Microrna signature for the early diagnosis of localized radiation-induced lesions

Abstract

The present invention relates to an in vitro method for predicting the occurrence and / or severity of a localized irradiation-induced lesion or an in vitro method for detecting localized irradiation in a subject and / or for determining a localized irradiation level in a subject, said methods comprising the following steps: a) obtaining an expression profile for at least the following 15 microRNAs in a biological sample from the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-mIR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, wherein the expression profile is measured beforehand in vitro using the biological sample from said subject; b) comparing the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, and c) on the basis of the comparison in step b), predicting the occurrence and / or severity of a lesion in the subject as a result of localized irradiation or detecting localized irradiation and / or determining the localized irradiation level.

Description

[0001] microRNA signature for the early diagnosis of localized radiation-induced lesions

[0002] FIELD OF INVENTION

[0003] The present invention relates to methods for predicting the occurrence and severity of musculocutaneous lesions following localized exposure to ionizing radiation and / or methods for detecting localized irradiation and / or determining a level of localized irradiation in a subject.

[0004] STATE OF THE ART

[0005] In the event of a radiological emergency involving a high number of potential victims, it is essential to improve the management of victims most at risk of developing severe injuries following external exposure to ionizing radiation (IR).

[0006] Medical care for victims comprises four key steps: identification of potential victims (triage), diagnosis / prognosis of pathologies, treatment of patients, and long-term follow-up. These steps are divided into three distinct phases: immediate emergency phase (triage of individuals and first aid), intermediate emergency phase (diagnosis and treatment of individuals exposed to radioactive materials), and convalescence phase (epidemiological surveillance, psychological support, and clinical follow-up of treated patients). For scenarios involving a large number of potential victims, triage of individuals within the affected area and the initial diagnostic steps can be carried out directly in the field to quickly identify victims requiring medical care, followed by laboratory analysis.In all cases, the objective is to minimize the time needed to definitively identify victims requiring medical attention. An initial categorization of exposed individuals can be carried out in the hospital, based on the analysis of clinical signs that appear within 24 hours of irradiation (vomiting, lymphopenia, etc.), supplemented by a more thorough clinical examination in a hospital setting, including molecular and functional diagnosis, and if possible, a reconstruction of the dose received by the patient.

[0007] In recent years, a limited number of circulating molecular markers have been described that are of interest for the diagnosis and prognosis of radiation syndromes, including Flt3-ligand (bone marrow involvement), or citrulline (gastrointestinal involvement).

[0008] To date, however, there are no operational biomarkers for the early diagnosis or prognosis of localized radiation-induced lesions. Yet, early detection of the most severely exposed victims would allow them to receive priority care and rapid access to appropriate therapy, compared to individuals irradiated at lower doses, and therefore at lower risk, as well as unexposed individuals present during the incident.

[0009] Several preclinical studies conducted in rodents and primates have identified different microRNA signatures mainly associated with exposure to lethal or sublethal doses of RI in the whole body, with the aim of developing new biodosimetric tools, or for the diagnosis and estimation of the degree of damage to the hematopoietic compartment (Acharya et al. (2015) Science translational medicine 7, 287ra269; Cui et al. (2011) PloS one 6, e22988; Fendler et al. (2017) Science translational medicine 9; Islam et al. (2015) PloS one 10, e0134827; Jacob et al. (2013) PloS one 8, e57603; Menon et al. (2016) PloS one 11, e0167333; Port et al. (2016) PloS one 11, e0165307; Wei et al. (2017) Radiation research 188, 342-354).

[0010] A Chinese patent (CN111197082) also proposes a combination of microRNAs taken from blood plasma to predict the level of radiation-induced damage within 12 hours following whole-body irradiation at a dose between 1 and 8 Gy.

[0011] However, in all these studies, the identified biomarkers are associated with whole-body, rather than localized, exposure to ionizing radiation. Yet, many microRNAs are markers of disruptions to specific biological functions, such as the functioning of the hematopoietic system. The specificity of these microRNAs, identified in "whole-body" signatures, makes their use difficult to extrapolate to the context of localized exposure, in which hematopoietic niches, for example, are less affected.

[0012] Furthermore, Ancel et al. (2024) Scientific Reports 14:2681 identified a microRNA signature in mice associated with lesions induced by localized irradiation. However, this signature was identified late, once the lesions were already observable (14 days after irradiation). Therefore, it does not allow for the detection of whether a subject has been exposed to localized irradiation and at what level, nor can it predict the occurrence and severity of radiation-induced lesions before they appear.

[0013] DESCRIPTION OF THE INVENTION

[0014] The present invention results from the inventors' identification of microRNAs whose plasma expression level is altered after localized irradiation at different doses of ionizing radiation. The use of this expression profile of only 23 specific microRNAs thus makes it possible, as early as 1 day after irradiation and therefore during the asymptomatic latency phase (before the possible appearance of radiation-induced lesions, which generally do not appear before 7 days following irradiation), to predict the occurrence and severity of musculocutaneous lesions resulting from localized exposure to ionizing radiation.

[0015] Advantageously, this signature simultaneously reveals the existence of localized irradiation, the level of exposure, and the severity of the resulting injury. Furthermore, it is obtained from blood biological samples, requiring minimally invasive access, thus facilitating the implementation of this early diagnosis, particularly when a large population is likely to have been exposed to localized irradiation and it is necessary to quickly differentiate between those who have actually been exposed (especially to high doses) and those who have not been, or have been exposed to very low doses.

[0016] The present invention thus relates to an in vitro method for predicting the occurrence and / or severity of a lesion induced by localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in a biological sample from the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from the biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, and c) based on the comparison in step b), predict the occurrence and / or severity of an injury in the subject following localized irradiation.

[0017] Another object of the invention relates to an in vitro method for detecting localized irradiation in a subject and / or determining a level of localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in a biological sample from the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from the biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, c) based on the comparison in step b), detect localized irradiation and / or determine the level of localized irradiation in the subject. In a particular embodiment of the methods according to the invention, the expression profile further includes the expression levels of the following 14 microRNAs in the biological sample of the subject:;

[0018] (xvi) hsa-miR-16-5p; (xvii) hsa-miR-19a-3p; (xviii) hsa-miR-29c-3p; (xix) hsa-miR-92a-3p; (xx) hsa-miR-93-5p; (xxi) hsa-miR-107; (xxii) hsa-miR-128-3p; (xxiii) hsa-miR-142-3p; (xxiv) hsa-miR-186-5p; (xxv) hsa-miR-208a-3p; (xxvi) hsa-miR-215-5p; (xxvii) hsa-miR-324-3p; (xxviii) hsa-miR-375-3p; and (xxix) hsa-miR-450a-5p; and possibly also the expression levels of the following 30 microRNAs in the subject's biological sample:

[0019] (xxx) hsa-let-7e-5p ; (xxxi) hsa-miR-18a-5p ; (xxxii) hsa-miR-23a-3p ; (xxxiii) hsa-miR-23b-3p ; (xxxiv) hsa-miR-26a-5p ; (xxxv) hsa-miR-27b-3p ; (xxxvi) hsa-miR-1249-3p ; (xxxvii) hsa-miR- 130b-3p ; (xxxviii) hsa-miR-142-5p ; (xxxix) hsa-miR-143-3p ; (xl) hsa-miR-144-3p ; (xli) hsa- miR-146a-5p ; (xlii) hsa-miR-146b-5p ; (xliii) hsa-miR-148b-3p ; (xliv) hsa-miR-150-5p ; (xlv) hsa-miR-185-5p ; (xlvi) hsa-miR-195-5p ; (xlvii) hsa-miR-199a-3p ; (xlviii) hsa-miR-200a-3p ; (xlixii) hsa-miR-200b-3p ; (I) hsa-miR-204-5p ; (li) hsa-miR-205-5p ; (lii) hsa-miR-222-3p ; (liii) hsa-miR-223-3p ; (color) hsa-miR-331-3p ; (Iv) hsa-miR-339-5p ; (Ivi) hsa-miR-342-3p ; (Ivii) hsa- miR-484 ; (Iviii) hsa-miR-497-5p ; et (lix) hsa-miR-532-5p.

[0020] In a particular embodiment, the expression profile obtained is compared, in step b), to at least one reference microRNA expression profile obtained from a biological sample of a non-irradiated subject and / or a subject who has undergone a given level of localized irradiation.

[0021] In one particular embodiment, the biological sample of said subject is a blood sample.

[0022] In a particular embodiment, the methods according to the invention include a preliminary step of determining the expression profile from the biological sample of said subject.

[0023] Preferably, microRNA expression profiles are determined by quantitative PCR.

[0024] In a particularly advantageous embodiment, the biological sample is obtained less than one week after the possible irradiation of said subject, preferably one day after the possible irradiation of said subject.

[0025] The present invention also relates to a computer program product comprising program code instructions for executing the steps of the methods according to the invention, when this program is executed by a computer. Another object of the invention relates to a kit for predicting the occurrence and / or severity of an injury induced by localized radiation in a subject and / or for detecting localized radiation in a subject and / or for determining a level of localized radiation in a subject, said kit comprising at least:

[0026] (i) a probe and / or primer specific to the hsa-let-7b-3p microRNA;

[0027] (ii) a probe and / or primer specific to the hsa-let-7d-3p microRNA;

[0028] (iii) a probe and / or primer specific to the hsa-miR-10a-5p microRNA;

[0029] (iv) a probe and / or primer specific to the hsa-miR-24-3p microRNA;

[0030] (v) a probe and / or primer specific to the hsa-miR-99b-5p microRNA;

[0031] (vi) a probe and / or primer specific to the hsa-miR-122-5p microRNA;

[0032] (vii) a probe and / or primer specific to the hsa-miR-139-5p microRNA;

[0033] (viii) a probe and / or primer specific to the hsa-miR-140-3p microRNA;

[0034] (ix) a probe and / or primer specific to the hsa-miR-191-5p microRNA;

[0035] (x) a probe and / or primer specific to the hsa-miR-214-3p microRNA;

[0036] (xi) a probe and / or primer specific to the hsa-miR-340-5p microRNA;

[0037] (xii) a probe and / or primer specific to the hsa-miR-361-5p microRNA;

[0038] (xiii) a probe and / or primer specific to the hsa-miR-501-5p microRNA;

[0039] (xiv) a probe and / or primer specific to the hsa-miR-652-3p microRNA; and

[0040] (xv) a probe and / or primer specific to the hsa-miR-744-5p microRNA; said kit comprising probes and / or primers specific to fewer than 200 different microRNAs; the kit optionally comprising further a computer program product including program code instructions for carrying out steps a) and b) of the methods according to the invention, when such program is executed by a computer.

