EP3548060A1 - Placental growth factor for the treatment of fetal alcohol syndrome disorders (fasd) - Google Patents

Abstract

The invention relates to a placental growth factor (PlGF) to be used as a drug in the prevention and/or treatment of fetal alcohol syndrome disorders (FASD) selected from the group comprising fetal alcohol syndrome (FAS), cerebrovascular disease, and growth retardation in a subject exposed to alcohol in utero. The invention also relates to a pharmaceutical composition or a product comprising the PlGF for the same therapeutic uses.

Description

 PLACENTAL GROWTH FACTOR FOR TREATMENT

 DISORDERS CAUSED BY FETAL ALCOHOLIZATION (FASD)

INTRODUCTION

 Alcohol is a teratogen responsible for physical and behavioral damage. In humans, prenatal exposure to alcohol can lead to alterations in brain development. Thus, alcohol consumption during pregnancy (fetal alcoholization) is the leading cause of disability and in particular mental retardation of non-genetic origin in the world and also in France.

 The damage varies according to the period in which the fetus was exposed, blood alcohol levels, genetic and environmental factors, and the mode of consumption (chronic, binge drinking).

 Fetal Alcohol Syndrome (FAS) is the most extreme and disabling manifestation of fetal alcohol spectrum disorder (FASD). Its incidence is estimated in France at 1, 5% of births. FAS associates physical abnormalities such as hypotrophy (growth retardation), craniofacial dysmorphism and neurobehavioral abnormalities resulting in cognitive function disorders (attention deficit, motor skills, learning disorders, memorization). Neurological sequelae are also present in FASD children whose incidence is estimated in France at nearly 1% of births.

 Cerebral angiogenesis is concomitant with the process of neurogenesis and contributes to good brain development by providing nerve cells with nutrients, oxygen and trophic factors. In particular, cerebral angiogenesis is a prerequisite for the proper development of the neural network. In addition, a direct impact of cerebral vessels on the migration process of neuronal populations and oligodendrocytes has recently been demonstrated. It has also been established previously by the inventors that alcohol in utero interferes with cerebral angiogenesis and that this effect contributes to brain abnormalities of alcoholization.

 Targeting vascular abnormalities of in utero exposure to alcohol therefore appears as a therapeutic strategy to reduce the neurodevelopmental effects of alcohol and to correct impaired brain functions. In fact, the current management of FASD children aims, by stimulating motor and cognitive functions, to reduce language, learning and attention disorders that are diagnosed late, often between 4 and 5 years of age. The scholarship. The results obtained by such monitoring are limited and the disorders

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FIRE I LLE OF REM PLACEM ENT (RULE 26) in utero alcohol will have a long-term impact on the social and professional integration of these future adults. To date, there is no curative treatment for the neurodevelopmental effects of alcohol in FASD children.

There is therefore a need to develop a novel therapy of alcohol-related disorders in utero for treating in particular an abnormal cerebral vasculature, to improve cerebral angiogenesis or developmental disorders associated therewith.

DESCRIPTION

The inventors have previously demonstrated that the placental determination of placental growth factor (PIGF) makes it possible to identify, among children exposed in utero to alcohol, those who have suffered brain damage. In particular, these children show a decrease in placental PIGF levels which corresponds to impaired cerebral angiogenesis.

Subsequently, in a pre-clinical model of in utero alcoholization, the inventors have discovered that placental PIGF supplementation corrects the action of alcohol on morphometric and anatomical indicators indicative of fetal growth such as the size body and head at birth. In addition, the inventors have demonstrated that increasing the amount of PIGF can greatly reduce the deleterious effect of alcohol on the cerebral vasculature of the fetus. In particular, it makes it possible to improve cerebral angiogenesis and to correct the disorganization of microvessels induced by alcohol. Fetal Growth Factor (PIGF) has been described in the prior art as a target for the treatment of cardiovascular diseases, retinopathies, skin diseases or even cancers (Lu et al., Discov Med, 2016 21: 349- 61, Aprile et al., Crit Rev Oncol Hematol, 2015 95: 165-78 and Shibuya, Endocr Metab Immune Disord Drug Targets, 2015, 15: 135-44). More specifically, PIGF has been described in the treatment of retinopathies in adults (US2015013359) and in children (Hard et al., Act Paediatrica Foundation 2011 100, pp. 1063-1065). Nevertheless, no study suggests the therapeutic effect of IGFP in the treatment of fetal alcohol spectrum disorder (FASD). In addition, treatments to correct the adverse effect of alcohol on brain vasculature and growth of a subject having been exposed in utero to alcohol have not been demonstrated. The present invention thus opens a new way in the prevention and / or treatment of FASD and in particular, in the prevention and / or treatment of neurological disorders (such as hyperactivity, loss of attention, depression, anxiety, emotional disturbances, excessive irritability, behavioral problems, etc.) due to abnormal neuronal and oligodendrocytic migraine-mediated migrations or growth disorders (hypotrophy) following alcohol consumption during the pregnancy. In a first aspect, the present invention thus relates to a placental growth factor (PIGF) for its use in the prevention and / or treatment of fetal alcohol spectrum disorder (FASD) in a subject having been exposed to alcohol. in utero.

The term "PIGF" or "Placental growth factor" or "placental growth factor" (all these terms are synonymous) means a protein of the family of vascular endothelial growth factors (VEGF). More particularly, PIGF within the meaning of the invention is a 149 amino acid protein highly similar to VEGF-A which is recognized by the same receptor as the latter, VEGF-R1, but which is not recognized by the VEGF receptor. -R2. PIGF is strongly expressed by the placenta, but not by the fetus and in particular by the fetal brain. N-terminal glycosylated PIGF is secreted and functions as a dimer to control angiogenesis. The term "PIGF" refers in particular to all 4 isoforms PIGF1-4: PlGF-1 and PlGF-3 are isoforms that do not bind heparin while PlGF-2 and PlGF-4 contain additional domains that allow to fix heparin. Even more preferentially, PIGF is understood to mean a human protein whose sequence is chosen from any of the isoforms identified by Uniprot accession numbers P49763-2 (PlGF-1, the amino acid sequence of which corresponds to SEQ ID No. NO: 1); P49763-3 (PlGF-2 whose amino acid sequence corresponds to SEQ ID NO: 2); P49763-1 (PlGF-3 whose amino acid sequence corresponds to SEQ ID NO: 3); P49763-4 (PlGF-4 whose amino acid sequence corresponds to SEQ ID NO: 4) presented in the sequence listing appended to the present application. According to one embodiment of the present invention, recombinant PIGF or protein analogs of human PIGF can also be used in the prevention and / or treatment of FASD.

