WO2019057972A1 - Procédés et kits permettant de déterminer si un patient souffrant de drépanocytose a des vaso-occlusions ou présente un risque d'avoir des vaso-occlusions - Google Patents

Procédés et kits permettant de déterminer si un patient souffrant de drépanocytose a des vaso-occlusions ou présente un risque d'avoir des vaso-occlusions Download PDF

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WO2019057972A1
WO2019057972A1 PCT/EP2018/075809 EP2018075809W WO2019057972A1 WO 2019057972 A1 WO2019057972 A1 WO 2019057972A1 EP 2018075809 W EP2018075809 W EP 2018075809W WO 2019057972 A1 WO2019057972 A1 WO 2019057972A1
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scd
vaso
cirdna
occlusions
dnase
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Olivier Blanc-Brude
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Descartes
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods and kits for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions.
  • Extracellular genomic DNA has been identified in human blood and in cell cultures 1_3 .
  • Circulating extracellular DNA in plasma benefits from renewed interest, as high affinity fluorescent nucleotide-intercalating probes facilitate its reliable quantification in body fluids.
  • CirDNA was quantified in human pathology and animal models. In healthy humans, cirDNA may occur between 1 and 27 ng/ml according to sample processing and DNA extraction techniques 4 ' 5 . CirDNA levels are increased during deep vein thrombosis 6"9 , infection and septic shock 10 ⁇ 12 , transfusion-related acute lung injury 13 ⁇ 15 , after severe trauma 11,16 , or in pre-eclamptic women 11 . Moreover, cirDNA levels increase up to 200 ng/ml in gastric and breast cancer 18 ' 19 .
  • Sickle cell disease is a single gene disorder characterized by mutant hemoglobin- S (HbS) and chronic intravascular hemolysis. It is characterized chronic anemia, increased levels of cell-free hemoglobin (Hb) and red blood cell (RBC) membrane microvesicles (MV), also called microp articles (MP), in plasma 20 23 . Painful vaso-occlusive crises (VOC) are typical of SCD and often associated to a further rise in hemolysis 20 ⁇ 24 ⁇ 25 .
  • cirDNA levels are upregulated in SCD, during VOC 5 ' 26 ' 27 ,, and associated to high nucleosome levels in plasma 27 ' 28 .
  • CirDNA may be released by activated leukocytes, as neutrophil extracellular traps (NETs).
  • NETs are genomic DNA-based net- like structures released by neutrophils after chromatin decondensation. NETs concentrate specific enzymatic activities, including neutrophil elastase (NE), myeloperoxidase (MPO) and a-defensins. NETs can participate in innate immunity and pathogen clearance by capturing and eliminating bacteria, fungi and viruses 29 0 .
  • NETs may also contribute to thrombosis 6 ' 7,9 , by recruiting and activating platelets, or mixing with and strengthening fibrin meshwork 8 . It is the DNA strands of NETs that mediates their integration into forming thrombi. Thrombi formed during deep vein thrombosis are rich in cirDNA 6 , which makes them particularly resistant to plasmin 7 ' 31 , but sensitive to DNase 6 ' 9,14 . In intensive care units, high cirDNA levels are thus considered a risk factor for thrombosis, systemic inflammation, organ failure and death. However, the pathophysiological sequelae and mechanism of action of cirDNA remain unknown in SCD.
  • NETs and cirDNA have yet to be studied with respect to their natural antagonist, endogenous DNase activity.
  • previous studies in SCD often failed to discriminate VOC devoid of, or associated with severe infection and, most prominently, acute chest syndrome (ACS).
  • ACS acute chest syndrome
  • NETs were studied after severe challenge with pro-inflammatory cytokines. As pathogens trigger NETosis, it remains unclear whether intravascular hemolysis can be responsible for increased cirDNA and NETs in SCD, independently from infection.
  • vaso-occlusions Some of the most severe consequence of SCD are thought to occur through repeated episodes of ischemia, leading to cumulative tissue injury and, eventually, organ failure. However, it is likely that not all vaso-occlusions and ischemic accidents lead to a recognized VOC. Some vaso-occlusion may remain too brief or silent. This is made even more complex by the highly heterogeneous reactions and interpretations of pain by SCD patients. Therefore, novel biological markers of vaso-occlusions, independent from the notion of pain or hospitalization, would be of great value to help clinicians identify SCD patients suffering from recurrent vaso-occlusions and ischemic episodes, and those most at risk of cardiovascular injury.