[0041] In one particular embodiment, the kit according to the invention further comprises at least:

[0042] (xvi) a probe and / or primer specific to the hsa-miR-16-5p microRNA;

[0043] (xvii) a probe and / or primer specific to the hsa-miR-19a-3p microRNA;

[0044] (xviii) a probe and / or primer specific to the hsa-miR-29c-3p microRNA;

[0045] (xix) a probe and / or primer specific to the hsa-miR-92a-3p microRNA;

[0046] (xx) a probe and / or primer specific to the hsa-miR-93-5p microRNA;

[0047] (xxi) a probe and / or primer specific to the hsa-miR-107 microRNA;

[0048] (xxii) a probe and / or primer specific to the hsa-miR-128-3p microRNA;

[0049] (xxiii) a probe and / or primer specific to the hsa-miR-142-3p microRNA;

[0050] (xxiv) a probe and / or primer specific to the hsa-miR-186-5p microRNA;

[0051] (xxv) a probe and / or primer specific to the hsa-miR-208a-3p microRNA; (xxvi) a probe and / or primer specific to the hsa-miR-215-5p microRNA;

[0052] (xxvii) a probe and / or primer specific to the hsa-miR-324-3p microRNA;

[0053] (xxviii) a probe and / or primer specific to the hsa-miR-375-3p microRNA; and

[0054] (xxix) a probe and / or primer specific to the hsa-miR-450a-5p microRNA; and possibly in addition at least:

[0055] (xxx) a probe and / or primer specific to the hsa-let-7e-5p microRNA;

[0056] (xxxi) a probe and / or primer specific to the hsa-miR-18a-5p microRNA;

[0057] (xxxii) a probe and / or primer specific to the hsa-miR-23a-3p microRNA;

[0058] (xxxiii) a probe and / or primer specific to the hsa-miR-23b-3p microRNA;

[0059] (xxxiv) a probe and / or primer specific to the hsa-miR-26a-5p microRNA;

[0060] (xxxv) a probe and / or primer specific to the hsa-miR-27b-3p microRNA;

[0061] (xxxvi) a probe and / or primer specific to the microRNA hsa-miR-1249-3p;

[0062] (xxxvii) a probe and / or primer specific to the hsa-miR-130b-3p microRNA;

[0063] (xxxviii) a probe and / or primer specific to the hsa-miR-142-5p microRNA;

[0064] (xxxix) a probe and / or primer specific to the hsa-miR-143-3p microRNA;

[0065] (xl) a probe and / or primer specific to the hsa-miR-144-3p microRNA;

[0066] (xli) a probe and / or primer specific to the hsa-miR-146a-5p microRNA;

[0067] (xlii) a probe and / or primer specific to the hsa-miR-146b-5p microRNA;

[0068] (xliii) a probe and / or primer specific to the hsa-miR-148b-3p microRNA;

[0069] (xliv) a probe and / or primer specific to the hsa-miR-150-5p microRNA;

[0070] (xlv) a probe and / or primer specific to the hsa-miR-185-5p microRNA;

[0071] (xlvi) a probe and / or primer specific to the hsa-miR-195-5p microRNA;

[0072] (xlvii) a probe and / or primer specific to the hsa-miR-199a-3p microRNA;

[0073] (xlviii) a probe and / or primer specific to the hsa-miR-200a-3p microRNA;

[0074] (xlix) a probe and / or primer specific to the hsa-miR-200b-3p microRNA;

[0075] (I) a probe and / or primer specific to the hsa-miR-204-5p microRNA;

[0076] (li) a probe and / or primer specific to the hsa-miR-205-5p microRNA;

[0077] (lii) a probe and / or primer specific to the hsa-miR-222-3p microRNA;

[0078] (liii) a probe and / or primer specific to the hsa-miR-223-3p microRNA;

[0079] (liv) a probe and / or primer specific to the hsa-miR-331-3p microRNA;

[0080] (iv) a probe and / or primer specific to the hsa-miR-339-5p microRNA;

[0081] (Ivi) a probe and / or primer specific to the hsa-miR-342-3p microRNA;

[0082] (Ivii) a probe and / or primer specific to the microRNA miR-484;

[0083] (iviii) a probe and / or primer specific to the hsa-miR-497-5p microRNA; and (lix) a probe and / or primer specific to the hsa-miR-532-5p microRNA. In one particular embodiment, the kit according to the invention further comprises at least one reagent for performing a nucleic acid amplification reaction.

[0084] DESCRIPTION OF THE FIGURES

[0085] Figure 1 presents a graphical summary of the experimental protocol. After irradiation at various doses, a blood sample was taken before euthanasia at the indicated times for microRNA expression profiling (broad spectrum, then targeted analysis on an independent cohort). In this model, mice are asymptomatic for the first 7 days, then develop a distinct radiological burn of significant severity, evident on day 14.

[0086] Figure 2 represents the performance (ROC curves) of the J1 (left) and J7 (right) prognostic models obtained from targeted analysis of plasma microRNAs in mice, using the miR-BR panel, with areas under the curve (outcome) overall > 0.75 for each group.

[0087] Figure 3 represents the performance (ROC curves) of the J1 (left) and J7 (right) prognostic models obtained from targeted analysis of plasma microRNAs in rats, using the miR-BR panel.

[0088] Figure 4 represents the correlation curves between the molecular signature identified here at J1 and the different lesion scores observed in the rats.

[0089] Figure 5 represents the correlation curves between the molecular signature identified here at day 7 and the different lesion scores observed in the rats.

[0090] DETAILED DESCRIPTION OF THE INVENTION

[0091] Definitions

[0092] Localized irradiation refers to localized exposure (as opposed to whole-body exposure) to ionizing radiation of an intensity and duration likely to cause an adverse effect, such as unwanted tissue damage. Sources of ionizing radiation exposure include, but are not limited to, radiotherapy (e.g., localized radiation exposure to a tissue or organ), involuntary exposure to ionizing radiation (e.g., a nuclear accident, an act of terrorism), and improper or unauthorized disposal of radioactive materials. Localized irradiation typically results from exposure to a source of ionizing radiation located close to the individual's body (e.g., in a pocket or in contact with the individual's hand).Conversely, exposure to a source of ionizing radiation located far from the subject's body and / or when the source is proportional to the size of the subject's body or when the subject moves around the source, is interpreted as whole-body exposure.

[0093] By "localized irradiation level" we mean here the amount of ionizing radiation to which a subject has been exposed, on average, over the surface of an area of ​​the body exposed to ionizing radiation.

[0094] The amount of ionizing radiation to which a subject is exposed can be expressed in different ways. The absorbed dose, expressed in grays (Gy), corresponds to the energy absorbed per unit mass. The equivalent dose (expressed in sieverts, Sv) takes into account the type of radiation. It is calculated by multiplying the absorbed dose by a radiological weighting factor that depends on the type of radiation (X-rays, gamma rays, etc.). This factor is 1 for X-rays, gamma rays, and beta rays; 20 for alpha particles; and it varies for neutrons (depending on their energy). Finally, the effective dose (also expressed in sieverts) takes into account the type of tissue or organ affected. Preferably, the level of localized radiation to which a subject has been exposed corresponds to the absorbed dose (in grays) as defined above.The level of localized radiation to which a subject has been exposed can also be defined according to the localized radiological lesions that it induces or can induce in the subject.

[0095] A localized radiation dose level can, for example, be categorized according to the absorbed dose as follows: low level for an absorbed dose between 1 and 5 Gy, moderate level for an absorbed dose between 5 and 10 Gy, high level for an absorbed dose between 10 and 20 Gy, and critical level for an absorbed dose greater than 20 Gy. Alternatively, a localized radiation dose level can, for example, be categorized according to the radiological lesion it induces or may induce as follows: low level for the occurrence of second-phase erythema or temporary hair loss, moderate level for the occurrence of permanent hair loss or dry desquamation (or radiodermatitis), high level for the occurrence of exudative desquamation (or radiodermatitis), and critical level for the occurrence of necrosis.

[0096] By "localized radiation-induced injury" or "radiation-induced injury" we mean here any disorder, disease or pathological condition which occurs as a result of or is induced by localized exposure of a subject to ionizing radiation of sufficient intensity and duration to cause an undesirable biological effect, for example undesirable tissue damage.

[0097] Radiation-induced damage in various tissues and organs generally follows a similar course after exposure to ionizing radiation. Depending on the dose of ionizing radiation to which the individual is exposed, they undergo an acute response phase that typically occurs at least 2 to 3 days and sometimes weeks after exposure. The acute response phase usually involves inflammatory components, is generally self-limiting, and in some patients may resolve relatively quickly. Depending on the dose of ionizing radiation to which the individual is exposed, the acute phase may be followed by a chronic phase, usually beginning one or more months after exposure. The chronic phase is often characterized by significant tissue remodeling and fibrosis. Radiation-induced disorders are well known and have been observed in a variety of tissues and organs.Depending on various aspects, the lesion induced by localized irradiation may be, but is not limited to, one or more of the following: second-phase erythema, temporary hair removal, permanent hair removal, dry desquamation (or radiodermatitis) (scaly and peeling skin), exudative desquamation (or radiodermatitis) (characterized by oozing / fluid loss), ulceration (appearing as an open wound), and necrosis (tissue death).

[0098] Radiation-induced injuries can be classified according to their severity.

[0099] The "severity" of a radiation-induced injury refers to the risk the injury poses to an individual. The severity of a radiation-induced injury also dictates the extent of treatment necessary to properly treat the individual. For example, a radiation-induced injury can be mild, moderate, severe, or critical.

[0100] A mild radiation-induced lesion may, for example, cause slight discomfort and typically resolves without treatment (e.g., second-phase erythema or temporary hair removal). A moderate radiation-induced lesion may cause more than mild discomfort and may require treatment to resolve (an example of a moderate radiation-induced lesion is permanent hair removal or dry desquamation). A severe radiation-induced lesion is qualitatively different from a moderate or mild lesion, causing significant discomfort and requiring intensive treatment, including interventions beyond those used to treat a moderate lesion. An example of a severe radiation-induced lesion is exudative desquamation. A critical radiation-induced lesion is life-threatening and would require hospitalization and intensive treatment, which may be unsuccessful and result in the subject's death.Examples of critical radiation-induced lesions include ulceration and necrosis.