In particular, the PIGF of the present invention is obtained by using a prokaryotic or eukaryotic recombinant protein production system, in particular by i) culturing a microorganism or transformed eukaryotic cells using a nucleotide sequence coding for the Human PIGF (accession number NCBI of gene 5228, accession number of transcripts NM_002632.5 (SEQ ID NO: 5), NM001207012.1 (SEQ ID NO: 6), NM_001293643.1 (SEQ ID NO: 5) ID NO: 7)) and ii) isolating the protein produced by said microorganism or said eukaryotic cells. This technique is well known to those skilled in the art. For more details about it, we can refer to the following work: Recombinant DNA Technology I, Editors Aie Prokop, Raskesh K Bajpai; Annals of the New York Academy of Sciences, Volume 646, 1991. The PIGF protein is preferably purified / isolated from cell lysates and / or cell supernatants by which it is expressed and / or secreted. This purification can be done by any means known to those skilled in the art. Many purification techniques are described in Voet D and Voet JG, Protein and Nucleic Acid Purification Techniques. The recombinant protein production systems use nucleic acid vectors comprising nucleic acids encoding the polypeptides to be synthesized, which are introduced into host cells that produce said polypeptides (for more details, refer to "Recombinant DNA Technology I", Editors Aies Prokop, Raskesh K Bajpai, Annals of the New York Academy of Sciences, Volume 646, 1991). Many vectors in which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form or integration into the chromosomal material of the host ), as well as the nature of the host cell (for example, plasmids are preferably introduced into bacterial cells, while YACs are preferably used in yeasts). These expression vectors may be plasmids, YACs, cosmids, retroviruses, episomes derived from EBV, and all the vectors that the person skilled in the art may judge appropriate for the expression of said chains. The vectors according to the invention comprise the nucleic acid encoding PIGF, or a similar sequence, as well as the means necessary for its expression. The term "means necessary for the expression of a peptide" means the term peptide being used for any peptide molecule, such as protein, polyprotein, polypeptide, etc. any means which makes it possible to obtain the peptide, such as in particular a promoter, a transcription terminator, an origin of replication and preferably a selection marker. The means necessary for the expression of a peptide are operably linked to the nucleic acid sequence encoding the polypeptide fragment of the invention. By "operationally related" is meant a juxtaposition said elements necessary for the expression of the gene encoding the polypeptide fragment of the invention, which are in a relationship such that it allows them to function in an expected manner. For example, there may be additional bases between the promoter and the gene encoding the polypeptide fragment of the invention as long as their functional relationship is preserved. The means necessary for the expression of a peptide can be homologous means, that is to say naturally understood in the genome of the vector used, or else be heterologous, that is to say artificially added to from another vector and / or organism. In the latter case, said means are cloned with the polypeptide fragment to be expressed. Examples of heterologous promoters include viral promoters such as SV40 promoter (Simian Virus 40), Herpes simplex virus thymidine kinase gene promoter (TK-HSV-1), sarcoma virus LTR of Rous (RSV), the immediate first promoter of cytomegalovirus (CMV) and the major adenoviral major promoter (MLP), as well as any cellular promoter that controls the transcription of genes encoding peptides in higher eukaryotes, such as the promoter of constitutive phosphoglycerate kinase (PGK) gene (Adra et al., Gene Volume 60, Issue 1, 1987, Pages 65-74), the liver specific gene promoter alphal-antitrypsin and FIX, and the SM22 specific promoter smooth muscle (Moessler et al., Development 1996 Aug; 122 (8): 2415-25). Methods for suppressing and inserting DNA sequences into expression vectors are widely known to those skilled in the art and include enzymatic digestion and ligation steps. The vectors of the invention may also include sequences necessary for targeting the peptides to particular cell compartments. An example of targeting may be targeting to the endoplasmic reticulum obtained using adenovirus E3 leader-type targeting sequences (Ciernik IF, et al., The Journal of Immunology, vol. 162, 1999, pages 3915 -3925). The term "transcription terminator" here designates a sequence of the genome that marks the end of the transcription of a gene or operon, into messenger RNA. The mechanism of transcription termination is different in prokaryotes and eukaryotes. Those skilled in the art know the signals to be used according to the different cell types. For example, if the cell into which the vector will be introduced is a bacterium, it will use a Rho-independent terminator (inverted repeat sequence followed by a series of T (uracils on transcribed RNA) or a Rho-dependent terminator ( consisting of a consensus sequence recognized by the Rho protein). The term "origin of replication" (also called ori) is a unique ^DNA sequence for ^the initiation of replication. ^It is from this sequence that unidirectional or bidirectional replication begins. The skilled person knows that the structure of ^the replication origin varies from ^one species ^to another; it is species specific well ^that it has some common features between species. A protein complex is formed in this sequence and allows ^the opening of ^the DNA and the start of the replication.

The vectors comprising the genetic sequence encoding PIGF are prepared by methods commonly used by those skilled in the art. The resulting clones can be introduced into an appropriate host by standard methods known to ^those skilled in the art to introduce polynucleotides into a host cell. Such methods may be processing ^using dextran, precipitation by calcium phosphate, transfection ^using polybrene, protoplast fusion, ^{electroporation, encapsulation} of the polynucleotide into liposomes, the ^'biolistic injection and direct microinjection ^of DNA into the nucleus. Said sequence (isolated or inserted in a plasmid vector) can also be combined with a substance which allows it to cross the host cell membrane, such that ^a carrier as a nanotransporter or a liposome preparation, or cationic polymers. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes.

As examples ^of microorganisms suitable for the purposes of the ^invention include yeast (Buckholz RG, Current Opinion in Biotechnology vol 4, No. 5, 1993, pages 538 -.. 42) and bacteria (Olins and Lee, Current Opinion in Biotechnology, Vol 4, No. 5, 1993, pp. 520-5). By way ^of examples of eukaryotic cells include cells from ^animals such as mammals (Edwards CP and Aruffo A, Current Opinion in Biotechnology vol 4, No. 5, 1993, pages 558 -.. 63), the reptiles, and the like. The plant cells can also be used. Among the mammalian cells, Chinese hamster ovary (CHO) cells, monkey (COS and Vero cells), dwarf hamster kidney (BHK cells), pig kidney (PK 1 cells ) and rabbit kidney (RK13 cells, human cell lines ^osteosarcoma (cell 143 B), HeLa human cell lines and human cell lines ^hepatoma (the Hep G2 cell type). It is also possible use of insect cells in which methods employing baculoviruses can be used, for example (Luckow VA, Journal of Virology 67, 8, 1993, pages 4566-79). preferred embodiment of the invention, the host cell used to produce the fragment of the invention is a bacterium, preferably the bacterium E. coli. The person skilled in the art knows the conditions under which these cells should be cultivated, as well as the experimental conditions necessary for the expression of the polypeptide fragments by these cells.

The method for producing recombinant PIGF may comprise the following steps: a) culturing in a suitable culture medium and conditions a host cell comprising the PIGF coding vector; and b) isolating the PIGF produced in step a).

PIGF can be isolated (purified) from the expressing cell. In this case, a preliminary step of lysis of said cells may be necessary. The media and culture conditions associated with each cell type used for the production of recombinant proteins are well known to those skilled in the art.

Isolation (or purification) of IGFP can be done by any means known to those skilled in the art. For example, differential precipitation or ultracentrifugation. It may also be advantageous to purify the PIGF by ion exchange chromatography, affinity chromatography, molecular sieving, or isofocalization. All of these techniques are described in Voet D and Voet JG, Protein and Nucleic Acid Purification Techniques, Chapter 6, Biochemistry, 2nd Edition.

More specifically, in a first step, the material from which the protein is to be extracted (animal or plant tissue, bacteria, etc.) is generally ground. Various devices ( ^" Waring Blender ^" , Potter-Eveljhem apparatus, ^" Polytron ^" , etc.) may be used for this purpose. This homogenization is done in a buffer of appropriate composition, well known to those skilled in the art. ^The homogenate thus obtained is then clarified, usually by centrifugation, to remove large particles poorly ground or to obtain the cellular fraction containing the desired protein. If the protein is precisely in a cellular compartment, a mild detergent (Triton X-100, Tween 20, sodium deoxycholate, etc.) is generally used to release it by dissolving the membranes of this compartment. ^The detergent use must often be done in a controlled manner because they can break lysosomes, releasing hydrolytic enzymes (proteases, nucleases, etc.) that can attack and destroy the proteins or other molecules ^that we want to isolate. Special precautions should be taken when working with degradation-sensitive or few proteins. A Frequent solution to this problem is the inclusion in solutions of protease inhibitors that are physiological (trypsin inhibitors, antipain, leupeptin, etc.) or artificial (E64, PMSF, etc.). Then various techniques exist to isolate the desired protein.

One of the most suitable methods for large volumes is differential precipitation with ammonium sulphate. The ion exchange chromatography or chromatography ^affinity, as applicable to large volumes of sample but having a fairly good separation power, are good intermediate methods. To finalize purification, molecular sieving or isofocalisation is often used. These techniques make it possible to refine the purity, but require very small volumes of concentrated proteins. It is often advantageous, between these steps, to eliminate the salts or products used in these chromatographies. This can be achieved by dialysis or ultrafiltration.

It may also be advantageous to use a vector bearing a sequence making it possible to identify the PIGF of the invention. In addition, it may be advantageous to facilitate secretion in a prokaryotic or eukaryotic system. Indeed, in this case, the recombinant protein of interest will be present in the supernatant of the cell culture rather than inside the host cells.

PIGF can be produced at levels of at least 1 mg per liter, preferably 2 mg per liter, even more preferably 5 mg per liter of cell culture.