  • the present invention relates to methods and kits for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions.
  • the present invention is defined by the claims.
  • Circulating cell-free DNA in plasma has gained interest as an element of innate immunity, thrombosis and coagulation.
  • VOC vaso-occlusions
  • SCD sickle cell disease
  • RBC red blood cell
  • the first object of the present invention relates to a method of determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions comprising i) determining level of circulating cell-free DNA and the DNAse activity in a blood sample obtained from the patient, ii) calculating the ratio between DNAse activity and level of circulating cell- free DNA, iii) comparing the ratio calculated a step ii) with a predetermined reference value and iv) concluding that the patient has or is at risk of having vaso-occlusions when the ratio is lower than the predetermined reference value.
  • the inventors also showed that the DNase activity would represent a reliable biomarker for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions, as a part of a clinically obvious vaso-occlusive crisis, or as clinically silent events.
  • the second object of the present invention relates to a method of determining whether a patient suffering from sickle cell disease has or is at risk of having vaso- occlusions comprising i) determining level of the DNAse activity in a blood sample obtained from the patient, ii) comparing the level obtained a step ii) with a predetermined reference value and iii) concluding that the patient has or is at risk of having vaso-occlusions when the level is lower than the predetermined reference value.
  • the term "sickle cell disease” or “SCD” has its general meaning in the art and refers to a hereditary blood disorder in which red blood cells assume an abnormal, rigid, sickle shape. Sickling of erythrocytes decreases the cells' flexibility and results in a risk of various life-threatening complications.
  • the term includes includes sickle cell anemia, hemoglobin SC disease and Hemoglobin sickle beta-thalessemia.
  • vaso-occlusion has its general meaning in the art and refers to a common complication of sickle cell disease which leads to the occlusion of capillaries and the restriction of blood flow to an organ, resulting in ischaemia, with vascular dysfunction, tissue necrosis, and often organ damage.
  • Vaso-occlusions are usually a constituent of vaso- occlusive crises, but they may also be more limited, clinically silent, and not cause hospitalization for vaso-occlusive crisis.
  • vaso-occlusive crisis has its general meaning in the art and refers to a common painful complication of sickle cell disease which leads to hospitalization, in association with occlusion of capillaries and restrict blood flow to an organ resulting in ischaemia, severe pain, necrosis, and most often with transient vaso-occlusions, and organ damage.
  • risk relates to the probability that an event will occur over a specific time period, as in the conversion to vaso-occlusion, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention, include time to vaso-occlusion conversion and therapeutic vaso-occlusion conversion risk reduction ratios.
  • Risk evaluation in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to vaso-occlusion or to one at risk of developing vaso- occlusion.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of vaso-occlusion, such as alcohol consumption or cigarette smoking, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to vaso-occlusion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for vaso-occlusion.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for vaso-occlusion.
  • the present invention may be used so as to discriminate those at risk for developing vaso-occlusion from those having vaso-occlusion, or those having vaso-occlusion from normal.
  • blood sample means a whole blood, serum, or plasma sample obtained from the patient.
  • the blood sample according to the invention is a plasma sample.
  • a plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about l,500*g for about 15-20 minutes (room temperature), followed by pipeting of the plasma layer. Platelet- free plasma (PFP) will be obtained following centrifugation at about 13,000*g for 5 min.
  • PFP Platelet- free plasma
  • the plasma sample may be centrifuged in a range of from about 15,000 to about 20,000*g.
  • the plasma sample is ultra-centrifuged at around 17,570*g at a temperature of about 4°C.
  • Different buffers may be considered appropriate for resuspending the pelleted cellular debris, which contains the microvesicles.
  • buffers include reagent grade (distilled or deionized) water and phosphate buffered saline (PBS) pH 7.4.
  • PBS buffer Sheath fluid
  • the blood sample obtained from the patient is a platelet free platelet sample (PFP) sample.
  • PFP may be separated from 10 ml citrated whole blood drawn from the fistula-free arm, 72 hours after the last dialysis. PFP may be obtained after citrate blood centrifugation at 1500*g (15 min), followed by 13000*g centrifugation (5 min, room temperature).
  • circulating cell-free DNA has its general meaning in the art and refers to the DNA released by the cell and present in the patient's blood stream. It is easy and routine for one of ordinary skill in the art to determining the level of circulating cell-free DNA in a blood sample obtained from the patient. In particular, the assay described in the EXAMPLE is particularly suitable for determining the level of circulating cell-free DNA.