[0101] The severity of a radiation-induced injury will depend, among other things, on the level of localized radiation to which the subject has been exposed.Thus, typically, a subject who has been exposed to a low level of localized irradiation (typically an absorbed dose between 1 and 5 Gy) may develop a mild radiation-induced lesion (e.g., second-phase erythema or temporary hair removal), a subject who has been exposed to a moderate level of localized irradiation (typically an absorbed dose between 5 and 10 Gy) may develop a moderate radiation-induced lesion (e.g., permanent hair removal or dry desquamation), a subject who has been exposed to a high level of localized irradiation (typically an absorbed dose between 10 and 20 Gy) may develop a severe radiation-induced lesion (e.g., exudative desquamation), and a subject who has been exposed to a critical level of localized irradiation (typically an absorbed dose greater than 20 Gy) may develop a critical radiation-induced lesion (e.g., ulceration and / or necrosis).

[0102] By "second phase erythema" we mean here an erythema which develops during the manifestation phase of the radio-induced lesion, and which is therefore distinct from the primary erythema which can appear within a few hours after exposure to ionizing radiation due to dilation of the capillaries before disappearing spontaneously.

[0103] “Temporary hair removal” here refers to temporary hair loss in an area of ​​the body due to reversible damage to the hair follicles in that area caused by ionizing radiation.

[0104] By "permanent hair removal" we mean here permanent hair loss on an area of ​​the body due to irreversible damage to the hair follicles in that area induced by ionizing radiation.

[0105] By "dry desquamation" we mean here an atypical keratinization of the skin caused by the decrease in the number of clonogenic cells in the basal layer of the epidermis.

[0106] Exudative desquamation refers to epidermal shedding caused by the loss of a high proportion of clonogenic cells in the basal layer of the epidermis. It is characterized by sensitive, red skin associated with serous exudate, hemorrhagic crusting, and a risk of blistering.

[0107] The term "ulceration" here refers to a wound on the skin or mucous membrane, accompanied by tissue disintegration. Ulcers can lead to complete loss of the epidermis and often parts of the dermis and even subcutaneous fat.

[0108] By "necrosis" we mean here a form of cellular damage that leads to the premature death of cells in living tissues by autolysis.

[0109] A "subject" suitable for use in the methods according to the invention can be any mammal, including a human, dog, cat, bovine, goat, pig, ovine, monkey, or rodent (rat, mouse, etc.). Preferably, the subject is a human subject. "MicroRNA" or "miRNA" refers to a single-stranded molecule of approximately 21 to 24 nucleotides, preferably 21 to 23 nucleotides in length, encoded by genes that are transcribed from DNA but not translated into proteins (non-coding RNA); instead, they are transformed from primary transcripts known as pri-miRNAs into short stem-loop structures called pre-miRNAs and finally into functional miRNAs. During maturation, each pre-miRNA gives rise to two distinct fragments with strong complementarity, one from the 5' arm and the other from the 3' arm of the gene encoding the pri-miRNA.Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and their main function is to downregulate gene expression.

[0110] An international nomenclature for microRNAs exists (see Ambros et al. (2003) RNA 9(3):277-279; Griffiths-Jones (2004) NAR 32 (Database Issue):D109-D111; Griffiths-Jones et al. (2006) NAR 34 (Database Issue):D140-D144; Griffiths-Jones et al. (2008) NAR 36 (Database Issue):D154-D158; and Kozomara et al. (2011) NAR 39 (Database Issue):D152-D157), which is available on miRBase (specifically version 22.1 as of December 6, 2024) at http: / / www.mirbase.org / . Each microRNA is assigned a unique name with a predefined format, as follows:

[0111] • For a mature microRNA: sss-miR-XY, where sss is a three-letter code indicating the microRNA species, hsa stands for human, and the capital R in miR indicates that it is a mature microRNA. However, some authors in the literature also incorrectly use "mir" for mature microRNAs. In this case, it can be identified as a mature microRNA by the presence of -Y. X is the unique, arbitrary number assigned to the microRNA sequence in the particular species, which may be followed by a letter if several highly homologous microRNAs are known. For example, 20a and 20b refer to highly homologous microRNAs. The letter Y indicates whether the mature microRNA, obtained by cleaving the pre-microRNA, corresponds to the 5' arm (Y is then "5p") or the 3' arm (Y is then "3p") of the gene encoding the pri-mRNA. In the previous international nomenclature for microRNAs, "-Y" was not present.The two mature microRNAs obtained from the 5' or 3' arm of the gene encoding the pri-microRNA were then distinguished by the presence or absence of an asterisk (*) immediately following the X. The presence of the asterisk indicated that the sequence corresponded to the less frequently detected microRNA. Since this classification has been subject to change, a new nomenclature using the codes "3p" and "5p" has been implemented.

[0112] • For a pri-microRNA: sss-mir-X, where o sss is a three-letter code indicating the species of the microRNA, “hsa” meaning human, o the lowercase “r” in mir indicates that it is a pri-microRNA and not a mature microRNA, which is confirmed by the absence of “-Y”, o X is the unique arbitrary number assigned to the microRNA sequence in the particular species, which may be followed by a letter if several highly homologous microRNAs are known.

[0113] Each microRNA is also assigned an access number for its sequence.

[0114] The sequences, SEQ ID NO: and miRBase database access number of the microRNAs mentioned in this application are presented in Table 1 below. [Table 1] Methods

[0115] The present invention relates to an in vitro method for predicting the occurrence and / or severity of a lesion induced by localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in the biological sample of the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from a biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, and c) based on the comparison in step b), predict the occurrence and / or severity of an injury in the subject following localized irradiation.

[0116] By "prediction," we mean making a forecast or prognosis as to whether an individual may suffer radiation-induced injury, or radiation-induced injury of a given severity, at a future date. In some cases, an individual is considered likely to develop radiation-induced injury if they are more likely to develop such injury than an individual in the general population who has not been irradiated. The prediction may not be 100% accurate.

[0117] By "occurrence" of a radiation-induced lesion, we mean here the fact that a radiation-induced lesion appears in a given subject, in particular within a given period of time, for example within a period of time of more than 6 days, more than 7 days, more than 10 days, more than 14 days, or more than one month after the possible irradiation.

[0118] The present invention also relates to an in vitro method for detecting localized irradiation in a subject and / or determining a level of localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in the biological sample of the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from a biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, c) based on the comparison in step b), detect localized irradiation and / or determine the level of localized irradiation in the subject. By "expression profile" of the at least 15 microRNAs, we mean the expression levels of the at least 15 microRNAs as defined herein.

[0119] The microRNA expression profile obtained in step a) of the methods according to the invention thus comprises the expression levels of the following 15 microRNAs in the biological sample of the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, as defined in Table 1 above.

[0120] In a particular embodiment, the expression profile further includes the expression level of one or more reference ncRNAs.

[0121] The term "reference non-coding RNA" or "reference ncRNA" refers to a small non-coding RNA that is expressed at a relatively constant level in healthy individuals, particularly those who have not been irradiated. Such reference ncRNAs can be selected from among miRNAs, transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs).

[0122] When such reference ncRNAs are added to the expression profile (which is not always necessary), they are used for normalization purposes. In this case, the number of reference ncRNAs used for normalization in the methods according to the invention is preferably between one and five, with a preference for one, two, or three.

[0123] In a particular embodiment, the expression profile further includes the expression levels of the following 14 microRNAs in the subject's biological sample: (xvi) hsa-miR-16-5p; (xvii) hsa-miR-19a-3p; (xviii) hsa-miR-29c-3p; (xix) hsa-miR-92a-3p; (xx) hsa-miR-93-5p; (xxi) hsa-miR-107; (xxii) hsa-miR-128-3p; (xxiii) hsa-miR-142-3p; (xxiv) hsa-miR-186-5p; (xxv) hsa-miR-208a-3p; (xxvi) hsa-miR-215-5p; (xxvii) hsa-miR-324-3p ; (xxviii) hsa-miR-375-3p ; and (xxix) hsa-miR-450a-5p, as defined in Table 1 above.

[0124] In a more specific embodiment, the expression profile further includes (in addition to the expression levels of the 29 microRNAs described above), the expression levels of the following 30 microRNAs in the subject's biological sample:

[0125] (xxx) hsa-let-7e-5p ; (xxxi) hsa-miR-18a-5p ; (xxxii) hsa-miR-23a-3p ; (xxxiii) hsa-miR-23b-3p ; (xxxiv) hsa-miR-26a-5p ; (xxxv) hsa-miR-27b-3p ; (xxxvi) hsa-miR-1249-3p ; (xxxvii) hsa-miR- 130b-3p ; (xxxviii) hsa-miR-142-5p ; (xxxix) hsa-miR-143-3p ; (xl) hsa-miR-144-3p ; (xli) hsa- miR-146a-5p ; (xlii) hsa-miR-146b-5p ; (xliii) hsa-miR-148b-3p ; (xliv) hsa-miR-150-5p ; (xlv) hsa-miR-185-5p ; (xlvi) hsa-miR-195-5p ; (xlvii) hsa-miR-199a-3p ; (xlviii) hsa-miR-200a-3p ; (xlixii) hsa-miR-200b-3p ; (I) hsa-miR-204-5p ; (li) hsa-miR-205-5p ; (iii) hsa-miR-222-3p ; (liii) hsa-miR-223-3p ; (color) hsa-miR-331-3p ; (Iv) hsa-miR-339-5p ; (Ivi) hsa-miR-342-3p ; (Ivii) hsa- miR-484 ; (Iviii) hsa-miR-497-5p ; and (lix) hsa-miR-532-5p, as defined in Table 1 above.

[0126] However, in each case, adding a small number of other microRNAs to the tested expression profile might not alter, or even improve, the sensitivity, specificity, positive predictive value (PPV), and / or negative predictive value (NPV), and thus decrease the error rate of the methods according to the invention. In the methods according to the invention, the expression profile may therefore include at least one, for example, one, two, three, four, or five other microRNAs than those listed above.

[0127] Furthermore, the methods according to the invention may further include the determination of at least one additional parameter useful for the prediction (of occurrence and possibly severity), detection or determination of an exposure level.

[0128] In step a) of the methods according to the invention, an expression profile of the 15 microRNAs mentioned above (and optionally of at least one reference ncRNA) is obtained in the biological sample of the subject, the expression profile being previously measured in vitro from a biological sample of said subject.

[0129] The term "biological sample" refers to any sample that can be taken from a subject, such as a sample of bodily fluid (including a serum sample, a plasma sample, a urine sample, a blood sample, particularly a peripheral blood sample, a lymph sample), or a tissue sample such as a biopsy. It also includes specific cell subtypes or derivatives extracted from them, such as peripheral blood mononuclear cells (PBMCs).