Alternatively, it is possible to prepare the PIGF by chemical synthesis. Those skilled in the art are familiar with chemical synthesis processes, for example techniques employing solid phases or using partial solid phases, by condensation of the protein or by synthesis in conventional solution. The fragments of the invention may for example be synthesized by synthetic chemistry techniques, such as Merrifield synthesis which is advantageous for reasons of purity, antigenic specificity, absence of undesired side products and for its ease of production. This chemical synthesis can be coupled to a genetic engineering or genetic engineering approach alone using techniques well known to those skilled in the art and described for example in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 1989. Reagents and starting materials are commercially available, or can be synthesized by well-known conventional techniques (see for example, WO 00/12508, WO 2005/085260). In particular, PIGF analogs can be synthesized using the technique described by Zheng et al. (Acta Ophthalmologica 2012 90 (7): e512 - e523). As indicated above, the present invention relates to a PIGF for its use in the prevention and treatment of FASD. "Fetal Alcohol Spectrum Disorder (FASD)" refers to all disorders in children resulting from exposure to alcohol during pregnancy. This term includes, among other things, all the behavioral disorders that will gradually become apparent with age. Children with these disorders are called "FASD children. In their most severe version, FASD is fetal alcohol syndrome (FAS). This results in craniofacial dysmorphia (including shortened palpebral fissures, a smooth, elongated, erased nasolabial fold, and a thin upper lip); nonspecific growth retardation (prenatal or postnatal height or weight or head circumference) or both; miscellaneous malformations (cardiopathies, urogenital malformations, digestive malformations or cerebral architectural disorders) and neurodevelopmental disorders sometimes expressed by mental retardation and more often by learning difficulties. Children with FAS are called "FAS children". The majority of children with FASD experience maladaptive behaviors and behavior that progressively become apparent with age (including breastfeeding and eating difficulties, sleep disorders, and then, at school age, behavioral abnormalities , cognitive disorders affecting learning, intellectual deficits with a Ql lowered by 20 points (1 to 2 standard deviations), difficulties in learning to read (dyslexia) and even more calculation (dyscalculia), disorders attention with hyperactivity or hyperkinesia.They are known as Attention Deficit Hyperkinesia Syndrome (SDAH) (or Attention Deficit Hyperactivity Disorder (ADHD) in English). substituted by the term Fetal Alcohol Spectrum Discord (FASD). A complete definition of FASD is given in the ACA Report. French National Medical School of Fetal Alcohol Syndrome (adopted March 22, 2016).

The inventors have previously shown that exposure to alcohol causes cerebrovascular damage. By "cerebrovascular disease" is meant here any alteration of the cerebrovascular system, including an alteration resulting in impaired or even defective operation of the system. A cerebrovascular disease in the context of the invention may in particular be a disorganization of the cerebrovascular system. More particularly, fetal alcoholization induces a random orientation of the cerebral vessels. In a particular embodiment, the fetal alcohol disorder is linked to cerebrovascular disease. Even more particularly, said fetal alcohol disorder is related to disorganization of the cerebrovascular system.

The inventors have also demonstrated by their previous work that fetal alcoholization causes a failure in cerebral angiogenesis. Cerebral angiogenesis is the process of forming blood vessels in the brain.

According to one embodiment of the present invention, PIGF is used to stimulate cerebral angiogenesis and thus improve brain function.

According to yet another embodiment of the invention, the PIGF can be used to prevent and / or treat at least one FASD selected from any of the above-mentioned maladjustment and behavior disorders. PIGF can also be used to prevent and / or treat fetal alcohol syndrome (FAS), especially when FAS is manifested as hypotrophy.

In the context of the present invention, the term "Fetal Alcohol Syndrome (FAS)" refers to the most extreme and disabling manifestation of fetal alcohol spectrum disorder (FASD). It combines physical abnormalities such as hypotrophy (growth retardation), cranio-facial dysmorphism and neurobehavioral abnormalities resulting in cognitive function disorders (attention deficit, motor skills, learning disorders, lock). Partial FAS is when the child has only a part of the FAS symptoms. Partial SAF children, however, always have one or more neurobehavioral abnormalities.

In the context of the invention, a SAF child, particularly a newborn, is considered "hypotrophic" if its weight and height are below the 10th percentile of the reference curves. This is often the case for premature newborns and newborns exposed in utero to alcohol. The hypotrophy can also be diagnosed before term, by ultrasound: one speaks about fetal hypotrophy or intrauterine growth retardation.

The inventors have furthermore demonstrated that increasing the amount of PIGF makes it possible to increase the size of the whole body of a fetus or of one or more of its parts, in particular the size of the skull, the size of the body. , abdomen size and depth. This allows partial or complete recovery of the normal morphometric appearance. The administration of PIGF thus makes it possible to compensate for the hypotrophy of SAF subjects who represent the most severe forms of FASD. According to another embodiment, the present invention thus relates to a PIGF for its use in the prevention and / or treatment of FAS, especially when FAS is manifested in the form of hypotrophy due to intrauterine exposure to FAS. 'alcohol. In particular, it is a hypotrophy of the whole body of a subject or one or more of its parts selected from the torso (or called the body), the abdomen and the skull. In particular, the subject is a fetus or a child, particularly a premature baby.

According to one embodiment of the present invention the subject having been exposed in utero to the alcohol is selected from an embryo, a fetus or a child, preferably a fetus or a child, in particular, a premature child. The period between the ^30th gestational week and the term of pregnancy is the period during which brain angiogenesis is the most important. It is thus particularly preferred to administer the PIGF to a fetus exposed in utero to alcohol at precisely this time.

According to a particular embodiment of the invention, when the subject TCAF is a premature child, the treatment will consist in supplementing in PIGF this subject on the exerter period corresponding to the lost weeks of intra-uterine life. Preferably, the treatment window can extend from the 30th gestational week to the theoretical term (40 gestational weeks), during which time cerebral angiogenesis in humans is particularly intense.

The term "subject" according to the invention means a human, and preferably an embryo, a fetus or a child. An "embryo" as it is understood here corresponds to a fertilized oocyte less than three months old. By "fetus" is meant an individual taken before birth and whose gestational age is between 3 and 9 months. After childbirth, the subject becomes a child. By "child" is meant according to the invention an individual whose age is less than 3 years. Thus included in the category of children according to the invention, newborns, whose age is between 0 and 1 month, infants, who are between 1 month and 2 years, and the children themselves, who are at least 2 years old. A "newborn", as we understand it here, may as well be born at term as it is premature. In the context of this application the term "premature" refers to a child born alive before 37 weeks of amenorrhea. This term covers three sub-categories: extreme prematurity (<28 weeks); extreme prematurity (between the ^28th and ^32nd week); average prematurity or late (between the ^32nd and ^37th week). In the present invention, premature children treated with PIGF are preferably in the category of medium or even late prematurity.

The expression "a subject with Fetal Alcohol Spectrum Disorder" or "FASD subject" as used herein refers to an embryo, fetus or subject, particularly human, who is exposed to or likely to be exposed to In utero alcohol with fetal alcohol impairment or who is at risk of developing maternal alcohol consumption as a condition related to fetal alcohol spectrum disorder, including the effects described above. In particular, a TCAF subject may have a whole body size or its parts below normal and a disorganized cerebral vascular network, said disorganization being notably related to a random orientation of the cerebral vessels. When a FASD subject is affected in particular by a FAS, it is referred to in this case as a "SAF subject".

By "treatment" is meant any action to reduce or eradicate the symptoms or causes of FASD. A treatment within the meaning of the invention may comprise the administration of PIGF, a pharmaceutical composition or a product comprising it with or without a psychotherapeutic follow-up.

By "prevention" is meant any action that completely or partially prevents the risk of symptoms or the causes of FASD. Prevention within the meaning of the present invention comprises the administration of PGIG, of a pharmaceutical composition, to a subject exposed in utero to alcohol or to a subject having been exposed in utero to alcohol but for which the symptoms of TCAF have not yet appeared and cerebral angiogenesis still in progress. The inventors take advantage of the fact that PIGF is an angiogenic factor naturally present at these stages of fetal development in a healthy subject, which has the advantage of easy use of IFIG by compensation in the prevention of FASD. According to one embodiment of the present invention, the placental growth factor is administered in a therapeutically effective amount to a subject having been exposed to the alcohol in utero.

In the context of the invention, "a therapeutically effective amount" means an amount sufficient to influence the therapeutic course of a particular disease state. A therapeutically effective amount is also that to which all the toxic effects or side effects of the agent are compensated by the therapeutically beneficial effects of the active ingredient used.

According to another embodiment of the present invention, the placental growth factor (PIGF) is administered in utero and / or ex utero. It is thus conceivable to start a treatment (or prevention) by the administration of IGFI during the intrauterine period and to continue it after delivery especially when the child is born prematurely and therefore loses the physiological contribution of placental PIGF.

Preferably, the PIGF will be administered alone or in a pharmaceutical composition systemically, particularly intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or orally. More preferably, the PIGF will be administered several times, spread over time.