  • circulating cell-free DNA may be quantified by colorimetric or fluorometric assays which are typically performed by adding reagents to the patient's blood sample, which produces a color change, the degree of which correlates with the level of circulating cell-free DNA.
  • Other assays include hemagglutinin inhibition, complement fixation, and diffusion in agarose.
  • Other assays involve RNA-DNA hybridization, RIA, and counter Immunoelectrophoresis assays that allow quantification of nanogram amounts of circulating DNA. With real-time PCR and PicoGreen double-stranded DNA quantification assays, picogram amounts of free DNA can be quantified.
  • DNase has its general meaning in the art and refers to all enzymes having a phosphodiesterase activity and the ability to hydrolyse DNA. Accordingly, the term “DNAse activity” refers to the ability of the DNAses present in the blood sample to hydrolyse DNA in particular circulating cell-free DNA. Accordingly, the term “DNAse activity” has its general meaning in the art and refers to the capacity of DNAse to hydrolyse DNA. It is easy and routine for one of ordinary skill in the art to determining DNAse activity in a blood sample obtained from the patient. Conventional DNase activity assays can consist in monitoring the increase in UV absorbance that occurs when the base pairs unstack as the DNA is degraded.
  • the assay mays consist in incubating the blood sample with an amount of DNA in a suitable buffer; separating the reaction products on an ethidium bromide agarose gel by electrophoresis and measuring the relative intensities of fluorescence of the DNA bands under UV light.
  • Alternative relative changes in fluorescence of dye e.g. SYBR green.
  • unitz assay unitz, M; 1950, S. Gen Physiol, 33:363 and the modified Kunitz assay devised by Yamamoto (Yamamoto, M; 1971, Biochim Biophys Acta, 228:95).
  • the predetermined reference value is a threshold value or a cut-off value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of ratios between DNAse activity and level of circulating cell-free DNA in properly banked historical patient samples may be used in establishing the predetermined reference value.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after calculating he ratio in a group of reference, one can use algorithmic analysis for the statistic treatment of the determined levels in samples to be tested, and thus obtain a classification standard having significance for sample classification.
  • ROC curve Receiver Operator Characteristic Curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • the method of the present invention is thus particularly suitable for stratifying the risk in the patients and make the choice of the most appropriate therapeutic strategy.
  • the agent is selected from the group consisting of DNase, antibodies (i.e. antibodies to histones or to a particular histone), and histone degrading enzymes (i.e. mast cell proteinase 1 (Gene ID: 1215)).
  • the agent is a DNA-hydrolysing antibody such as described in ozyr AV, Gabibov AG.
  • DNA-hydrolyzing Ab is catalytic activity a clue for physiological significance? Autoimmunity. 2009 May;42(4):359-61.
  • the agent is a DNase. Any suitable DNase may be used in the present invention.
  • the DNase will most preferably be a DNase I (EC 3.1.21.1). It may, however, in some embodiments be a DNase II (EC 3.1.21.1). DNases occur in a number of species and any DNase capable of cleaving DNA may be used in the invention.
  • the DNase may be from an animal source such as of bovine or porcine origin. It may be of plant, fungal, or microbial origin. However, typically and most preferably the DNase is of human origin and is preferably a recombinant human DNase. Commercially available DNase preparations such as DornaseTM and PulmozymeTM may be used in embodiments of the invention.
  • the DNase has DNA hydrolytic activity, for example in the case of DNase I it may hydro lyse DNA to give '-phosphate nucleotides and in the case of DNase II it may hydro lyse DNA to give 3' phosphate nucleotides.
  • a fluorescence-based assay using, for example, Hoechst Stain may be used such as that which was detected in Labarce & Paiden, 1980, Anal. Biochem., 102:344-352 to assay for DNA hydrolysis.
  • Hydrolytic activity may be assessed in a variety of ways known in the art including analytical polyacrylamide and agarose gel electrophoresis, hyperchromicity assay (Kunitz, J. Gen. Physiol.
  • Asparagine residues and in particular the asparagine at amino acid positions 7 and 74 of the mature human DNase I are prone to deamidation. This process converts the asparagine residues in question to aspartic acid or iso-aspartate residues. Deamidation reduces the activity of the enzyme and this is particularly the case for deamidation at the asparagine at amino acid position 74 of mature human DNase I.