[0130] In a preferred embodiment, the biological sample of said subject is a blood sample.

[0131] The term "blood sample" refers to a volume of whole blood or a fraction thereof containing microRNAs, for example serum, plasma, etc. Preferably, the blood sample is a plasma sample.

[0132] In a preferred embodiment, the biological sample is obtained less than one week after the possible irradiation of said subject, preferably between 12 hours and 1 week after the possible irradiation, in particular between 24 hours and 6 days after the possible irradiation, between 1 day and 5 days after the possible irradiation, between 1 day and 4 days after the possible irradiation, between 1 day and 3 days after the possible irradiation, or between 1 day and 2 days after the possible irradiation, particularly preferably one day after the possible irradiation of said subject.

[0133] The methods according to the invention may or may not include the prior step of in vitro measurement of the expression profile of the 15 microRNAs mentioned above, and optionally of at least one reference ncRNA from the biological sample of said subject.

[0134] In one embodiment, the expression profile has been previously measured in vitro from a biological sample of said subject by a third party, recorded in a memory, and the profile is obtained in step a) by accessing the memory.

[0135] In another embodiment, the method according to the invention further comprises a preliminary step of in vitro measurement of the expression profile of the 15 microRNAs mentioned above, and optionally of at least one reference ncRNA from the biological sample of said subject.

[0136] The expression profile of microRNAs can be measured by any technology known to those skilled in the art. In particular, each level of reference microRNA or ncRNA expression can be measured using any technology known to those skilled in the art, including nucleic acid microarrays, quantitative PCR, next-generation sequencing, and hybridization with a labeled probe.

[0137] In one particular embodiment, microRNA expression profiles are determined by quantitative PCR. Quantitative PCR, or real-time PCR, is a well-known and readily available technology for those skilled in the art and does not require a detailed description.

[0138] In one particular embodiment, which should not be considered as limiting the scope of the invention, the determination of microRNA expression profiles using quantitative PCR can be performed as follows. Briefly, in a total volume reaction of 40 pL, reverse transcription (RT) is performed on an RNA mixture extracted from the biological sample to be analyzed using the miRCURY LNA RT Kit (Qiagen). The RT products are used to prepare 8200 pL of PCR reaction mixture with the miRCURY LNA SYBR Green PCR Kit (Qiagen). Quantitative PCR is typically performed on an ABI 7900HT or QuantStudio 12K Flex instrument (Applied Biosystems) for 40 cycles. The number of RNA molecules in a sample is estimated by recording the amplification cycle in the exponential phase (cycle threshold or CT), at which point the fluorescence signal can be detected above the background fluorescence.Thus, the initial number of RNA molecules is inversely proportional to the CT value. The expression level of one or more microRNAs or one or more reference ncRNAs can be measured by their CT value. The expression level of microRNAs can be normalized using these reference microRNAs or ncRNAs by subtracting the CT values ​​of the microRNAs of interest from the CT values ​​of the reference microRNAs or ncRNAs to obtain the deltaQr. In another embodiment, the expression profile of microRNAs can be determined by sequencing or by using a nucleic acid microarray.

[0139] According to the invention, a "nucleic acid chip" consists of various nucleic acid probes attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. A microchip may be made of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. The probes may be nucleic acids such as cDNA ("cDNA chip") or RNA, or DNA or RNA oligonucleotides ("oligonucleotide chip"), and the oligonucleotides may be approximately 25 to 60 bases or less in length.

[0140] To determine the expression profile of a target nucleic acid sample, the sample is labeled and placed in contact with the chip under hybridization conditions. This leads to the formation of complexes between the target nucleic acids, which are complementary to the probe sequences attached to the chip surface. The presence of these labeled hybridized complexes is then detected. Numerous variations of on-chip hybridization technology are available to those skilled in the art.

[0141] Appropriate chip oligonucleotides specific to any microRNA useful in the invention can be designed, based on the sequence of each microRNA (see Table 1 above), using any chip oligonucleotide design method known in art. In particular, any available software developed for chip oligonucleotide design can be used, such as, for example, OligoArray software (available at http: / / berry.engin.umich.edu / oligoarray / ), GoArrays software (available at http: / / www.isima.fr / bioinfo / goarrays / ), Array Designer software (available at http: / / www.premierbiosoft.com / dnamicroarray / index.html), Primer3 software (available at http: / / frodo.wi.mit.edu / primer3 / primer3_code.html), or Promide software (available at http: / / oligos.molgen.mpg.de / ).

[0142] In step b) of the methods according to the invention, the microRNA expression profile obtained in step a) is compared with at least one reference microRNA expression profile.

[0143] By "reference microRNA expression profile" we mean a predetermined microRNA expression profile, obtained from a biological sample of a subject with a known particular condition.

[0144] In a preferred embodiment, the reference microRNA expression profile is obtained from a biological sample of a non-irradiated subject and / or a subject who has undergone a given level of localized irradiation. By "non-irradiated subject" is meant a subject who has not been exposed, either locally or whole-body, to a level of ionizing radiation exceeding 1 mSv per year, in addition to natural radioactivity (the level of which may vary depending on the region where the subject lives, but the level of natural radioactivity in each region is known or can be measured by a person skilled in the art).

[0145] By "subject having undergone a given level of localized irradiation", we mean here a subject who has been exposed to localized irradiation of a level resulting in deterministic effects, in particular radiation-induced lesions as defined above.

[0146] Preferably, at least one reference microRNA expression profile is a reference expression profile from a non-irradiated subject. Alternatively, at least one reference microRNA expression profile may be a reference expression profile from a subject who has received a given level of localized irradiation. Even more preferably, the prediction of the occurrence / severity of a radiation-induced injury or the detection of localized irradiation / determination of a localized irradiation level in a subject is performed by comparison with at least one reference microRNA expression profile from a non-irradiated subject and at least one reference expression profile from a subject who has received a given level of localized irradiation.Predicting the occurrence / severity of a radiation-induced lesion or detecting localized irradiation / determining a localized irradiation level in a subject can thus be achieved using a reference microRNA expression profile from a non-irradiated subject and a reference microRNA expression profile from a subject who has undergone a given level of localized irradiation. Advantageously, to obtain a more reliable and precise prediction of the occurrence / severity of a radiation-induced lesion or a more reliable and precise detection of localized irradiation / determining a localized irradiation level, said prediction / detection / determination is performed using several reference microRNA expression profiles from non-irradiated subjects and several reference microRNA expression profiles from subjects who have undergone a given level of localized irradiation.

[0147] Reference microRNA expression profiles of subjects who have undergone a given level of localized irradiation may advantageously include (particularly when the method aims to predict the severity of radiation-induced injury or to determine the level of exposure) reference microRNA expression profiles of subjects who have undergone different levels of localized irradiation (e.g., low / high, low / moderate / high, or low / moderate / high / critical, as defined above).

[0148] Comparing a microRNA expression profile of a tested subject with reference microRNA expression profiles, which allows either the prediction of the occurrence / severity of a radiation-induced lesion, or the detection of localized irradiation / determination of a localized irradiation level in a subject based on their expression profile, can be performed by a person skilled in the art using various methods well known to the person skilled in the art.

[0149] Thus, the comparison of a microRNA expression profile from the tested subject with a reference microRNA expression profile can be performed using statistical models or machine learning technologies. Preferably, PLS (Partial Least Squares or Projection to Latent Structures) regression is used to implement the comparison step.

[0150] The PLS regression preferentially used to implement the comparison step is typically the DIABLO approach as described in Singh et al. (2019) Bioinformatics 35:3055-3062.

[0151] In short, DIABLO extends the regularized sparse generalized canonical correlation analysis (sGCCA) method to a classification or supervised framework. sGCCA is a multivariate dimensionality reduction technique that uses singular value decomposition and selects co-expressed (correlated) variables from multiple omics datasets. sGCCA maximizes the covariance between linear combinations of variables (latent component scores) and projects the data into the smaller dimensional subspace spanned by the components. The selection of correlated molecules across the omics levels is performed internally with a penalty h on the vector of variable coefficients defining the linear combinations. Since all latent components are scaled within the algorithm, sGCCA maximizes the correlation between the components.The terms "covariance" and "correlation" are, however, used interchangeably here.

[0152] We denote Q as the normalized, centered, and scaled datasets, X <1> (N x Pi), X <2> (N x p2), ..., X Q> (N xp Q measuring the expression levels of Pi, PQ "omics variables on the same N samples". sGCCA solves the optimization function for each dimension h = 1, ..., H: [Math 1] where dh (q> is the variable coefficient or loading vector on dimension h associated with the residual matrix X h (q> of the Xf dataset q) C = {cij is a (Q x Q) design matrix that specifies whether the datasets should be connected. Elements in C can be set to zero when the datasets are unconnected and to zero when the datasets are fully connected. Furthermore, in the equation above, A(q) is a non-negative parameter that controls the amount of shrinkage and therefore the number of undamaged coefficients in ah (q) Penalization allows the selection of a subset of variables with non-null coefficients that define each ttf component score q) = X h <q) has h <q) The result is the identification of variables that are strongly correlated between and within omics datasets.

[0153] The sGCCA model of the above equation is iterative; a first set of coefficient vectors (ai <1> ...ai <q>< / q> ) is obtained by maximizing the equation above for h = 1 with Xi <q>< / q> =X <q>< / q> , before maximizing the equation for h = 2 using the residual matrices X2 (q) =Xi <q) -ti <q>< / q> ai <q>< / q>1 < q < Q. This process is repeated until a sufficient number of dimensions (or sets of components) is obtained. The optimization problem is solved using a monotonically convergent algorithm.

[0154] In the DIABLO approach, a set of omics data X <q>< / q> in the equation above is substituted with a matrix of dummy indicators Y (N x G) to indicate the class membership of each sample, where G is the number of phenotypic groups. Furthermore, for ease of use, the penalty parameter h is <q>< / q> has been replaced by the number of variables to select in each dataset and each component, because there is a direct correspondence between the two parameters.

[0155] PLS regression is particularly relevant for making a prediction in the case of small samples (e.g. n = 10 per group).

[0156] Alternatively, the comparison can be performed using support vector machines (SVMs), linear regression or derivatives thereof (such as the generalized linear model abbreviated as GLM, including logistic regression), linear discriminant analysis (LDA), random forests, k-NN (“Nearest Neighbour”) or PAM (“Predictive Analysis of Microarrays”) statistical methods.

[0157] computer program product

[0158] The present invention also relates to a computer program product comprising program code instructions for executing the steps of the methods as described in the "Methods" section above, when this program is executed by a computer.