In particular, when the administration of IGF is carried out in utero, it preferably takes place directly at the placental level in order to reach the fetal brain more quickly and prevent the degradation of the protein by the maternal organism. When the administration of IFIG takes place after birth, preferably it is administered parenterally, preferably intravenously, especially if it is a premature child or a newborn.

Optimal modes of administration, dosages and dosage forms may be determined according to the criteria generally considered in establishing a patient-specific treatment such as, for example, the age or body weight of the patient, the severity of his or her general condition, tolerance to treatment and side effects noted.

When PIGF is administered as pharmaceutical compositions, for subcutaneous, intramuscular, intravenous, transdermal local administration, PIGF can be administered in unit dosage forms, in admixture with conventional pharmaceutical carriers to the subject in need thereof. . Suitable unit dosage forms include intramuscular, intravenous forms.

As indicated above, the choice of the most appropriate administrative voice will depend on when this administration is performed. In particular, when the administration of a cosmetic composition comprising IGF is carried out in utero, this will be done intermuscularly or intravenously, preferably at the placental level. administration of the pharmaceutical composition comprising PIGF to a newborn child or a premature child is preferably made intravenously.

For parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing dispersing agents and / or pharmacologically compatible wetting agents are used.

Forms for parenteral administration are conventionally obtained by mixing PIGF with buffers, stabilizing agents, preservatives, solubilizing agents, isotonic agents and suspending agents. In accordance with known techniques, these mixtures are then sterilized and then packaged in the form of intravenous injections.

As a buffer, those skilled in the art may use buffers based on organic phosphate salts. Examples of suspending agents include methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, acacia and sodium carboxymethylcellulose. In addition, useful stabilizers according to the invention are sodium sulfite and sodium metasulfite, while sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol can be mentioned as preservatives.

The pharmaceutical compositions of the invention may be formulated to be administered to the patient by a single route or by different routes. The dosage depends of course on the form in which the PIGF will be administered, the mode of administration, the therapeutic indication, the age of the patient and his condition.

The dose to be administered is preferably from 0.001 to 250 mg / kg of PIGF per day, preferably from 0.01 to 100 mg / kg of PIGF per day, more preferably from 0.1 to 50 mg / kg of PIGF per day. day and even more preferably from 1 to 25 mg / kg of PIGF per day. The unit dose of PIGF preferably comprises from 0.1 to 50 mg / kg of this compound.

According to one embodiment of the present invention, the initially administered dose of PIGF may be adjusted if necessary during treatment depending on the response to this treatment of the subject being treated. The skilled person based on this general knowledge and on the present description will adjust the dose of PIGF so as to optimize its therapeutic effect.

As mentioned above in the description, the placental growth factor (PIGF) can also be used in the form of active ingredient in a pharmaceutical composition.

According to a second aspect, the present invention thus relates to a pharmaceutical composition comprising a placental growth factor (PIGF) as defined above and a pharmaceutically acceptable vehicle for its use in the prevention and / or treatment of disorders caused by the fetal alcoholization (FASD). The pharmaceutical composition according to the present invention can be used in the prevention and / or treatment of different types of FASD as described for the PIGF below. In particular, these FASD are selected from the group consisting of disorders of maladjustment and behavior or fetal alcohol syndrome (FAS), especially when manifested as hypotrophy and cerebrovascular disease due to exposure to alcohol in utero.

The composition of the present invention can also be used to enhance cerebral angiogenesis in a subject having been exposed to alcohol in utero.

For the purposes of the present invention, the term "pharmaceutically acceptable carrier" means any material which is suitable for use in a pharmaceutical product. By way of example of a pharmaceutically acceptable vehicle, mention may be made of lactose, optionally modified starch, cellulose, hydroxypropylmethylcellulose, mannitol, sorbitol, xylitol, dextrose, calcium sulphate, sodium phosphate and the like. calcium, calcium lactate, dextrates, inositol, calcium carbonate, glycine, bentonite and mixtures thereof.

The pharmaceutical composition according to the invention can be in various forms and be administered in different ways as indicated above in detail.

According to one embodiment, the pharmaceutical composition of the present invention comprises a PIGF as an active ingredient in a concentration of between 0.001 mg / kg and 250 mg / kg by weight relative to the total weight of the subject to whom the pharmaceutical composition will be administered. . Preferably, the concentration of PIGF is between 0.01 mg / kg and 100 mg / kg by weight relative to the weight of the subject to which the pharmaceutical composition will be administered and even more particularly, between 0.15 mg / kg and 50 mg / kg. kg by weight relative to the weight of the subject to which the pharmaceutical composition will be administered or between 1 mg / kg and 25 mg / kg by weight relative to the total weight of the subject to which the composition will be administered.

The inventors have demonstrated that the increase in the amount of PIGF is particularly effective in restoring the attainment of cerebral vasculature thus making it possible to reduce the neuronal function to a normal state.

According to one embodiment of the present invention, the PIGF or the pharmaceutical composition comprising it is therefore used for the treatment and / or prevention of cerebral vasculature damage due to exposure to alcohol in utero.

In addition, the inventors have also demonstrated that increasing the amount of PIGF makes it possible to increase the size of the entire body of a fetus or one or more of its parts, in particular the size of the skull, the size of the body, abdomen size and depth after exposure to alcohol during the intrauterine period. This allows partial or complete recovery of the normal morphometric appearance.

According to another embodiment, the present invention thus relates to a PIGF or a pharmaceutical composition comprising it for its use in the prevention and / or treatment of hypotrophy due to intrauterine exposure to alcohol, especially when this hypertrophy is a manifestation of FAS. In particular, it is a hypotrophy of the whole body of a subject or one or more of its parts selected from the torso (or called the body), the abdomen and the skull. In particular, the subject is a SAF subject who is a fetus or newborn, particularly a premature baby.

A fourth aspect of the invention relates to a method of treating fetal alcohol disorder in a subject. The method comprises a step of administering or overexpressing PIGF or administering a pharmaceutical composition comprising same to a subject having FASD including in particular fetal alcohol syndrome (FAS), particularly when manifested in the form of FASD. hypotrophy and / or cerebrovascular disease of a subject who has been exposed to alcohol in utero. This method of treatment may comprise a prior stage of diagnosis of FASD. According to a fifth aspect, the present invention relates to the placental growth factor (PIGF), a pharmaceutical composition or a product comprising it according to the invention for use in the treatment of FASD, said use comprising a step prior to identification of the subject, said identification comprising the steps of: a) measuring the amount of PIGF in a biological sample of said subject, preferably from the placenta or cord blood; b) comparing the amount of PIGF of step a) with a reference that is a measure of the amount of PIGF in a healthy individual, and c) determining a FASD or risk of developing FASD in said subject. The term "biological sample" according to the invention any sample that can be taken from a subject. Alternatively, the biological sample is a sample from the placenta, including the umbilical cord. Indeed, PIGF is expressed by placental cells throughout pregnancy. This makes it possible to assay the PIGF without compromising the integrity of the subject, in particular when the subject is an embryo or a fetus. In general, the biological sample must allow the determination of the level of expression of PIGF. The test sample may be used as obtained directly from the biological source or as a result of pretreatment to change the character of the sample. For example, such pretreatment may include plasma preparation from blood, dilution of viscous fluids, and so on. Pre-treatment methods may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of disrupting components, addition of reagents, lysis, etc. In addition, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.

PIGF protein is a secreted protein (DeFalco, Exp Mol Med 44 (1): 1-9, 2012). Preferred biological samples for determining the level of expression of said protein include in particular blood, plasma, or lymph samples. Preferably, the biological sample is a blood sample. Even more preferably, the biological sample is a sample of placental blood or cord blood. This is usually collected during childbirth. Blood from placental vessels can then be obtained to measure the rate of PIGF. According to one embodiment, when the amount of PIGF measured in step a) is less than the reference, the subject is determined to be suffering from or having a risk of developing a FASD and in particular an impairment of cerebral vasculature or FAS. , especially hypotrophy. According to the present invention, the amount of PIGF is measured when a subject has at least one disorder that may be related to intrauterine alcohol exposure. The amount of PIGF will also be measured when a subject does not present one or more particular disorders related to intrauterine alcohol exposure but for whom exposure to alcohol during that period has been proven or suspected. Thus, measuring the amount of PIGF as described above will make it possible to confirm or refute a FASD or the risk of developing one prior to the therapeutic management.

According to one embodiment, the amount of PIGF is determined by measuring the amount of transcripts encoding the PIGF or the amount of the polypeptide.