  • Techniques are available for removing deamidated forms of the enzymes to leave the amidated forms and these may be employed to prepare DNases for use in the invention. These techniques may include tentacle cation exchange (TCX) or affinity purification using DNA to purify the amidated forms of the enzyme which are still capable of binding DNA.
  • a DNase preparation for use in the invention may typically comprise from 85 to 100%, preferably from 90 to 100%), more preferably from 95 to 100% and even more preferably from 99 to 100%) amidated, or partially amidated, enzyme by weight.
  • these figures refer to the amount of wholly amidated enzyme i.e. with all of the residues which are naturally amidated being amidated. They will typically have more than 95%>, preferably more than 99%> and even more preferably more than 99.9% of the DNase in an amidated form.
  • these values refer to values at the time of production or to their values from one month to a year, preferably from two to six months and more preferably from three to four months after production. They may refer to the value during any point of the shelf-life of the product.
  • variants of naturally occurring or known DNases may be used in the invention.
  • DNase encompasses such variants.
  • variants refers to polypeptides which have the same essential character or basic biological functionality as DNase.
  • the essential character of a DNase is phosphodiesterase activity and the ability to hydrolyse DNA. Assays for measuring DNA cleavage are described herein and these may be used to determine whether a variant has hydro lytic activity.
  • the sequence of the DNase may be modified so that it has extended half-life. For example the sequence of the enzyme may be changed to remove recognition sequences for certain proteases and in particular those derived from inflammatory cells.
  • the DNase employed in the invention may also be chemically modified to alter its properties such as, for example, its biological half-life.
  • Residues which in particular may be derivatised include cysteinyl residues (most commonly by reaction with a-haloacetates), histidyl residues (by reaction with diethylpyrocarbonate at pH 5.5-7.0), lysinyl and amino terminal residues (by reaction with succinic or other carboxylic acid anhydrides), arginyl residues (by reaction with reagents such as phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin).
  • cysteinyl residues most commonly by reaction with a-haloacetates
  • histidyl residues by reaction with diethylpyrocarbonate at pH 5.5-7.0
  • lysinyl and amino terminal residues by reaction with succinic or other carboxylic acid anhydrides
  • arginyl residues by reaction with reagents such as phenylglyoxal, 2,3-
  • Carboxyl side groups may be selectively modified by reaction with carbodiimides or may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • the covalent attachment of agents such as polyethylene glycol (PEG) or human serum albumin to the DNases may reduce their immunogenicity and/or toxicity of the variant and/or prolong its half-life and hence may be used in the invention.
  • the DNase may be directly conjugated to the glycosaminoglycan or joined through an intermediate molecule.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a “therapeutically effective amount” of the agent is meant a sufficient amount of the agent to treat vaso-occlusion in the subject. It will be understood, however, that the total daily usage of the agent is decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific agent; and like factors well known in the medical arts.
  • the daily dosage of the agent may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the agent for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 CirDNA, NETs and cirDNase activity levels in SCD plasma.
  • Sytox-GreenTM a high affinity cell- impermeant cyanine nucleic acid-intercalating fluorescent dye, and subtracted background values of unlabeled plasma.
  • FIG. 2 Anti-DNA therapeutic intervention in a model of SCD vaso-occlusions
  • Spontaneous reperfusion started to increase significantly 2 hours post injection of vehicle (saline, lline). However, injection of DNase-1 (iv.) accelerated renal reperfusion. Velocity increased within 10 minutes and returned to normal within 30 minutes. (*) indicate significance, p ⁇ 0.05 vs. velocity after microvesicle injection.
  • CirDNA and cirRNA were quantified by fluorometry using cyanine nucleic acid- intercalating fluorescent dyes Sytox-GreenTM and SytoRNA-selec (ThermoFisher, France) 6,7,i5,3 i ci rcu i a u g NETs were quantified as DNA-NE complexes in plasma, using an in house- designed immunosorbant assay.
  • Polyclonal anti-human NE antibody (Santa Cruz, SC-9521, Clinisciences, France) was immobilized in 96 well plates as published 4 . PFP (50% in buffer) were then incubated for 1 h at 37°C. Supernatants were eliminated.
  • NE-bound DNA was revealed with Sytox GreenTM (ThermoFisher, France) and expressed in relative fluorescence units (R.F.U.) to represent NETs.
