT, and a decreased risk of severe PGD following LT. These findings should be confirmed in further analyses performed on another retrospective series or in the frame of a prospective study. If confirmed, these findings would identify donor CCSP G38A status as an important risk factor and potential therapeutic target to predict and prevent the occurrence of PGD.

Table 1. Characteristics of lung transplant donors and recipients (study group, n=104).

Study Group 1 - no Group 2 - P- group PGD PGD value

(n=104) (n=84) (n=20)

Recipient

Male gender 53 (51%) 43 (51%) 10 (50%) 0.92

Age at transplant 50 (30.2- 52 (29.0- 44 (32.0- 0.10

58.7) 59.5) 52.0)

Height (cm) 166,5 ± 8,3 166,6 ± 8,5 165,8 ± 7,4 0.74

Weight (kg) 56 (48.0- 55 (47-70.5) 60 (50-65) 0.91

68.7)

BMI (kg.m-2) 19.8 (17.9- 19.6 (17.8- 21.7 (18.9- 0.68

24.0) 24.0) 24.0)

Diagnosis 0.53

Cystic fibrosis 41 (39%) 34 (40%) 7 (35%)

COPD/emphysema 38 (37%) 32 (38%) 6 (30%)

Interstitial lung disease 20 (19%) 15 (18%) 5 (25%)

Other 5 (5%) 3 (4%) 2 (10%)

Donor

Recipient-Donor Gender 0.65

Male - Male 44 (42%) 36 (43%) 8 (40%)

Male - Female 18 (17%) 15 (18%) 3 (15%)

Female - Male 9 (9%) 6 (7%) 3 (15%)

Female - Female 33 (32%) 27 (32%) 6 (30%)

Age 48 (29.7- 48 (27.0- 51 (34.0- 0.55

58.0) 59.0) 56.0)

Tobacco use 39 (38%) 27 (33%) 12 (63%) 0.02 3

Cause of death 0.18

Cerebrovascular/stroke 43 (41%) 33 (39%) 10 (50%)

Anoxia/hypoxia 14 (13%) 14 (17%) 0

Haemorrhage/aneurysm 13 (13%) 9 (11%) 4 (20%)

Other 34 (33%) 28 (33%) 6 (30%)

ICU length of stay (days) 2 (1-3) 2 (1-3) 1 (1-3) 0.06

9

PaO2/Fi02 (mmHg) 358 (183- 365 (176- 343 (273- 0.73

422) 421) 441)

Procedure

Graft allocation 0.10

Regular 83 (81%) 68 (81%) 15 (75%)

High emergency 10 (9%) 6 (7%) 4 (20%)

Non available 11 (10%) 10 (12%) 1 (5%)

Ischemic time (min) 345 (287- 340 (289- 338 (258- 0.30

379) 378) 420)

Transplant type 0.39

Double lung transplant 87 (84%) 69 (82%) 18 (90%)

Single lung transplant 17 (16%) 15 (18%) 2 (10%)

Outcome

Postoperative ECMO 11 (11%) 0 11 (55%) <0.0

01

ICU length of stay (days) 15 (7-29) 14 (6-21) 42.5 (21- <0.0

47) 01

90-day mortality 5 (4.8%) 1 (1%) 4 (20%) <0.0

01

1-year mortality 10 (9.6%) 6 (7%) 4 (20%) 0.08

0

Table 2. Allele and genotype of CCSP (G38A) polymorphism in donors and recipients group, n=104, and number of DNA duets available, n=49). Study Group Group

p- group 1 - no PGD 2 - PGD

value (n=104) (n=84) (n=20)

DNA duets available 49 36 13

2

AA

5 (10%) 3 (8%) (15%)

18 17

Donors AG 0.030

(37%) (47%) 1 (8%)

26 16 10

GG

(53%) (45%) (77%)

5

AA

6 (12%) (14%) 1 (8%)

24 17 7

Recipients AG 1

(49%) (47%) (54%)

19 14 5

GG

(39%) (39%) (38%)

p-value 0.25 0.73 0.072

Table 3. Multivariate analysis of risk factors associated with severe PGD, and including donor, recipient, and procedure variables (study group, n=104). Akaikc Information Criterion (AIC) = 59.4. OR=Odds Ratio. 95CI=95% confidence interval.

Vari Clas Univar Multiva OR 95CI able sed iate p-value riate p-value

Don No 0.023 0.18 2.63 0.65 or smoking Yes -11.5

Don GG 0.011 Referen Refer

or CCSP AG ce ence 0.00 polymorph! AA 0.026 0.082 41-0.52 sm 0.96 1.05 0.12

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Claims

CLAIMS:

1. A method of identifying lungs harvested from a donor and to be grafted to a recipient, having or at risk of having or developing a Primary Graft Dysfunction (PGD), comprising determining, in a sample obtained from said donor or said lungs, the presence or absence of a single nucleotide polymorphism (SNP) located in SCGB1A1 gene.

2. The method according to claim 1, wherein the SNP is selected from the group consisting of SCGB1A1 dbSNP rs3741240 G>A and wherein:

- the presence of the allele (A) of SCGB1A1 dbSNP rs3741240 G>A indicates a decreased risk of having or developing primary graft dysfunction.

3. The method according to claim 1 or 2, wherein the sample is a blood sample or a lung biopsy.

4. The method according to claim 1 to 2, wherein the presence or absence of said SNP is determined by nucleic acid sequencing or by PC analysis.

5. A method for treating or preventing primary graft dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

6. A method for treating or preventing primary graft dysfunction comprising delivering to ex vivo lungs a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

7. A method for preparing ex vivo lungs comprising delivering to said ex vivo lungs a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof.

8. A method of grafting lungs comprising the following steps: detecting the presence or the absence of the allele (A) iSCGBlAl dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs; grafting said lungs on the recipient when the presence of SCGBIA1 dbSNP rs3741240 G>A polymorphism in said sample is detected.

9. A method of grafting lungs comprising the following steps:

administering to said donor a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1A1 dbSNP rs3741240 G>A polymorphism in said sample is detected;

- grafting said lungs on the recipient.

10. A method of grafting lungs comprising the following steps:

detecting the presence or the absence of the allele (A) of SCGBIA1 dbSNP rs3741240 G>A in a sample obtained from a potential donor or lungs;

- grafting said lungs on the recipient;

administering to said recipient a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1A1 dbSNP rs3741240 G>A polymorphism in said sample is detected.

11. A method of grafting lungs comprising the following steps:

delivering to said ex vivo lungs a therapeutically effective amount of CCSP protein or functional equivalent thereof, vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof when the absence of SCGB1A1 dbSNP rs3741240 G>A polymorphism in said sample is detected;

- grafting said lungs on the recipient.

12. The method according to claims 5 to 11 wherein the transduced or transfected cells are mesenchymal stem cells.

13. The method according to claims 5 to 12 wherein the CCSP protein or functional variant thereof, the vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof, or the transduced or transfected cells with an amino acid sequence encoding CCSP protein or functional equivalent thereof or a vector comprising a nucleic acid encoding for CCSP protein or functional equivalent thereof are administered to the donor or delivered to the ex vivo lungs, before the lungs transplantation.