The level of gene expression or protein can be measured by many methods that are available to those skilled in the art. There may be several intermediate steps between taking the biological sample and measuring the PIGF expression, said steps corresponding to extracting from said sample an mRNA sample (or corresponding cDNA) ) or a protein sample. This can then be directly used to measure the expression of PIGF. Preparation or extraction of mRNA (as well as retrotranscription thereof into cDNA) or proteins from a cell sample are only routine procedures well known to those skilled in the art.

Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of PIGF at either mRNA level (i.e., all mRNAs or cDNAs) present in the sample), or proteins (i.e., all the proteins present in the sample), can be measured. The method used to do this depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available.

When the expression of PIGF is measured at the level of the mRNA (or corresponding cDNA), any technology usually used by those skilled in the art can be implemented. These technologies for analyzing the level of gene expression, such as, for example, transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, based on DNA) and RT-PCR (reverse transcription). PCR transcription, if you start from RNA) or quantitative RT-PCR or nucleic acid chips (including DNA chips and oligonucleotide chips) for higher throughput.

By "nucleic acid chips" is meant herein several different nucleic acid probes that are attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip may consist of polymers, plastics, resins, polysaccharides, silica or a material based on silica, carbon, metals, inorganic glass, or nitrocellulose.

The probes can be nucleic acids such as cDNAs ("cDNA chips"), mRNAs ("mRNA chips") or oligonucleotides ("oligonucleotide chips"), said oligonucleotides typically having a length of between about 25 and 60 nucleotides.

To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labeled and then brought into contact with the chip under hybridization conditions, leading to the formation of complexes between said nucleic acid. labeled target and probes attached to the surface of the chip that are complementary to this nucleic acid. The presence of labeled hybridized complexes is then detected.

These technologies make it possible to monitor the level of expression of a particular gene or of several genes, or even of all genome genes (full genome or full transcriptome) in a biological sample (cells, tissues, etc.). These technologies are used routinely by those skilled in the art and therefore there is no need to detail them here.

Alternatively, it is possible to use any current or future technology to determine gene expression based on the amount of mRNA in the sample. For example, one skilled in the art can measure the expression of a gene by hybridization with a labeled nucleic acid probe, for example by Northern blot (for mRNA) or by Southern blot (for cDNA) but also by techniques such as the Serial Analysis Method of Gene Expression (SAGE) and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. It is also possible to use fabric chips (also known as TMAs: "tissue microarrays"). The tests usually used with tissue chips include immunohistochemistry and fluorescent in situ hybridization. For mRNA analysis, the tissue chips can be coupled with fluorescent in situ hybridization. Finally, it is possible to use mass sequencing in parallel to determine the amount of mRNA in the sample (RNA-Seq or "Whole Transcriptome Shotgun Sequencing"). For this purpose, several methods of mass sequencing in parallel are available. Such methods are described in, for example, US 4,882,127, US 4,849,077; US 7,556,922; US 6,723,513; WO 03/066896; WO 2007/11 1924; US 2008/0020392; WO 2006/084132; US 2009/0186349; US 2009/0181860; US 2009/0181385; US 2006/0275782; EP-B1-1141399; Shendure to Ji, Nat Biotechnol., 26 (10): 1135-45. 2008; Pihlak et al. Nat Biotechnol., 26 (6): 676-684, 2008; Fuller et al. , Nature Biotechnol., 27 (11): 1013-1023, 2009; Tuesdays, Genome Med., 1 (4): 40, 2009; Metzker, Nature Rev. Broom., 1 1 (1): 31 -46, 2010.

Preferably, the expression of the PIGF is measured at the protein level by a method selected from immunohistology, immunoprecipitation, western blot, dot blot, ELISA or ELISPOT, protein chips, microarray chips. antibodies, or tissue microarrays coupled with immunohistochemistry, FRET or BRET techniques, microscopic or histochemical methods, including methods of confocal microscopy and electron microscopy, methods based on the use of one or more excitation wavelengths and a suitable optical method, such as an electrochemical method (voltammetry and amperometry techniques), the atomic force microscope, and radiofrequency methods, such as multipolar resonance spectroscopy , confocal and non-confocal, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (eg, surface resonance resonance, ellipsometry, resonant mirror method, etc.), flow cytometry, radioisotopic or magnetic resonance imaging, polyacrylamide gel electrophoresis (SDS-PAGE) analysis; by HPLC-Mass spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS).

More preferably, the amount of PIGF is determined by a method chosen from immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled with immunohistochemistry. Antibodies directed against PIGF are commercially available (see, for example, RaD Systems, Santa Cruz, Abcam, etc.) and may be used in the methods of the invention. Even more preferably, the expression of PIGF is measured by Western Blot or ELISA. In addition, the amount of PIGF is normalized with respect to a control marker which may be a gene selected from B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IP08 and HMBS, or a polypeptide selected from the product of said genes.

The rate of PIGF thus measured is then compared to an expression level of reference PIGF to determine whether it is a FASD subject.

By "a level of expression of reference PIGF" is meant within the meaning of the present application any rate of expression of said factor used for reference. For example, a reference expression level can be obtained by measuring the level of expression of PIGF in a biological sample, for example a placenta or umbilical blood, of a healthy subject, i.e. a subject who has not been exposed to alcohol in utero.

The invention will be described more precisely by means of the examples below. Said examples are provided here by way of illustration and are not, unless otherwise indicated, intended to be limiting.

LEGENDS OF FIGURES

Figure 1. Effects of in utero alcohol exposure on cortical angiogenesis in mouse E20 embryos. A, B: Effects of Fetal Alcohol Exposure from GD15 to GD20 on the Organization of Cortical Microvessels in Control (A) Animals and Exposed to Alcohol (B). Microvessels of the brain were visualized by immunohistochemistry against CD31. The arrows indicate the microvessels of the brain with a radial orientation in the "control" group. It should be noted a loss of the radial organization in the group "Alcohol". I -VI: Cortical layers; CC: Corpus callosum. C: Distribution of orientation (angle categories) of cortical microvessels in the immature cortex of fetus GD20. Statistical analysis was performed using the x ² test. D: Western blot quantification of effects of fetal alcohol exposure during the last week of pregnancy on cortical expression of CD31 at GD20. vs the "Witness" group using an unpaired t-test.

Figure 2. Effects of In utero Alcohol Exposure on Expression of VEGF / PIGF Family Members in Mouse E20 Embryos. AE: Western blot quantification of protein levels of VEGFA (A), PIGF (B), sVEGF-RI (C), mVEGF-R1 (D) and VEGF-R2 in the cortex of the "Control" and "Alcohol" groups. F: Western blot comparison of PIGF protein levels in the cortex and placenta of the E20 embryos of the "control" group. ^* p <0.05; ^*** p <0.001 vs the "Witness" group using an unpaired t-test. Figure 3. Effects of in utero alcohol exposure on the ultrastructural characteristics of the placenta in GD20 mice. A: Observation by Cresyl violet staining of the effect of alcoholic exposure on the laminar structure of the placenta. The maternal side of the placenta is upward. Alcohol affects the segregation of junction areas and labyrinth (dotted lines). B: Quantification by image analysis of the effects of alcohol on the thickness of Reichert's membrane. C, D: Low magnification observation of the giant trophoblast layer in the "Control" (C) and "Alcohol" (D) groups. The arrows indicate giant trophoblasts. These have a typical rectangular shape in the placenta of the group "Witness", while in the group "Alcohol" they have a rounded shape. EH: Images acquired by medium (E, F) and strong (G, H) electron microscopy showing the cellular morphology of giant trophoblasts and the presence of zonula occludens (arrows) in the "control" (E, G) and "Alcohol" (F, H). Zonula occludens (stars) is no longer visible in animals treated with alcohol. The inserts in E and F indicate the area observed at higher magnification at G and H, respectively. D: maternal decidua; J: junction area; L: labyrinth area; Tg: layer of giant trophoblasts. *** p <0.001 vs the "Witness" group using an unpaired t-test.