[0159] In particular, the program product includes code instructions for executing steps a) and b) described in the "Methods" section above, when this program is executed by a computer, and, where applicable, step c).

[0160] To obtain the expression profile in step a), the program reads from a memory location where the expression profile was previously stored. For the comparison in step b), the program also accesses a memory location where each reference microRNA expression profile has been stored. This could be the memory location where the expression profile obtained in step a) was stored or a different memory location.

[0161] Kit

[0162] Another object of the invention relates to a kit for predicting the occurrence and / or severity of an injury induced by localized irradiation in a subject and / or for detecting localized irradiation in a subject and / or determining a level of localized irradiation in a subject, said kit comprising at least:

[0163] (i) a probe and / or primer specific to the hsa-let-7b-3p microRNA;

[0164] (ii) a probe and / or primer specific to the hsa-let-7d-3p microRNA;

[0165] (iii) a probe and / or primer specific to the hsa-miR-10a-5p microRNA;

[0166] (iv) a probe and / or primer specific to the hsa-miR-24-3p microRNA;

[0167] (v) a probe and / or primer specific to the hsa-miR-99b-5p microRNA;

[0168] (vi) a probe and / or primer specific to the hsa-miR-122-5p microRNA;

[0169] (vii) a probe and / or primer specific to the hsa-miR-139-5p microRNA;

[0170] (viii) a probe and / or primer specific to the hsa-miR-140-3p microRNA;

[0171] (ix) a probe and / or primer specific to the hsa-miR-191-5p microRNA;

[0172] (x) a probe and / or primer specific to the hsa-miR-214-3p microRNA;

[0173] (xi) a probe and / or primer specific to the hsa-miR-340-5p microRNA;

[0174] (xii) a probe and / or primer specific to the hsa-miR-361-5p microRNA;

[0175] (xiii) a probe and / or primer specific to the hsa-miR-501-5p microRNA;

[0176] (xiv) a probe and / or primer specific to the hsa-miR-652-3p microRNA; and

[0177] (xv) a probe and / or primer specific to the hsa-miR-744-5p microRNA; said kit comprising probes and / or primers specific to fewer than 200 different microRNAs; the kit may further comprise a computer program product including program code instructions for carrying out steps a) and b) of the methods as described in the "Methods" section above, when such program is executed by a computer.

[0178] In one particular embodiment, the kit according to the invention further comprises at least:

[0179] (xvi) a probe and / or primer specific to the hsa-miR-16-5p microRNA;

[0180] (xvii) a probe and / or primer specific to the hsa-miR-19a-3p microRNA;

[0181] (xviii) a probe and / or primer specific to the hsa-miR-29c-3p microRNA; (xix) a probe and / or primer specific to the hsa-miR-92a-3p microRNA;

[0182] (xx) a probe and / or primer specific to the hsa-miR-93-5p microRNA;

[0183] (xxi) a probe and / or primer specific to the hsa-miR-107 microRNA;

[0184] (xxii) a probe and / or primer specific to the hsa-miR-128-3p microRNA;

[0185] (xxiii) a probe and / or primer specific to the hsa-miR-142-3p microRNA;

[0186] (xxiv) a probe and / or primer specific to the hsa-miR-186-5p microRNA;

[0187] (xxv) a probe and / or primer specific to the hsa-miR-208a-3p microRNA;

[0188] (xxvi) a probe and / or primer specific to the hsa-miR-215-5p microRNA;

[0189] (xxvii) a probe and / or primer specific to the hsa-miR-324-3p microRNA;

[0190] (xxviii) a probe and / or primer specific to the hsa-miR-375-3p microRNA; and

[0191] (xxix) a probe and / or primer specific to the hsa-miR-450a-5p microRNA.

[0192] In another particular embodiment, the kit according to the invention further comprises at least:

[0193] (xvi) a probe and / or primer specific to the hsa-miR-16-5p microRNA;

[0194] (xvii) a probe and / or primer specific to the hsa-miR-19a-3p microRNA;

[0195] (xviii) a probe and / or primer specific to the hsa-miR-29c-3p microRNA;

[0196] (xix) a probe and / or primer specific to the hsa-miR-92a-3p microRNA;

[0197] (xx) a probe and / or primer specific to the hsa-miR-93-5p microRNA;

[0198] (xxi) a probe and / or primer specific to the hsa-miR-107 microRNA;

[0199] (xxii) a probe and / or primer specific to the hsa-miR-128-3p microRNA;

[0200] (xxiii) a probe and / or primer specific to the hsa-miR-142-3p microRNA;

[0201] (xxiv) a probe and / or primer specific to the hsa-miR-186-5p microRNA;

[0202] (xxv) a probe and / or primer specific to the hsa-miR-208a-3p microRNA;

[0203] (xxvi) a probe and / or primer specific to the hsa-miR-215-5p microRNA;

[0204] (xxvii) a probe and / or primer specific to the hsa-miR-324-3p microRNA;

[0205] (xxviii) a probe and / or primer specific to the hsa-miR-375-3p microRNA;

[0206] (xxix) a probe and / or primer specific to the hsa-miR-450a-5p microRNA;

[0207] (xxx) a probe and / or primer specific to the hsa-let-7e-5p microRNA;

[0208] (xxxi) a probe and / or primer specific to the hsa-miR-18a-5p microRNA;

[0209] (xxxii) a probe and / or primer specific to the hsa-miR-23a-3p microRNA;

[0210] (xxxiii) a probe and / or primer specific to the hsa-miR-23b-3p microRNA;

[0211] (xxxiv) a probe and / or primer specific to the hsa-miR-26a-5p microRNA;

[0212] (xxxv) a probe and / or primer specific to the hsa-miR-27b-3p microRNA;

[0213] (xxxvi) a probe and / or primer specific to the microRNA hsa-miR-1249-3p;

[0214] (xxxvii) a probe and / or primer specific to the hsa-miR-130b-3p microRNA;

[0215] (xxxviii) a probe and / or primer specific to the hsa-miR-142-5p microRNA; TJ

[0216] (xxxix) a probe and / or primer specific to the hsa-miR-143-3p microRNA;

[0217] (xl) a probe and / or primer specific to the hsa-miR-144-3p microRNA;

[0218] (xli) a probe and / or primer specific to the hsa-miR-146a-5p microRNA;

[0219] (xlii) a probe and / or primer specific to the microRNA hsa-miR-146b-5p; (xliii) a probe and / or primer specific to the microRNA hsa-miR-148b-3p; (xliv) a probe and / or primer specific to the microRNA hsa-miR-150-5p; (xlv) a probe and / or primer specific to the microRNA hsa-miR-185-5p; (xlvi) a probe and / or primer specific to the microRNA hsa-miR-195-5p; (xlvii) a probe and / or primer specific to the microRNA hsa-miR-199a-3p; (xlviii) a probe and / or primer specific to the microRNA hsa-miR-200a-3p; (xlix) a probe and / or primer specific to the microRNA hsa-miR-200b-3p;

[0220] (I) a probe and / or primer specific to the hsa-miR-204-5p microRNA;

[0221] (li) a probe and / or primer specific to the hsa-miR-205-5p microRNA;

[0222] (lii) a probe and / or primer specific to the hsa-miR-222-3p microRNA;

[0223] (liii) a probe and / or primer specific to the microRNA hsa-miR-223-3p; (liv) a probe and / or primer specific to the microRNA hsa-miR-331-3p; (iv) a probe and / or primer specific to the microRNA hsa-miR-339-5p; (ivi) a probe and / or primer specific to the microRNA hsa-miR-342-3p; (ivii) a probe and / or primer specific to the microRNA miR-484;

[0224] (iviii) a probe and / or primer specific to the hsa-miR-497-5p microRNA; and (lix) a probe and / or primer specific to the hsa-miR-532-5p microRNA.

[0225] The term "probe" here refers to a nucleic acid connected to at least one marker. A marker here refers to a marker or identifier for identification and / or differentiation connected to or incorporated within any entity, such as a compound, a biological particle (e.g., a cell, bacterium, spore, virus, or organelle), or a partition. A marker might, for example, be a dye that makes an entity optically detectable and / or optically distinguishable. Examples of dyes used for labeling include fluorescent dyes (fluorophores) and fluorescence extinguishers. A probe might be a sequence-specific binding partner for a nucleic acid target and / or an amplicon. Amplification of the target sequence might involve the use of at least one probe capable of binding to at least one target sequence.

[0226] In a PCR method, the amplified nucleic acid product can be detected in several ways. Several fluorescence-based detection methods can be performed in real time and include the use of a fluorescent intercalating agent such as SYBR® Green I or a probe that hybridizes to the amplification products. Examples of such fluorescence-based methods include adjacent hybridization probes (Wittwer, CT. et al., 1997, BioTechniques 22:130-138), molecular beacon probes (Tyagi S. and Kramer FR 1996, Nat. Biotech. 14:303-308), and scorpion probes (Whitcomb et al., 1999, Nat. Biotech. 17:804-807). Adjacent hybridization probes are typically designed to be internal to the amplification primers. The 3' end of a probe is labeled with a donor fluorophore while the 5' end of an adjacent probe is labeled with an acceptor fluorophore.When two probes are specifically hybridized in close proximity (spaced 1 to 5 nucleotides apart), the donor fluorophore, excited by an external light source, emits light that is absorbed by a second acceptor. This second acceptor emits more fluorescence, producing a fluorescence resonance energy transfer (FRET) signal. Molecular tag probes have a stem-and-loop structure in which the loop is the probe, and at the base of the stem, a fluorescent group is located at one end while a quenching group is at the other. Molecular tags undergo a fluorogenic conformational change upon hybridization to their targets, thus separating the fluorochrome from its quenching agent. Real-time PCR amplicon detection devices are capable of performing rapid PCR cycles combined with fluorescent intercalating agents such as SYBR® Green I or with FRET detection.

[0227] The probe(s) used may thus include a component comprising at least one detectable marker. The detectable marker may be capable of producing an optical signal. Alternatively, the detectable marker may include a fluorophore. Examples of fluorophores include, but are not limited to, fluorescent proteins, for example GFP (green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein); non-protein fluorophores selected from the group consisting of xanthene derivatives (for example, fluorescein, rhodamine, Oregon green, eosin, 6-carboxyfluorescein and Texas red); cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine), squaraine derivatives and cyclic-substituted squaraines, including Seta, SeTau and Square dyes, naphthalene derivatives (dansyl and prodan derivatives), coumarin derivatives,Oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), anthracene derivatives (e.g., anthraquinones, DRAQ5, DRAQ7, and CyTRAK Orange), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin), and their derivatives. The detectable marker(s) are advantageously capable of producing a variable signal. The variable signal can be generated upon hybridization of the probe to the target sequence. For example, the signal may be detectable before the probe binds to the target sequence, and during the hybridization of the probe to the target sequence,The signal is reduced in intensity or becomes completely undetectable. In another example, the detectable signal may be produced only during probe hybridization to the target sequence, or the strength of the detectable signal may be increased during probe hybridization to the target sequence.