  • Circulating cell-free DNase (cirDNase) activity was measured using an in house-designed radial diffusion assay. Calf thymus DNA (Sigma- Aldrich, D-4522) was used as DNase substrate, diluted in 1% agarose gels. PFP samples (5 ⁇ ) were loaded in wells, and left to diffuse and digest DNA for 18 to 48 hours at 37°C. DNA was stained with fluorescent Sybr GreenTM (Sigma-Aldrich). Images were collected under ultraviolet illumination. Digested DNA disks were measured using Image-J freeware and expressed in arbitrary units (A.U.).
  • RBC aggregation properties were studied in whole blood within 4 hours of collection, by syllectometry (i.e., laser backscatter versus time) using Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, The Netherlands), after adjusting Hct to 40%, as previously described 5 ⁇ 39 .
  • syllectometry i.e., laser backscatter versus time
  • LORCA Laser-assisted Optical Rotational Cell Analyzer
  • Healthy or SCD human blood neutrophils depleted from platelets, monocytes, lymphocytes and RBC were cultured in Hanks-buffered saline and treated with heme or purified control and SCD RBC microvesicles (prepared as published 3 ' 40 ), or synthetic multilayer vesicles (MLV).
  • Extracellular DNA was stained with Sytox Green and NETs were expressed as a proportion of nuclei (%) with outward extracellular DNA filaments, distinguished from resting horseshoe- shaped nuclei, or nuclei full of decondensed chromatin.
  • purified RBC were cocultured with NETs. After washing, RBC bound to NETs, horseshoe-shaped nuclei, or decondensed nuclei were counted.
  • SAD mice are a validated model for inducible VOC, particularly suited to the study of RBC degradation products, as their spontaneous hemolysis remains moderate.
  • SAD mice like SCD patients, also present neutrophil hyperleukocytosis 4 ⁇ 45 , a relevant environment for NETs stimulation. Renal vaso-occlusions were characterized as before by Echo-Doppler 33 ' 40 ' 46 .
  • NE -bound cirDNA levels ie. NETs
  • NETs were mostly absent from blood donors and matched healthy volunteer plasma ( Figure lb).
  • steady state SCD patients displayed a 2.5 fold increase in NETs versus controls (p ⁇ 0.005), and a further rise (+2.6 fold) during early VOC (ie. +600% over controls; p ⁇ 0.005).
  • nucleated cells stained vivid green (white circles) seemed to occupy the capillary immediately up- or down-stream of the RBC stack. Similar CD235a-DNA colocalization was not seen in non-SCD nephrons (data not shown), but could be observed in some SCD nephrons (data not shown).
  • Adding purified DNA (5 ⁇ g/ml) increased ⁇ by about 50% in blood from SCD patients at steady state (p ⁇ 0.001), but had no effect during VOC, whose ⁇ was already increased 2.5 fold over steady state (pO.001).
  • adding recombinant DNase-1 to VOC blood (10 g/ml, 20 minutes pre-incubation) strongly reduced Y (-50%; /? ⁇ 0.001).
  • adding DNA or DNase-1 to control blood failed to modulate significantly ⁇ or AI.
  • CirDNA gained intense attention since NETosis has been described, bringing the description of new and unexpected pathophysiological effects of genomic DNA during immune responses, thrombosis and coagulation. Our data reveal a novel pathological contribution of cirDNA to hemorheology, related to RBC aggregation and vaso-occlusions.
  • cirDNA levels also increased transiently during acute phase VOC (within 48 hours), and returned to steady state levels before patient discharge. This was consistent with a putative contribution of cirDNA in the vascular manifestations of SCD.
  • increased cirDNA levels might be explained by the induction of NETs in response to heme 15,27 and heme-loaded microvesicles, as all are elevated.
  • the drop in cirDNA levels before hospital discharge also implies that current handling of VOC helps limit the deleterious effects of cirDNA. Targeted anti-cirDNA therapy might however accelerate the process.
  • a subgroup of VOC patients with ACS displayed high and particularly variable levels of cirDNA. Infection is thus able, but not necessary, to upregulate cirDNA and NETs during vaso-occlusive events. Conversely, cirDNA levels might correlate with disease severity, as ACS is one of the most deleterious manifestations of SCD. Finally, the rise in cirDNA levels observed during VOC remained modest compared to the difference between steady state SCD and controls. We hypothesize that cirDNA may operate mostly as co-factor to RBC modifications thought to participate in vaso-occlusions, such as hemoglobin deoxygenation, sickle formation or other forms of RBC injury.
  • CirDNA may thus, at least partly, relate to NETs in SCD.