Figure 4. Effects of in utero alcohol exposure on the expression of proteins involved in the placental barrier and placental energy metabolism. A, B: Immunohistochemical observation of the ZO-1 protein in the mouse placenta maze area of the "Control" (A) and "Alcohol" (B) groups. The ZO-1 protein appears to form groups of points (arrows) in the "Control" group while the labeling is diffuse in the "Alcohol" group. The trophoblast layers were demonstrated by immunoreactivity with the Glut-1 glucose transporter. The nuclei were marked at Hoechst. C: Double labeling with antibodies against MCT-1 monocarboxylate transporters and glucose in the labyrinth zone of a "control" placenta. In contrast to Glut-1, the expression of MCT-1 is associated with the maternal layer of the syncytiotrophoblast. The nuclei were marked at Hoechst. D: Western blot quantification of expression levels of ZO-1 and MCT-1 proteins in placentas of the "control" and "alcohol" groups. * p <0.05, ** p <0.01 vs the "control" group using an unpaired t-test. The Western blot analyzes showed that the ZO-1 levels decreased significantly in the placentas of animals exposed to ^alcohol while the rate of the MCT-1 protein was significantly increased. * P <0.05, ** p <0.01 compared to the control group using the unpaired t-test. EH: Immunohistochemistry tests illustrate the distribution of VEGF-R1 (E), Glut-1 (F, G) and PIGF (H) in layers of syncytiotrophoblasts from the mouse placenta. The nuclei were marked at Hoechst.

Figure 5. Effects of in utero alcohol exposure on the expression of VEGF / PIGF family members in murine placenta. AF: Western blot quantification of effects of alcohol exposure during the last week of gestation on placental expression of VEGF-A (A), PIGF (B), sVEGF-RI (C), mVEGF-R1 (D), VEGF-R2 (E) and CD31 (F) to GD20. G, H: Immunohistochemical labeling showing the distribution of VEGF-R2 (G) in Glut-1-labeled (H) labeled placenta syncytiotrophoblast layers. The nuclei were marked at Hoechst. ^* p <0.05 vs the "control" group using an unpaired t-test. Figure 6. Diffusion of Evans blue and recombinant human PIGF injected in utero from the placenta into the fetal brain and effect of placental repression of PIGF on cerebral vasculature. A, B: Time-course visualization of Evans Blue administered by microinjection into the placenta of a GD15-gravid mouse. The fluorescence was detected by UV illumination (A) and is represented using a dummy color scale (B). C, D: Time-based visualization of Evans Blue fluorescence in the fetal brain following GD15 placental microinjection. The fluorescence was detected by UV illumination (C) and is represented using a dummy color scale (D). E, F: Quantification over time by spectrophotometry of the absorbance at 595 nm of the Evans blue signal injected into the placentas (E) and subsequently into the brains of the corresponding fetuses (F). G: ELISA quantification of human PIGF in mouse fetal brain 30 min after injection of hPIGF into placentas of GD15 pregnant mice. ^* p <0.05 vs the "control" group using an unpaired t-test. H: Photomicrograph visualizing the expression of eGFP 48 hours after in utero transfection of GD15 pregnant mouse placenta with a plasmid encoding an eGFP. I, J: Triple staining eGFP / Glut-1 / Hoechst indicating that the fluorescence of eGFP (I) is mainly associated with Glut-1-tagged fetal trophoblastic layer (J) (arrowheads). The fetal part of the trophoblastic layers is identified by the presence of nucleated red blood cells specific to the fetal circulation (arrows). K: Western blot visualization of PIGF, GFP and actin proteins in placentas of non-transfected animals (sh- / GFP-), GFP-transfected (sh- / GFP +) and shPLGF / GFP transfected (sh + / GFP +). L, M: Western blot quantification of PIGF (L) levels and expression of GFP (M) in placentas of non-transfected animals (sh- / GFP-), GFP-transfected (sh- / GFP +) and shPLGF / GFP transfected (sh + / GFP +) four days after transfection. N: Western transfer quantification of VEGF-R1 expression levels in fetal brain from non-transfected (sh- / GFP-), GFP-transfected (sh- / GFP +) and shPLGF / GFP transfected placenta (sh + / GFP +) four days post-transfection. * P <0.05 vs group ^"sh- / ^GFP" using one-way ANOVA followed by ^a post-hoc Tukey test. OR: Visualization of the vascular system in the fetal cortex from untransfected placenta (Sh- / GFP-) (0), transfected GFP (Sh- / GFP +) (P) and transfected shPLGF / GFP (Sh + / GFP +) (Q) statistical analysis of the disruption of cortical vessels performed using ^the x2 test (R).

Figure 7. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta from gestational weeks 20 to 25. A, B: Immunohistochemical labeling against CD31 and counterstaining with toluidine blue to visualize microvessels (chestnuts) present in the placental villi (blue) of the "control" (A) and "FAS / pFAS" (B) groups at gestational ages [20-25 WG [. C: Percentage of villi classified by size in the placentas of the "control" and "FAS / pFAS" groups at gestational ages [20-25 WG [. D: Distribution of vessels by villous size in placentas of the "control" and "FAS / pFAS" groups at gestational ages [20-25 WG [. E: Village vascular vascular surface area in the placentas of the "control" and "FAS / pFAS" groups at gestational ages [20-25 WG [. * P <.05 vs the control group using an unpaired t-test.

Figure 8. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta from gestational weeks 25 to 35. A, B: Immunohistochemical labeling against CD31 and toluidine blue staining to visualize microvessels (chestnuts) present in Placental (blue) villi of the "control" (A) and "FAS / pFAS" (B) groups at gestational ages [25-35 WG [. C: Percentage of villi classified by size in the placentas of the "control" and "FAS / pFAS" groups at gestational ages [25-35 WG [. D: Distribution of vessels by villous size in placentas of the "control" and "FAS / pFAS" groups at gestational ages [25-35 WG [. E: Vascular villous surface area in placentas of the "control" and "FAS / pFAS" groups at gestational age [25-35 WG [. * p <0.05 vs the "control" group using an unpaired t-test.

Figure 9. Morphometric characterization of the effects of in utero alcohol exposure on the human placenta from gestational weeks 35 to 42. A, B: Immunohistochemical labeling against CD31 and toluidine blue staining to visualize the microvessels (chestnuts) present in placental villi (blue) groups' Witness "(A) and" FAS / pFAS "(B) at gestational ages ranging from [35-42 WG [. The luminal region of the microvessels is greatly reduced in the "FAS / pFas" group. C: Percentage of villi classified by size in placentas of the "control" and "FAS / pFAS" groups at gestational ages ranging from [35-42 WG [. D: Distribution of vessels by villous size in placentas of the "control" and "FAS / pFAS" groups at gestational ages ranging from [35-42 WG [. E: Village-size vascular area in placentas of the "control" and "FAS / pFAS" groups at gestational ages ranging from [35-42 WG [. ^* p <0.05 vs the "control" group using an unpaired t-test.

Figure 10. Time-course effects of in utero alcohol exposure on villous and vessel densities in human placentas and western blot characterization of pro-angiogenic proteins and energy metabolism. A: Evolution of villous densities in placentas of the "control" (A) and "FAS / pFAS" (B) groups at gestational age [20-25 WG [, [25-35 WG [and [35-42 WG [. B: Evolution of vessel densities in placentas of the "control" and "FAS / pFAS" groups at gestational ages [20-25 WG [, [25- 35 WG [and [35-42 WG [. p <0.05, p <0.01 vs the "Control" group as shown on the graph. ^* p <0.05, ^*** p <0.001 for the "Control" vs. "Alcohol" groups for a given gestational age class. CH: Quantification by western blot of protein levels of ZO-1 (C), MCT-1 (D), PIGF (E), VEGFA (F), VEGF-R1 (G) and VEGF-R2 (H) in placentas "Witness" and "FAS / pFAS" groups. ^* p <0.05 vs the "control" group using an unpaired t-test.

Figure 1 1. Comparison of cerebral and placental damage observed in human fetuses and induced by in utero alcohol exposure and statistical correlation. A-H:

Vascular organization in the brains (A, D) and placentas (E, H) of patients from the "control" group to WG22 (A, E) and WG31 (C, G) and vascular organization in the brains (B, D) and placentas (F, H) of patients from the group "FAS / pFAS" to WG21 (B, F) and WG33 (D, H). I, J: Statistical correlation between cortical vascular disorganization and placental vascular density in patients in the "Control" (I) and FAS / pFAS (J) groups.