[0228] In some cases, it may be advantageous to use a component comprising two detectable markers. The two detectable markers can operate independently. Alternatively, the two detectable markers can be a pair of interactive markers, preferably capable of generating a variable signal. For example, the signal might be detectable before the probe binds to the target sequence, and upon hybridization of the probe to the target sequence, the signal's intensity decreases or it becomes completely undetectable. Alternatively, the detectable signal might be produced only upon hybridization of the probe to the target sequence, or the intensity of the detectable signal might increase upon hybridization of the probe to the target sequence. In one specific example, the detectable signal is not generated when the two detectable markers are bound together by the probe sequence.Once at least one detectable marker is cleaved from the probe, the detectable signal is generated.

[0229] Preferably, the interactive marker pair may include a fluorophore and a snuffer. In one specific aspect, the fluorophore may be located at the 5' end of the probe, and the snuffer may be located at the 3' end of the probe. Examples of snuffers include, but are not limited to, TAMRA (tetramethylrhodamine), TaqMan® MGB, and BHQ™ (Black Hole Quencher™).

[0230] The probe(s) used in the context of the invention preferably have a length of at least 5, 10, 15, or 20 nucleotides. Alternatively, the length of the probe(s) used in the context of the present invention is less than 40, 35, 30, or 25 nucleotides. The length of the probe(s) may also be between 5 and 40 nucleotides, between 10 and 35 nucleotides, between 15 and 30 nucleotides, or between 20 and 25 nucleotides. In another aspect, the probe(s) used in the context of the present invention have a length of 8 nucleotides, or 9 nucleotides, or 10 nucleotides, or 12 nucleotides, or 14 nucleotides, or 16 nucleotides, or 18 nucleotides, or 20 nucleotides, or 22 nucleotides, or 24 nucleotides, or 26 nucleotides, or 28 nucleotides, or 30 nucleotides, or 32 nucleotides, or 34 nucleotides, or 36 nucleotides, or 38 nucleotides, or 40 nucleotides.In one particular embodiment, the probes used in the invention may advantageously comprise locked nucleic acids (LNAs) (modified nucleic acids) in order to increase the melting temperature (Tm) of the probes, thereby optimizing their specificity to the targeted DNA sequence. The publication by Matthew P. Johnson et al. (Nucleic Acids Research, 2004, Vol. 32, No. 6 e55) on LNA technology is a relevant reference. Thus, in a particular and preferred embodiment, the probes used in the present invention are LNA probes.

[0231] The term "primer" here refers to a single-stranded oligonucleotide or DNA fragment that hybridizes with a nucleic acid strand in such a way that the 3' end of the primer can act as a polymerization and extension site with the aid of a DNA polymerase enzyme. The primer may consist of any combination of nucleotides or their analogs, which may optionally be linked to form a linear polymer of any suitable length. A primer can have any suitable length, such as at least approximately 10, 15, 20, or 30 nucleotides. In some embodiments, primer lengths are in the range of approximately 10 to 60 nucleotides, approximately 12 to 50 nucleotides, approximately 15 to 30 nucleotides, and approximately 15 to 40 nucleotides. Preferably, the primer length is between 15 and 30 nucleotides.

[0232] In a preferred embodiment, the primers used in the invention are produced by synthesis. In some embodiments, a primer can be combined with a compatible primer in an amplification or synthesis reaction to form a primer pair consisting of a sense primer and an antisense primer. "Primer pair" thus refers to two primers comprising a sense primer that hybridizes to a single strand at one end of the DNA sequence to be amplified, and an antisense primer that hybridizes to the complementary strand at the other end of the DNA sequence to be amplified. Optionally, the sense primer initiates the synthesis of a first strand of nucleic acid, and the antisense primer initiates the synthesis of a second strand of nucleic acid, the first and second strands being substantially complementary to each other, or capable of hybridizing to form a double-stranded nucleic acid molecule.In some embodiments, one end of an amplification or synthesis product is defined by the sense primer, and the other end is defined by the antisense primer. Consequently, a primer pair can be a sense primer and an antisense primer, which together define the opposite ends (and thus the length) of a resulting amplicon. Typically, a primer is capable of hybridizing to a corresponding target sequence and undergoing primer extension when exposed to amplification conditions in the presence of dNTPs and a polymerase.

[0233] Primers can be prepared by various methods, including cloning of suitable sequences and direct chemical synthesis using well-established methods in the field (Narang et al., Methods Enzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)). Primers can also be obtained from commercial sources such as Qiagen or Applied Biosystems / Thermo Fisher.

[0234] In the context of the present invention, a "microRNA X-specific primer / probe" and its derivatives generally refer to a single-stranded or double-stranded polynucleotide, generally an oligonucleotide, which comprises at least one sequence that is complementary to at least 50%, generally complementary to at least 75% or at least 85%, more generally complementary to at least 90%, more generally complementary to at least 95%, more generally complementary to at least 98% or at least 99%, or identical, to at least a portion of a microRNA X. In such cases, the microRNA X-specific primer / probe and the microRNA X sequence are described as "matching" each other.In some embodiments, the microRNA X-specific primer / probe is substantially non-complementary to other microRNA sequences present in the sample; optionally, the microRNA X-specific primer / probe is substantially non-complementary to other nucleic acid molecules present in the sample. In some embodiments, the microRNA X-specific primer / probe may include minimal cross-hybridization with other specific primers / probes in the amplification reaction. In some embodiments, the microRNA X-specific primers / probes include minimal cross-hybridization with non-specific sequences in the amplification reaction mixture.In some embodiments, a microRNA X-specific sense primer and a microRNA X-specific antisense primer define a microRNA X-specific primer pair that can be used to amplify the microRNA X sequence via template-dependent primer extension. Preferably, the microRNA X-specific primers exhibit minimal self-complementarity.

[0235] When the kit is intended for use in a method of measuring an expression level that does not use a probe (e.g., quantitative PCR using a fluorescent intercalating agent), the kit advantageously includes only primers (no probes).

[0236] When the kit is intended for use in a method of measuring an expression level that uses a probe (e.g., quantitative PCR using a probe, such as those described above), the kit may include only primers (no probes, these can be ordered separately), only probes (no primers, these can be ordered separately), or advantageously both primers and probes.

[0237] In the context of the present invention, the kit comprises probes and / or primers specific to fewer than 200 different microRNAs, preferably fewer than 190 different microRNAs, fewer than 180 different microRNAs, fewer than 170, fewer than 160, fewer than 150, fewer than 140, fewer than 130, fewer than 120, fewer than 110, fewer than 100, fewer than 90, fewer than 80, fewer than 70, or fewer than 65 different microRNAs. In a particular embodiment, the kit comprises fewer than 200 pairs of different primers, in particular fewer than 190 pairs of different primers, fewer than 180 pairs of different primers, fewer than 170, fewer than 160, fewer than 150, fewer than 140, fewer than 130, fewer than 120, fewer than 110, fewer than 100, fewer than 90, fewer than 80, fewer than 70, or fewer than 65 pairs of different primers.In another particular embodiment, the kit comprises fewer than 200 different probes, in particular fewer than 190 different probes, fewer than 180 different probes, fewer than 170, fewer than 160, fewer than 150, fewer than 140, fewer than 130, fewer than 120, fewer than 110, fewer than 100, fewer than 90, fewer than 80, fewer than 70, or fewer than 65 different probes.

[0238] In a particular embodiment, the kit according to the invention further includes at least one reagent for carrying out a nucleic acid amplification reaction.

[0239] The reagents used to perform nucleic acid amplification are well known to those skilled in the art and include, for example, polymerases, reverse transcriptases, and nucleotides such as deoxyribonucleotide triphosphates (dNTPs).

[0240] In a particular embodiment, one or more of said primers or probes comprise a marker, as defined above.

[0241] In one particular embodiment, the marker comprises a fluorophore.

[0242] The term "fluorophore" refers to molecules capable of emitting light when excited by a light source. Fluorophores are well-known molecules to those skilled in the art and include, for example, Alexa Fluor type fluorophores (Alexa 350, Alexa 488, Alexa 555, Alexa 647, etc.), Cyanine type fluorophores (CY3, CY3.5, CY5, etc.), FAM type fluorophores (fluorescein amidite), Fluorescein Isothiocyanate (FITC), HEX type fluorophores, Texas Red type fluorophores, and ATTO type fluorophores.

[0243] The term "quencher" refers to a chemical species capable of deactivating an excited state created in a molecular entity by energy or electron transfer, or by a chemical mechanism. Quenchers are molecules well known to those skilled in the art, the most commonly used being Dabcyl, Eclipse Dark Quencher, and Black Hole Quencher. A fluorophore can also act as a quencher. For this to occur, the emission spectrum of the fluorophore attached to the 5' end must not overlap with the excitation spectrum of the fluorophore-quencher attached to the 3' end. This non-exhaustive list is intended to illustrate the concept of a quencher, but it should in no way restrict the present invention to the use of these quenchers alone.

[0244] In the present invention, the probes used can be probes corresponding to the definition of Taqman technology. Such probes are labeled at their 5' end with a fluorophore, for example FAM, and at their 3' end with a quencher, for example TAMRA, which inhibits fluorophore emission when they are in close proximity. During PCR, if the probe hybridizes to its target, it is hydrolyzed by DNA polymerase. The fluorophore thus separated from the quencher emits a signal proportional to the number of probes hydrolyzed, measurable at the time of elongation.

[0245] In a particular embodiment, the kit according to the invention further comprises an intercalating agent.

[0246] The term "intercalating agent" refers to a molecule capable of reversibly intercalating into DNA and becoming fluorescent when located within the double helix. This may include SYBRGreen, a SYTO-type fluorophore (SYTO9, SYTO82, etc.), EvaGreen, ethidium bromide, or any other DNA intercalating agent used in real-time monitoring of nucleic acid amplification. For the purposes of this invention, said intercalating agent may be selected from: SYBRGreen, a SYTO-type fluorophore (SYTO9, SYTO82, etc.), EvaGreen, and ethidium bromide.

[0247] In one particular embodiment, the intercalating agent is a fluorescent intercalating agent.

[0248] The present invention will be illustrated in more detail by the example below.

[0249] EXAMPLE

[0250] The present invention is based on the use of omics-type technologies for broad-spectrum screening of molecules (microRNAs) present in blood, allowing minimally invasive access to biological samples containing the molecules of interest, using a preclinical model of localized irradiation established in mice and rats.

[0251] Materials and methods

[0252] The preclinical model used consisted of exposing a 2x2 cm area of ​​the hind leg of 10-week-old male C57BL / 6 mice to different doses of ionizing radiation (IR) using a medical accelerator. Several cohorts of mice (n=30 / dose group / analysis time) were exposed under gaseous anesthesia (isoflurane) to X-ray doses (10 MV) of 0, 20, 40, and 80 Gy, leading, after an asymptomatic latency period of 7 days, to the development of radiation-induced lesions of distinct severities after 14 days (Figure 1).

[0253] On days 1 and 7 post-exposure, blood was collected by intracardiac puncture in the presence of 0.105 M sodium citrate (anticoagulant). Plasma was obtained by successive centrifugation at 1500 x g, 15 min, 4°C, and then platelet-poor plasma by centrifugation at 13000 x g, 2 min, 4°C.

[0254] For each animal, a complete blood count (CBC, standard analyzer), plasma C-reactive protein level (enzyme-linked immunosorbent assay ELISA), cutaneous blood flow (laser Doppler imaging) and cutaneous barrier function (intransient water loss, IWL) were recorded before euthanasia.

[0255] An observational lesion score was also established based on clinical criteria including the presence and intensity of erythema, the extent of the lesion, the degree of dryness or moisture of the wound, and paw retraction. All these elements were integrated into a multivariate analysis with microRNA expression data to generate the most relevant mathematical models and select the most robust microRNAs to constitute prognostic signatures.

[0256] The level of microRNA expression can be obtained by various techniques commonly used by people skilled in the art, including nucleic acid chips (DNA, oligonucleotides, LNA) and next-generation high-throughput sequencing and quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction).

[0257] RNAs were extracted from plasma using commercial kits such as miRNeasy Plasma / serum Advanced (Qiagen) or Trizol (Invitrogen), according to procedures well known to those skilled in the art. The RT step was performed using a method employing a modified oligo-dT after 5' polyadenylation of the microRNAs (miRCURY® system, Qiagen).

[0258] Quality controls were added during the RNA purification and RT steps using artificial oligonucleotides (UniSp6 as a reverse transcription quality control and UniSp3 as an amplification quality control). The PCR step was performed using commercial miRCURY® primers specific to each microRNA, validated internally by the supplier, and listed in Table 2.

[0259] [Table 2]

[0260]

[0261] Real-time quantification of the amplification product was achieved by using a fluorescent double-stranded DNA intercalator (SYBRGreen present in the miRCURY® kit).

[0262] Results

[0263] The “miR-BR” panel, composed of 62 microRNAs (+ 2 internal controls) was constructed from the broad-spectrum microRNA signatures obtained at times J1 and J7 on 2 independent mouse cohorts (n=10 / group / cohort), as well as the 8 microRNAs of the diagnostic signature previously established at J14 (Ancel et al. 2024), and some additional reference microRNAs for normalization of expression levels and comparison of individuals (Table 3).

[0264] [Table 3]

[0265] : microRNA not identified in humans

[0266] The relative expression levels of the microRNAs of interest were quantified using the α-ACt method against the expression levels of reference microRNAs, a method well-known to those skilled in the art. The α-ACt values ​​were integrated with the other pathophysiological variables listed above to perform a supervised multivariate analysis in order to model a multiscale signature capable of simultaneously discriminating between different dose groups and highlighting the most significant correlations between the different entities on each scale.

[0267] More specifically, two covariate blocks were defined for each sample: a "microRNA" block comprising all microRNA expression measurements, and a "clinical" block consisting of CRP data, CBC, lesion score values ​​(where applicable), weight, cutaneous blood perfusion, and PIE.

[0268] To conduct an integrative study by simultaneously analyzing the two different blocks, a discriminant analysis using the partial least squares method (sPLS-DA) was performed using the DIABLO method from the "mixOmics" package (version 6.18.1) in R. DIABLO extends the correlation analysis of covariate blocks measured as continuous values ​​by including an additional block corresponding to the dose group of each sample. This model is particularly useful in the context of this study because it avoids the subjective categorization of lesion scores (clinical block), which are maintained on their original continuous scale, and also takes into account the categorical nature of the dose groups.

[0269] DIABLO maximizes the covariance (correlation after data normalization) between different blocks by constructing components that exhibit maximum correlations conditional on the dose, thus enabling the simultaneous performance of association and classification tasks. In this study, the model's hyperparameters (used to control the learning process) were defined to achieve an equitable weighting between the correlation and classification tasks. The multiscale signature produced by this model is therefore optimal for simultaneously classifying dose groups and maximizing the correlation between different blocks, and in particular between microRNA expression and lesion score.

[0270] The mathematical models generated after multivariate analyses on the measurements made allow, on the one hand, to identify individuals according to their level of exposure to ionizing radiation, and on the other hand, to predict the risk of developing a radiation-induced lesion and to estimate its severity.

[0271] The miR-BR panel (Table 3) was used for targeted analysis of plasma microRNAs in two new independent mouse cohorts to confirm the relevance of these microRNAs and their potential for segregation of mice according to dose groups.

[0272] Thirty-one microRNAs were selected by the model for these two analysis time points, with 24 microRNAs common to the J1 and J7 signatures, including 9 microRNAs with a stability score greater than 0.5 (Table 4). The performance of these models is highly advantageous for categorizing mice according to their dose group (Figure 2).

[0273] [Table 4]

[0274] : microRNA not identified in humans

[0275] To test the interoperability of these microRNA signatures in another animal species, the inventors conducted a similar study in a preclinical model of radiological burns in Sprague Dawley rats. Two independent cohorts of 3-month-old male rats were used (N=43, n=10⁻¹¹ rats / group) in this study. Similar to the mouse model, the rats' left hind legs (2.5 cm x 4 cm) were exposed to 20, 40, or 80 Gy (plus an unexposed control group), and the animals were then monitored for 3 months with weekly assessments of injury score and measurements of weight, cutaneous blood flow, and PIE. A blood sample was taken on day 1 and day 7 post-exposure to extract RNAs and quantify the plasma expression levels of microRNAs present using the miR-BR panel (Table 3), according to the same protocol as in mice, described above.

[0276] In this study, multivariate analyses were able to take into account, in addition to microRNA expression levels on days 1 and 7, all clinical monitoring measurements performed weekly during the 12 weeks following exposure to RI. Thus, each rat was associated with specific microRNA expression measurements at two time points during the asymptomatic latency phase (days 1 and 7), and with the evolution of its lesion at subsequent time points, allowing the generation of a predictive model at days 1 and 7 associated with the actual severity of the lesion (Figure 3).

[0277] A total of 42 microRNAs with a stability score >0.5 were selected by the model for these 2 analysis times, with 11 microRNAs common to the 2 signatures J1 and J7 (Table 5).

[0278] [Table 5]

[0279] : microRNA not identified in humans

[0280] Comparing the results obtained in mice and rats, 17 of the 31 microRNAs identified in mice (Table 4) were also identified in the rat model (Table 6). For each microRNA, the identifier number and nucleotide sequence are provided according to the latest version of the miRBase reference database (mirbase.org, v22). Of these 17 microRNAs, 15 exist in humans and can therefore be included in a molecular signature applicable to humans.

[0281] [Table 6]

[0282] : microRNA not identified in humans

[0283] Figures 4 and 5 illustrate the correlations between the molecular signature identified in the example above on day 1 or day 7 and the different lesion scores observed in rats. Briefly, for each clinical monitoring parameter, correlation analyses were performed between the temporal functions of changes over 12 weeks post-irradiation of these functional parameters, on the one hand, and the microRNA signatures on day 1 (Figure 4) or day 7 (Figure 5), on the other. Significant correlations were thus obtained for both microRNA signatures with the 12-week post-irradiation changes in erythema intensity, degree of lesion moisture or edema, paw retraction, overall lesion score, and transepidermal water loss.

[0284] These results confirm the value of these molecular signatures for predicting the occurrence or severity of radiation-induced injury. In conclusion, the molecular signatures established in these preclinical models (mouse and rat) make it possible to distinguish exposed individuals according to the radiation dose group received and the degree of severity experienced (measurements taken during the asymptomatic latency phase) or confirmed (measurements taken during the manifestation phase of lesions).

[0285] REFERENCES

[0286] • Acharya, SS et al. (2015) Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury. Science translational medicine 7, 287ra269.

[0287] • Cui, W., Ma, J., Wang, Y. & Biswal, S. (2011) Plasma miRNA as biomarkers for assessment of total-body radiation exposure dosimetry. PloS one 6, e22988.

[0288] • Fendler, W. et al. (2017) Evolutionarily conserved serum microRNAs predict radiation- induced fatality in nonhuman primates. Science translational medicine 9.

[0289] • Islam, A., Ghimbovschi, S., Zhai, M. & Swift, J. M. (2015) An Exploration of Molecular Correlates Relevant to Radiation Combined Skin-Burn Trauma. PloS one 10, e0134827.

[0290] • Jacob, N. K. et al. (2013) Identification of sensitive serum microRNA biomarkers for radiation biodosimetry. PloS one 8, e57603.

[0291] • Menon, N. et al. (2016) Detection of Acute Radiation Sickness: A Feasibility Study in Non-Human Primates Circulating miRNAs for Triage in Radiological Events. PloS one 11 , e0167333.

[0292] • Port, M. et al. (2016) MicroRNA Expression for Early Prediction of Late Occurring Hematologic Acute Radiation Syndrome in Baboons. PloS one 11, e0165307.

[0293] • Wei, W. et al. (2017) Serum microRNAs as Early Indicators for Estimation of Exposure Degree in Response to Ionizing Irradiation. Radiation research 188, 342-354.

[0294] • Ancel et al. (2024) MicroRNA blood signature for localized radiation injury. Scientific Reports 14 :2681.

[0295] • Ambros et al. (2003) A uniform system for microRNA annotation. RNA 9(3) :277-279.

[0296] • Griffiths-Jones (2004) The microRNA Registry. NAR 32(Database Issue) :D109-D111.

[0297] • Griffiths-Jones et al. miRBase: microRNA sequences, targets and gene nomenclature. (2006) NAR 34(Database Issue) :D140-D144.