  • NETs DNA- bound NE
  • the proportion of cirDNA present as NETs, or in other forms, is challenging to establish. In our opinion, this remains a technical challenge, as classical release of NE, myeloperoxidase and other neutrophil-specific activities in plasma would provide multiple opportunities for their association with DNA released by other cell types, thereby confusing the issue.
  • the DNA/DNase ratio is off balance during VOC
  • CirDNA In health and disease, cirDNA is virtually completely bound to cell surfaces 1 ⁇ 3 ' 18 ' 47 ; as magnesium stimulates DNA-membrane interactions 4S . CirDNA may thus contribute to RBC aggregation by providing binding material between stressed membranes, strengthening aggregates that would become more difficult to disperse with shear stress/rate. This is illustrated by the higher ⁇ (i.e., increased aggregate robustness) in aggregometry and the DNA-covered stacks of RBC in renal biopsy capillaries. Stronger RBC aggregates may increase blood flow resistance, as aggregates need to be completely dispersed to negotiate the smaller capillaries at the appropriate velocity 49 . Moreover, increased RBC aggregate robustness has been associated to a higher risk of ACS in SCD 38 .
  • cirDNA did not affect RBC aggregation in control blood. SCD RBC injury, exacerbated by hypoxia or other stimuli, may thus synergize with cirDNA to re-inforce RBC- RBC interactions.
  • cirDNA release including NETs
  • cirDNA may relate to hemordynamic perturbations typical of SCD, and that cirDNA can impact SCD hemorheology by modifying RBC aggregation properties and adhesion.
  • significant plasma DNase activity might constitute a healthy protective barrier against deleterious effects of cirDNA. This mechanism is downregulated in SCD, allowing sudden increases in cirDNA to exert pathological effects, typically during VOC. Consequently, complementing plasma DNase activity by intravenous infusion can be viewed as a potential curative approach in SCD.
  • Hebbel RP Reconstructing sickle cell disease: a data-based analysis of the "hyperhemolysis paradigm" for pulmonary hypertension from the perspective of evidence- based medicine. Am J Hematol. 201 1 ;86(2): 123-154.
  • Camus SM De Moraes JA, Bonnin P, et al. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease. Blood. 2015; 125(24):3805-3814.
  • Trudel M Saadane N, Garel MC, et al. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J. 1991 ; 10(1 1):3157-3165.

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Abstract

L'ADN acellulaire circulant dans le plasma (ADNcir) est devenu intéressant en tant qu'élément de l'immunité innée, de la thrombose et de la coagulation. Les inventeurs ont supposé que l'ADNcir pourrait favoriser des vaso-occlusions (VOC) et participer à celles-ci dans la drépanocytose (SCD). Les taux d'ADNcir se sont avérés être accrus de 300% chez des patients atteints de drépanocytose (n = 87) à l'état stationnaire par rapport à des témoins (n = 22) et une augmentation supplémentaire de 100% a été observée pendant des VOC aiguës. Cette dernière augmentation a disparu en moins d'une semaine d'hospitalisation, une fois les VOC terminées. Dans le plasma de patients atteints de drépanocytose, des taux d'ADNcir accrus ont été corrélés avec une activité ADNase plasmatique endogène réduite, pendant un état stationnaire de VOC et des VOC en phase aiguë. L'utilisation thérapeutique d'ADNase-1 a diminué la robustesse d'agrégats d'érythrocytes dans le sang de patients atteints de drépanocytose et a dégagé des vaso-occlusions rénales chez des souris transgéniques drépanocytaires. Par conséquent, un déséquilibre entre l'activité ADNase-cir et les taux d'ADNcir limite les vaso-occlusions en cas de drépanocytose et représenterait ainsi un biomarqueur fiable pour déterminer si un patient souffrant de drépanocytose a des vaso-occlusions ou présente un risque d'en avoir.
PCT/EP2018/075809 2017-09-25 2018-09-24 Procédés et kits permettant de déterminer si un patient souffrant de drépanocytose a des vaso-occlusions ou présente un risque d'avoir des vaso-occlusions WO2019057972A1 (fr)

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WO2021105137A1 (fr) * 2019-11-25 2021-06-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Inhibiteurs de trem-1 pour le traitement d'occlusions vasculaires et de lésions tissulaires chez les patients souffrant de drépanocytose

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WO2021105137A1 (fr) * 2019-11-25 2021-06-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Inhibiteurs de trem-1 pour le traitement d'occlusions vasculaires et de lésions tissulaires chez les patients souffrant de drépanocytose

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