Figure 12. Effects of overexpression of IFN in utero on fetal growth and cortical vascularization during intrauterine exposure to alcohol. A, B: A PGF / CRISPR / dCas9 activation approach coupled to electroporation of the placenta in utero was performed at GD13 (A) and overexpression of PIGF was monitored at GD20 (B). In the "Alcohol" group, exposure to alcohol in utero takes place between GD15 and GD20. C, D: Visualization of E20 fetuses from pregnant mice exposed to NaCl (C) or alcohol (D). It is worth noting the small size of fetuses exposed to alcohol. Bars indicate morphometric measurements that have been made (head size (a), body size (b), abdomen size (c), and size of whole fetus (a + b)). E, F: Visualization of E20 fetuses from pregnant mice after electroporation in utero PGF / CRISPR-dCas9 plasmids in placentas of "control" groups (E) or pregnant mice exposed to alcohol (F). G, H: Quantification of the size of the abdomen (G) and the whole fetus (H) in the group "Control" (NaCl) and in the group "Alcohol". In the same uterine horn some placentas did not undergo electroporation (black bars), others underwent electroporation with control plasmid CRISPR-cas9 (gray bars) or electroporation with plasmids PGF / CRISPR-dCas9 (white bars) ## p <0.01;### p <0.001;#### p <0.0001 vs the group "Witness" and ^* p <0.05; ^** p <0.01; ^**** p <0.0001 as indicated using the two-way ANOVA test followed by a Tukey post-hoc test. IK: Visualization of the vascular system in the fetal cortex E20 from control (NaCl) / non-transfected (I), transfected alcohol / CRISPR-cas9 (J) and alcohol PGF / CRISPR-dCas9 placentas transfected. It is noted that overexpression of PIGF at the placental level corrects the disorganization of the cerebral vasculature induced by alcoholization in utero. L: Quantification of the percentage of radial vessels in the cortex of fetuses E20 from electroporated placentas (black bars), electroporated with CRISPR-cas9 control plasmids (gray bars) and electroporation with PGF CRISPR-dCas9 plasmids (white bars). #p <0.05;## p <0.01 vs the "control" group and ^* p <0.05 as indicated using the two-way ANOVA test followed by a Tukey post-hoc test.

Figure 1 3. Effects of placental overexpression of PIGF on the size of the head and body of the fetus E20 in the "control" and "alcohol" groups. A, B: Quantification of the size of the head (A) and the body (B) in the group "Control" (NaCl) and the group "Alcohol". In the same uterine horn some placentas did not undergo electroporation (black bars), others were electroporated with CRISPR-cas9 control plasmids (gray bars) or electroporated with PGF CRISPR-dCas9 plasmids ( white bars). ## P <0.01;### P <0.001;#### P <0.0001 compared to the "Control" group and ^* p <0.05; ^** P <0.01 as indicated using the two-way ANOVA test followed by a Tukey post-hoc test. EXAMPLES

Examples A: Abnormalities following alcohol abuse in utero Examples A below include several results of tests carried out prior to the present invention demonstrating that in mice and humans:

• fetal alcoholization affects cerebral angiogenesis and the organization of the cerebrovascular network,

• These brain changes are correlated with vascular anomalies of the placenta,

• a placental pro-angiogenic factor is able to reach the fetal brain,

• neurodevelopmental abnormalities of cerebral angiogenesis in FASD children are associated with deregulation of the placental PIGF / cerebral VEGF-R1 system,

• a placental invalidation of the PIGF reproduces the effects of fetal alcoholization on cerebral VEGF-R1, and

• Deregulation of placental IGFI rates following fetal alcoholization can predict brain damage.

Anomalies of cerebral angiogenesis following alcohol in utero

Effects of in utero exposure to alcohol on the development of the cerebrovascular network

The present inventors have previously demonstrated that prenatal alcohol alcoholization induces cerebral vascular disorganization. In particular, the effect of alcohol is associated with a significant decrease in the number of cortical vessels having a radial orientation in favor of the number of microvessels having a random orientation (Figure 1). In parallel with the study conducted in mice, an analysis of the cerebral microvascular system in humans has shown that, as in mice, the cortical microvessels which have a radial orientation in the "control" group are totally disorganized in the group. FAS / pFAS "(Figure 11 and Jegou et al., Volume 72, No. 6, 31 December 2012, pages 952-960).

Effects of in utero exposure to alcohol on the expression of representative genes of the vascular system in mice

Quantitative RT-PCR (mRNA) and Western blot (protein) studies revealed marked dysregulation of VEG F-R1 and VEGF-R2 receptor levels, which angiogenic factors such as VEGFA or PIGF. The abnormalities of the cerebrovascular network are thus associated with a deregulation of the expression of pro-angiogenic receptors in the brain (Figure 2 and Jegou et al., Volume 72, No. 6, December 31, 2012, pages 952-960). Abnormalities of placental angiogenesis following alcohol in utero

Various placental parameters have been studied in the mouse (Figures 3-5) and in humans (Figures 7-9) by an immunohistochemical approach coupled with a morphometric analysis including in particular the density and size of the placental villi, the density and the vascular surface or the proportion of vessels per villus. In humans, these parameters were measured and compared between 34 placentas of control individuals and 36 placentas from individuals exposed in utero to alcohol. The placentas were divided into three age classes comparable to those of the brain study (Jegou et al., Vol.72, No. 6, December 31, 2012, pages 952-960). This paper presents the results for the age classes [20-25GW [, [25-35GW] and [35-42GW]. In particular, morphometric analysis indicates that the distribution of placental vessels by villous size and vascular area are significantly impacted by alcoholization (Figure 10). In addition, a longitudinal analysis of vascular density taking into account the "age" factor indicates that in the "control" group, placental angiogenesis strongly increases between age groups [20-25GW [and [25-35GW [. This strong increase in placental vasculature is explained by significant brain development during the third trimester of pregnancy that requires increased oxygen and nutrient requirements. In contrast, fetal alcoholization induces stagnation or even a decrease in placental vascular density (Figure 10).

In conclusion, the present results indicate that there are vascular abnormalities in the human placenta as well as in the cerebral cortex in subjects who have been exposed to alcohol. These results therefore support the hypothesis of a correlation between brain disorders and placental deficits of angiogenesis.

Demonstration of a correlation between placental and cerebral vascular abnormalities The placental and cerebral vascular abnormalities observed in humans following in utero alcoholization may be the result of totally unrelated processes cause-effect or, on the contrary, closely intertwined. The fact that the source of PIGF is unique and of placental origin argues in favor of the second hypothesis. However, in order to demonstrate a link between cerebrovascular and placental disorders, we carried out a correlation study on the one hand in the subjects of the "control" group and, on the other hand, in individuals of the "FAS / pFAS" group. "(Figure 1 1).

The results demonstrate that in the "control" group, the increase in placental vascularization does not affect the radial organization of the cortical vessels (R ² 0.4719). On the other hand, the lack of placental vascularization observed in the "FAS / pFAS" group is closely correlated with the random orientation of the cortical vessels (R ² 0.9995). There is therefore a very significant interaction between placental and cerebral vascular changes.

Demonstration of a functional link between placental PIGF and its brain receptor

In utero administration of a fluorescent molecule at the placental level in pregnant mice (GD15) is found after 20-30 min in the fetal brain (Figure 6). In addition, human recombinant PIGF injected into the mouse at the placenta is detected after 30 min by ELISA in the fetal brain (FIG. 6). These data indicate that placental molecules and in particular the PIGF are able to reach the brain of the fetus.

Invalid utero transfection inhibition of murine PIGF by shRNA resulted in repression of placental PIGF protein levels after 48 hours (Figure 6). This effect is associated with the brain level by a fall in the protein levels of the VEGF-R1 receptor (Figure 6). These results indicate that i) the specific repression of placental PIGF directly impacts brain receptor expression, ii) the specific repression of placental PIGF mimics the effects of alcohol on brain VEGF-R1 expression (Figures 2 and 6). ).

Expression levels of proteins known to be either actors of angiogenesis or proteins specific to the vascular system have been quantified by western blot. This work was done in animals (mice, placenta / brain) and humans (placenta).

In mice, the quantification of placental VEGF _A and PIGF expression levels demonstrates a significant decrease only in PIGF (of which the placenta is the only source in the body, Figure 5). At the same time, the quantification of VEGF _A and PIGF receptors indicates that the expression of VEGFR1 (unique receptor of PIGF) is decreased in both the placenta and the brain (Figures 2 and 5). This decrease, very marked, is the order of 50%. The expression of VEGFR2 at the cerebral level is not affected. In addition, the quantification of the vascular protein ZO-1, involved in the establishment of the placental and blood-brain barrier, is strongly diminished in the placenta (Figure 4) · In parallel with the work carried out in mice, protein expression analysis was conducted on human placentas with proven maternal alcohol and live children. We collected 7 placentas "Witnesses" and 6 placentas "Alcohol" and quantified by western blot the candidate markers identified in the Mouse. The results indicate that in the group "Alcohol" the expressions of PIGF and ZO-1 are very strongly diminished as in the mouse (Figure 10). These data indicate that the fetal alcohol effects observed at the placental and cerebral levels are found in two different species, mice and humans.