[0298] • Griffiths-Jones et al. miRBase: tools for microRNA genomics. (2008) NAR 36 (Database Issue) :D154-D158. • Kozomara et al. (2011) miRBase: integrating microRNA annotation and deep- sequencing data. NAR 39(Database Issue) :D152-157.

[0299] • Fliedner, I. Friesecke, et K. Beyrer, éditeurs, Medical management of radiaion accidents. Manual on the acute radiation syndrome. Londres : The Bitish Institute of Radiology, 2001.

[0300] • Singh et al. (2019) DIABLO : an integrative approach for identifying key molecular drivers from multi-omics assays Bioinformatics 35:3055-3062.

[0301] • Gueguen et al. (2024) Les micro-ARN comme biomarqueurs des lesions radio-induites médecine / sciences 40 : 634-42. • International Atomic Energy Agency, Medical Management of Radiation Injuries Safety

[0302] Reports Series No. 101

Claims

42 DEMANDS 1. An in vitro method for predicting the occurrence and / or severity of an injury induced by localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in the biological sample of the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from a biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, and c) based on the comparison in step b), predict the occurrence and / or severity of an injury in the subject following localized irradiation.

2. An in vitro method for detecting localized irradiation in a subject and / or determining a level of localized irradiation in a subject, said method comprising the following steps: a) obtaining an expression profile of at least the following 15 microRNAs in the biological sample of the subject: (i) hsa-let-7b-3p; (ii) hsa-let-7d-3p; (iii) hsa-miR-10a-5p; (iv) hsa-miR-24-3p; (v) hsa-miR-99b-5p; (vi) hsa-miR-122-5p; (vii) hsa-miR-139-5p; (viii) hsa-miR-140-3p; (ix) hsa-miR-191-5p; (x) hsa-miR-214-3p; (xi) hsa-miR-340-5p; (xii) hsa-miR-361-5p; (xiii) hsa-miR-501-5p; (xiv) hsa-miR-652-3p; and (xv) hsa-miR-744-5p, the expression profile being previously measured in vitro from a biological sample of said subject;b) compare the microRNA expression profile obtained in step a) with at least one reference microRNA expression profile, c) based on the comparison in step b), detect localized irradiation and / or determine the level of localized irradiation in the subject.

3. Method according to claim 1 or 2, wherein the expression profile further comprises the expression levels of the following 14 microRNAs in the biological sample of the subject: (xvi) hsa-miR-16-5p; (xvii) hsa-miR-19a-3p; (xviii) hsa-miR-29c-3p; (xix) hsa-miR-92a-3p; (xx) hsa-miR-93-5p; (xxi) hsa-miR-107; (xxii) hsa-miR-128-3p; (xxiii) hsa-miR-142-3p; (xxiv) hsa-miR-186-5p; (xxv) hsa-miR-208a-3p; (xxvi) hsa-miR-215-5p; (xxvii) hsa-miR-324-3p; (xxviii) hsa-miR-375-3p; and (xxix) hsa-miR-450a-5p; and possibly further the expression levels of the following 30 microRNAs in 43. The subject's biological sample: (xxx) hsa-let-7e-5p ; (xxxi) hsa-miR-18a-5p ; (xxxii) hsa-miR-23a-3p ; (xxxiii) hsa-miR-23b-3p ; (xxxiv) hsa-miR-26a-5p ; (xxxv) hsa-miR-27b-3p ; (xxxvi) hsa-miR-1249-3p ; (xxxvii) hsa-miR- 130b-3p ; (xxxviii) hsa-miR-142-5p ; (xxxix) hsa-miR-143-3p ; (xl) hsa-miR-144-3p ; (xli) hsa- miR-146a-5p ; (xlii) hsa-miR-146b-5p ; (xliii) hsa-miR-148b-3p ; (xliv) hsa-miR-150-5p ; (xlv) hsa-miR-185-5p ; (xlvi) hsa-miR-195-5p ; (xlvii) hsa-miR-199a-3p ; (xlviii) hsa-miR-200a-3p ; (xlixii) hsa-miR-200b-3p ; (I) hsa-miR-204-5p ; (li) hsa-miR-205-5p ; (lii) hsa-miR-222-3p ; (liii) hsa-miR-223-3p ; (color) hsa-miR-331-3p ; (Iv) hsa-miR-339-5p ; (Ivi) hsa-miR-342-3p ; (Ivii) hsa- miR-484 ; (Iviii) hsa-miR-497-5p ; et (lix) hsa-miR-532-5p.

4. Method according to any one of claims 1 to 3, wherein the expression profile obtained is compared, in step b), to at least one reference microRNA expression profile obtained from a biological sample of a non-irradiated subject and / or a subject who has undergone a given level of localized irradiation.

5. Method according to any one of claims 1 to 4, wherein the biological sample of said subject is a blood sample.

6. Method according to any one of claims 1 to 5, comprising the step of determining the expression profile from the biological sample of said subject.

7. Method according to claim 6, wherein the expression profiles of microRNAs are determined by quantitative PCR.

8. Method according to any one of claims 6 and 7, wherein the biological sample is obtained less than one week after possible irradiation of said subject, preferably less than one day after possible irradiation of said subject.

9. Product computer program comprising program code instructions for executing the steps of the method according to any one of claims 1 to 5, when such program is executed by a computer.

10. Kit for predicting the occurrence and / or severity of an injury induced by localized irradiation in a subject and / or for detecting localized irradiation in a subject and / or determining a level of localized irradiation in a subject, said kit comprising at least: (i) a probe and / or primer specific to the hsa-let-7b-3p microRNA; (ii) a probe and / or primer specific to the hsa-let-7d-3p microRNA; 44 (iii) a probe and / or primer specific to the hsa-miR-10a-5p microRNA; (iv) a probe and / or primer specific to the hsa-miR-24-3p microRNA; (v) a probe and / or primer specific to the hsa-miR-99b-5p microRNA; (vi) a probe and / or primer specific to the hsa-miR-122-5p microRNA; (vii) a probe and / or primer specific to the hsa-miR-139-5p microRNA; (viii) a probe and / or primer specific to the hsa-miR-140-3p microRNA; (ix) a probe and / or primer specific to the hsa-miR-191-5p microRNA; (x) a probe and / or primer specific to the hsa-miR-214-3p microRNA; (xi) a probe and / or primer specific to the hsa-miR-340-5p microRNA; (xii) a probe and / or primer specific to the hsa-miR-361-5p microRNA; (xiii) a probe and / or primer specific to the hsa-miR-501-5p microRNA; (xiv) a probe and / or primer specific to the hsa-miR-652-3p microRNA; and (xv) a probe and / or primer specific to the hsa-miR-744-5p microRNA; said kit comprising probes and / or primers specific to fewer than 200 different microRNAs; the kit further comprising optionally a computer program product including program code instructions for carrying out steps a) and b) of the method according to any one of claims 1 to 8, when such program is executed by a computer.

11. Kit according to claim 10, further comprising at least: (xvi) a probe and / or primer specific to the hsa-miR-16-5p microRNA; (xvii) a probe and / or primer specific to the hsa-miR-19a-3p microRNA; (xviii) a probe and / or primer specific to the hsa-miR-29c-3p microRNA; (xix) a probe and / or primer specific to the hsa-miR-92a-3p microRNA; (xx) a probe and / or primer specific to the hsa-miR-93-5p microRNA; (xxi) a probe and / or primer specific to the hsa-miR-107 microRNA; (xxii) a probe and / or primer specific to the hsa-miR-128-3p microRNA; (xxiii) a probe and / or primer specific to the hsa-miR-142-3p microRNA; (xxiv) a probe and / or primer specific to the hsa-miR-186-5p microRNA; (xxv) a probe and / or primer specific to the hsa-miR-208a-3p microRNA; (xxvi) a probe and / or primer specific to the hsa-miR-215-5p microRNA; (xxvii) a probe and / or primer specific to the hsa-miR-324-3p microRNA; (xxviii) a probe and / or primer specific to the hsa-miR-375-3p microRNA; and (xxix) a probe and / or primer specific to the hsa-miR-450a-5p microRNA; and possibly comprising in addition at least: (xxx) a probe and / or primer specific to the hsa-let-7e-5p microRNA; (xxxi) a probe and / or primer specific to the hsa-miR-18a-5p microRNA; (xxxii) a probe and / or primer specific to the hsa-miR-23a-3p microRNA; (xxxiii) a probe and / or primer specific to the hsa-miR-23b-3p microRNA; (xxxiv) a probe and / or primer specific to the hsa-miR-26a-5p microRNA; (xxxv) a probe and / or primer specific to the hsa-miR-27b-3p microRNA; (xxxvi) a probe and / or primer specific to the microRNA hsa-miR-1249-3p; (xxxvii) a probe and / or primer specific to the hsa-miR-130b-3p microRNA; (xxxviii) a probe and / or primer specific to the hsa-miR-142-5p microRNA; (xxxix) a probe and / or primer specific to the hsa-miR-143-3p microRNA; (xl) a probe and / or primer specific to the hsa-miR-144-3p microRNA; (xli) a probe and / or primer specific to the hsa-miR-146a-5p microRNA; (xlii) a probe and / or primer specific to the hsa-miR-146b-5p microRNA; (xliii) a probe and / or primer specific to the hsa-miR-148b-3p microRNA; (xliv) a probe and / or primer specific to the hsa-miR-150-5p microRNA; (xlv) a probe and / or primer specific to the hsa-miR-185-5p microRNA; (xlvi) a probe and / or primer specific to the hsa-miR-195-5p microRNA; (xlvii) a probe and / or primer specific to the hsa-miR-199a-3p microRNA; (xlviii) a probe and / or primer specific to the hsa-miR-200a-3p microRNA; (xlix) a probe and / or primer specific to the hsa-miR-200b-3p microRNA; (I) a probe and / or primer specific to the hsa-miR-204-5p microRNA; (li) a probe and / or primer specific to the hsa-miR-205-5p microRNA; (lii) a probe and / or primer specific to the hsa-miR-222-3p microRNA; (liii) a probe and / or primer specific to the hsa-miR-223-3p microRNA; (liv) a probe and / or primer specific to the hsa-miR-331-3p microRNA; (iv) a probe and / or primer specific to the hsa-miR-339-5p microRNA; (Ivi) a probe and / or primer specific to the hsa-miR-342-3p microRNA; (Ivii) a probe and / or primer specific to the microRNA miR-484; (Iviii) a probe and / or primer specific to the hsa-miR-497-5p microRNA; and (lix) a probe and / or primer specific to the hsa-miR-532-5p microRNA.

12. Kit according to claim 10 or 11, further comprising at least one reagent for carrying out a nucleic acid amplification reaction.

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