Examples B: Therapeutic Effect of PIGF

These examples correspond to the results obtained by tests of overexpression of PIGF at the placental level thus making it possible to reverse the deleterious effect of alcohol on the morphology and size of the fetus as well as on cerebral angiogenesis.

1. Materials and methods

Placental Overexpression of PIGF by In Vivo Activation of the PGF Gene in a CRISPR-dCas9 System Associated with in vivo electroporation, the CRISPR-dCas9 approach is an innovative gene overexpression method for identifying the role of endogenous proteins in processes of development. The PGF CRISPR-dCas9 activation plasmids (sc-42221 1 -ACT) constituting a complex of synergistic activation mediators (SAM) were designed and provided by Santa Cruz Biotechnology. SAM binds to a specific site upstream of the transcription initiation site of the PGF gene, thereby activating the endogenous transcription of the target gene. In practice, the PGF-CRISPR dCas9 activation plasmids are transfected by in utero electroporation to GD13 in two groups of mice ("Control" and "Alcohol"). Exposure to alcohol occurs between GD15 to GD20. A two-day delay between transfection of PGF CRISPR-dCas9 activation plasmids and alcohol treatment is required to allow for plasmid expression and overexpression of PLGF. For a given pregnant mouse, 3 placentas were transfected with CRISPR-dCas9 PGF activation plasmids, 3 placentas were transfected with the CRISPR-cas9 control plasmids (sc-418922) targeting a non-specific 20 nt guide RNA (negative control). The other placentas are not transfected and are used as a control ("control" group).

2. Results Placental overexpression of the PGF gene makes it possible to restore the cerebral angiogenesis of the fetus impaired by exposure to alcohol in utero.

A CRISPR-dCas9 activation strategy coupled with in utero transfection was used to induce high expression of the endogenous PGF gene in the placenta of pregnant mice not treated with alcohol ("Control" group) and pregnant mice exposed to alcohol ("Alcohol" group) (Fig. 12, A and B). At GD20, it has been observed that exposure to alcohol in utero leads to a decrease in intrauterine growth of the fetus (Figure 12, C and D.) with a significant decrease in the size of the head (p. <0.01, Fig. 13), body size (p <0.0001, Fig. S2), abdomen size (p <##, Fig. 12, G) and the size of the abdomen. whole fetus (p <####; Figure 12, H). In the "control" group, overexpression of PGF induces a macromorphic evolution of the fetus (Figure 12, C and E), with a significant increase in the size of the abdomen (p <0.01, Figure 12, G) and the size of the entire fetus (p <0.01, Figure 12H). In the "Alcohol" group, overexpression of PGF significantly increased body size (p <0.05, Figure 13) and whole fetal size (p <0.01, Figure 12, H). In comparison with the control group, PGF overexpression suppressed the deleterious effects of alcohol on head size (Figure 13) and abdominal size (Figure 12, G) and reduced 38.6 ± 2.8% and 46.8 ± 2.9% the effects of alcohol on body size (Figure 13) and the size of whole fetuses (Figure 12, H), respectively. No effect on fetal morphology was observed in pregnant mice transfected with the CRISPR-cas9 control plasmid (FIG. 12, G and H). As, it has been previously demonstrated, in the fetal brain of E20 mice, exposure to alcohol in utero leads to disruption of the cortical vascular system (Figures 1, A and B). Transfection of the placenta with a CRISPR-cas9 control plasmid has no effect on the angiogenic alterations induced by in utero alcohol exposure (Figures 12, I, J and L). On the other hand, in pregnant mice whose placenta was transfected with a CRISPR-PGF activation plasmid dCas9, the radial organization of the cortical microvessels was significantly restored (p <0.05, Fig. 4, K, L). These data are the first demonstration that overexpression of PLGF in the placenta can partially or completely restore deficiencies Morphological and alterations of cerebral angiogenesis induced by exposure to alcohol in utero.

Conclusion

In view of the various results obtained by the inventors in the pregnant mouse, it appears that: "the overexpression of PIGF in the placenta of a pregnant mouse exposed to alcohol allows the complete disappearance of morphometric abnormalities in the abdomen and of the skull of the fetus caused by exposure to alcohol. Thus, the size of the skull and abdomen that is reduced following exposure to alcohol returns to normal after overexpression of PIGF, overexpression of PIGF in the placenta of a pregnant mouse exposed to Alcohol allows the reduction of morphometric abnormalities in the body of the fetus and the whole fetus caused by exposure to alcohol. Thus, the size of the fetal body as well as the size of the whole fetus reduced during exposure to alcohol returns to almost normal after the overexpression of PIGF, the overexpression of PIGF at the placental level allows the improvement of the cerebral angiogenesis of the fetus altered by exposure to alcohol of a pregnant mouse. The neuronal function of this fetus will thus be improved,

• overexpression of PIGF can be used effectively as a drug in the treatment of FASD-like abnormalities, particularly to improve cerebral angiogenesis and / or restore the normal morphological appearance of the vascularization of the fetus with FASD.

Claims

1. Placental Growth Factor (PIGF) for use in the prevention and / or treatment of fetal alcohol spectrum disorder (FASD) in a subject who has been exposed to alcohol in utero.

2. PIGF for use according to claim 1, characterized in that the TCAF is related to cerebrovascular disease.

3. PIGF for use according to claim 1 or claim 2, characterized in that said PIGF stimulates cerebral angiogenesis.

4. PIGF for its use according to claim 1, characterized in that the FASD is fetal alcohol syndrome (FAS), especially when it manifests itself in the form of hypotrophy in a subject who has been exposed to alcohol in utero.

5. PIGF for use according to claim 4, characterized in that the hypotrophy is a hypotrophy of the whole subject or one of its selected parts among the torso, the abdomen and the skull.

6. PIGF for its use according to any one of claims 1 to 5, characterized in that said PIGF has the sequence represented by one of SEQ ID NO: 1 to SEQ ID NO: 4.

7. PIGF for use according to any one of claims 1 to 6, characterized in that it is obtained by genetic engineering or chemical synthesis.

8. PLGF for use according to any one of claims 1 to 7, characterized in that the subject having been exposed to the alcohol in utero is selected from an embryo, a fetus and a child, in particular a premature child.

9. PLGF for use according to any one of claims 1 to 8, characterized in that it is administered in utero or ex utero or in utero and ex utero.

10. PIGF for use according to any one of claims 1 to 9, characterized in that it is administered ex-utero to a premature child.

1 1. A pharmaceutical composition for use in the prevention and / or treatment of fetal alcohol spectrum disorder (FASD) comprising a PIGF as defined in any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. PIGF for its use according to any one of claims 1 to 10 or pharmaceutical composition for use according to claim 1 1, said use comprising a prior step of identifying the subject, said identification comprising the following steps: a) measurement the amount of PIGF in a biological sample of said subject, preferably from the placenta or umbilical cord blood; b) comparing the amount of PIGF from step a) with a reference that is a measure of the amount of PIGF in a healthy subject, and c) determining a FASD or a risk of developing FASD in said subject.

13. PIGF or composition for use according to claim 12 characterized in that the subject suffers from a FASD or has been identified at risk of developing a FASD if the measured amount of PIGF in step a) is less than the reference of step b).

14. PIGF or composition for their use according to claim 12, characterized in that the amount of PIGF is determined by measuring the amount of PIGF nucleic acid or the amount of PIGF polypeptide.

PIGF or composition for their use according to any one of claims 12 to 14 characterized in that the amount of PIGF is measured by a method selected from Northern blot, Southern blot, PCR, RT-PCR, quantitative RT-PCR, SAGE and its derivatives, nucleic acid chips, especially cDNA chips, oligonucleotide chips and mRNA chips, tissue chips and RNA-Seq and / or by a method selected from immunohistology, immunoprecipitation, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled to immunohistochemistry, FRET or BRET, microscopy or histochemistry methods, including confocal microscopy and electron microscopy methods, methods based on the use of one or more wavelengths of excitation and a suitable optical method, such as an electrochemical method (voltammetry and amperometry techniques), the atomic force microscope, and radiofrequency methods, such as multipole resonance spectroscopy, confocal and non-confocal, fluorescence detection, luminescence , chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (eg, surface plasmon resonance, ellipsometry, resonant mirror method, etc.), flow cytometry, radioisotopic or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); by HPLC-Mass spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS).