EP4127026A1

EP4127026A1 - Linear copolymer for gene transfection

Info

Publication number: EP4127026A1
Application number: EP21714144.9A
Authority: EP
Inventors: Julia BENSEMHOUN; Hervé Cheradame
Original assignee: Polytheragene
Current assignee: Polytheragene
Priority date: 2020-03-23
Filing date: 2021-03-23
Publication date: 2023-02-08
Also published as: WO2021191208A1; FR3108330B1; FR3108330A1

Abstract

The invention relates to a linear polyethyleneimine copolymer comprising monomer units of formula (I) and monomer units of formula (II), and to the use thereof in gene transfection.

Description

LINEAR COPOLYMER FOR GENE TRANSFECTION

The present invention relates to polyethylenimine-based polymers and their use in gene transfer for therapeutic application.

In some therapies it is desirable to correct genetic defects in target cells. To do this, it is necessary to transfer genes into the target cells. The main problem with in-vivo gene transfer is to ensure efficient transfer into target tissues or cells accompanied by a high level of gene expression. Transfection uses complexes comprising two parts: an active element which is generally a nucleic acid, a plasmid, a DNA fragment, an RNA fragment and a gene transfer vector which may also include functions to help in the transformation. localization of the complex on the target cell or tissue. The gene transfer vectors are typically viral vectors or synthetic vectors. Among the synthetic vectors, mention may be made of cationic lipids or polymeric vectors. Positive polyelectrolytes constitute a set of polymer vectors particularly suitable for gene transfer, insofar as their positive charge gives them properties of complexing DNA and plasmids, and of interaction with the target cell surfaces.

Cationic polymers have already been described in WO2009 / 112402. However, there is still an interest in proposing new copolymers allowing in particular improved transfection, in particular of genes.

Thus, an objective of the present invention is to provide a copolymer useful for transfection and allowing improved transfection compared to existing vectors.

Another objective of the present invention is to provide such a copolymer which exhibits reduced toxicity.

Still other objectives will become apparent in the light of the description of the invention which follows. The present invention relates to a linear polyethyleneimine copolymer (IPEI) comprising monomeric units of formula (I) and monomeric units of formula (II) wherein :

R ¹ and R ² , identical or different, represent a hydrogen atom, an alkyl group, linear or branched, comprising from 1 to 10 carbon atoms, preferably from 1 to 2 carbon atoms, at least one of R ¹ or R ² represents H;

R, identical or different, is chosen from H or an amine protecting group, for example Boc (tert-butoxycarbonyl) or Fmoc (fluorenylemethoxycarbonyl); n is a number between 1 and 99% of the total monomers; and m is a number between 1 and 99% of the total monomers.

Advantageously, the copolymer of the invention is a linear copolymer.

Preferably, R ¹ and R ² represent H.

In a first embodiment, R represents H.

Preferably R ¹ , R ² and R represent H.

In a second embodiment, R represents an amine protecting group, for example Boc (tert-butoxycarbonyl) or Fmoc (fluorenyl methoxycarbonyl).

Preferably, R ¹ , R ² represent Fl and R represents an amine protecting group, for example Boc (tert-butoxycarbonyl (CFl ₃ ) ₃ -O-C0-).

In a third embodiment, R, identical or different, represents Fl or an amine protecting group, for example Boc (tert-butoxycarbonyl) or Fmoc (fluorenyl methoxycarbonyl), at least one of R is an amine protecting group , for example Boc (tert-butoxycarbonyl) or Fmoc (fluorenylemethoxycarbonyl). Preferably, the copolymer of the invention is a random copolymer. The copolymer of the invention can correspond to one of the following formulas: in which R, R ¹ , R ² , m and n are as defined above.

Preferably, in the copolymer of the invention, m is less than or equal to 70% of the total monomers, preferably m is between 1 and 70%, more preferably between 2 and 50%, preferably between 4 and 40%, for example between 5 and 35%, in particular between 10 and 30%, for example between 1 and 14%, of the total monomers.

Preferably, in the copolymer of the invention, n is greater than or equal to 30%, preferably n is between 30 and 99%, more preferably between 50 and 98%, preferably between 60 and 96%, for example between 65 and 95%, in particular between 80 and 90%, preferably between 70 and 90%, for example between 86 and 99%, of the total monomers.

The copolymer of the invention can also comprise an additional monomer unit of the following formula III: in which

R ³ represents a C1-C6 alkyl group, preferably a methyl or ethyl group; r is a number between 0 and 95% of the total monomers, advantageously between 0 and 10% of the total monomers.

This monomer unit is also distributed randomly in the copolymer according to the invention.

Advantageously m + n + r = 100%.

The random copolymer according to the invention can have the following formula IX:

In which m, n, r, R ¹ , R ² and R ³ are as defined above, or the following formula X:

Or the following formula XI:

The copolymer of the invention can also comprise monomer units comprising saccharide residues or poly (ethylene oxide) residues or peptide units, advantageously polylysine.

The saccharide residues are advantageously chosen from residues of lactose, tetraglucose and mannose.

The saccharide residues and / or the poly (ethylene oxide) residues are grafted onto the nitrogen atom of the PEI unit of formula (I) advantageously via a CO group.

Advantageously, the proportion of these residues is such that the polyethylenimine is always generally linear in structure. Advantageously, each of these residues is present in the copolymer in a proportion of less than 70% of the total monomers, advantageously in a proportion of less than 50% of the total monomers and even more advantageously in a proportion of less than 30% of the monomers. total, even more advantageously in a proportion of less than 10% of the total monomers. Advantageously, each of these units is arranged randomly in the copolymer.

Preferably, the copolymer has a molar mass of between 1000 and 200000 Da, preferably between 3000 and 50,000 Da.

Advantageously, the copolymer according to the invention has the capacity to be positively charged by the amine function. The present invention also relates to a process for preparing the copolymers of the invention comprising the reaction between a polyethylene imine compound of formula (I) and a compound of formula (IV) and the deprotection of the amine functions. in which R1 is as defined above and R 'represents a protective group for an amine function, for example Boc (COOtBu) or Fmoc, p being greater than n and m + n, or where appropriate m + n + r, is equal to p.

Preferably, the compound of formula (IV) is the following compound:

( ^{| V} ) Boc-his (1-boc) -OSu

The process of the invention may preferably correspond to grafting of a compound of formula (IV) onto a compound of formula (I), preferably the activated ester function (succidinimyl function).

The process of the invention can be implemented in a solvent, the solvent must allow the solubilization of the PEI, in particular methanol, water, preferably with a control of the pH at neutral pH (approximately 7), DMSO or chloroform, from preferably DMSO.

The deprotection step is optional, it can be total or partial, and is carried out in any manner known to those skilled in the art, in particular by the action of trifluoroacetic acid (TFA). In the context of partial deprotection, at least one of the Rs is different from Fl.

Preferably, the process of the present invention is carried out at a temperature between 25 and 100 ° C, preferably between 50 and 80 ° C.

The product obtained can be purified by any technique known to those skilled in the art, in particular by extraction, for example with dichloromethane and then dialysis of the aqueous phases recovered. The process of the invention therefore consists of a random reaction of a histidine derivative with polyethylenimine.

The degree of modification of the polyethylenimine and therefore the value of m in the copolymer according to the invention depends on the duration of the step of bringing the compound of formula (IV) into contact with the IPEI.

Advantageously, the duration of this step is at most 5 days, advantageously at most 4 days, more advantageously at most 3 days, more advantageously between 12 hours and 2 days, preferably between 24 h and 2 days .

Preferably, the starting polyethylenimine has a molar mass of between 500 and 200,000 Da, preferably between 1,000 and 100,000 Da.

Particularly advantageously, the copolymer of the invention can be used as a pharmaceutically acceptable carrier, in particular as a carrier for an active substance.

In the context of the present invention, by active substance is meant any active substance that can be used:

- in pharmacies. Mention may in particular be made of analgesics, antipyretics, aspirin and derivatives, antibiotics, anti-inflammatory drugs, antiulcer drugs, antihypertensives, neuroleptics, antidepressants, oligonucleotides, peptides, proteins;

- in cosmetics. Mention may in particular be made of anti-UV and self-tanning compounds;

- in nitraceptic. Mention may be made, for example, of proteins;

- in food;

- in agrochemistry; in veterinarian.

Advantageously, the active substance is charged or capable of being negatively charged. The copolymer according to the invention, as mentioned above, is cationic or potentially cationic, thus an electrostatic type bond is created between the copolymer of the invention (i) and the active substance (ii).

Preferably, the copolymer according to the invention can be used as a vector in transfection. Thus, the present invention also relates to the copolymer according to the invention as an active substance carrier. Preferably, the present invention relates to the copolymer according to the invention for its use for the transfection of genes.

The present invention relates to the use of the copolymer according to the invention as a pharmaceutically acceptable carrier of an active substance in a pharmaceutical composition.

The present invention also relates to a pharmaceutical composition comprising a copolymer according to the invention, an active substance and optionally a pharmaceutically acceptable excipient.

Thus, the present invention also relates to the pharmaceutical composition according to the invention for its use for the transfection of genes.

The dosage may vary within wide limits depending on the therapeutic indication and the route of administration, as well as the age and weight of the subject.

The identification of the patient in need is defined by the person skilled in the art. By patient is meant a human being or an animal. A physician or veterinarian can identify, through clinical tests, physical examination, laboratory tests or diagnoses, and family and / or medical history, those individuals who need such treatment.

The term “sufficient amount” means an amount of compound according to the present invention effective for preventing or treating pathological conditions. The sufficient amount can be determined by one skilled in the art, by means of conventional technique and by observation of the results obtained under like circumstances. To determine the sufficient quantity, various factors must be taken into account by a person skilled in the art, in particular and without being limited thereto: the subject, his size, his age, his general state of health, the disease involved and his degree of severity; subject response, type of compound, mode of administration, bioavailability of the composition administered, dosage, concomitant use of other drugs, etc.

The compositions of the invention are intended for administration by the oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal route. Mention may in particular be made of tablets, capsules, powders, granules, oral solutions or suspensions, forms of sublingual and buccal administration, forms of subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and the forms rectal administration.

Among the pharmaceutically acceptable excipients, mention may be made of gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like, a sweetener, an antiseptic, a flavoring agent, a coloring agent, dispersion, wetting agent, suspending agent or diluent. The tablets can in particular be coated with sucrose or any other suitable material or else can be treated so as to have a prolonged or delayed activity and continuously release a predetermined amount of the active substance and of the copolymer according to the invention. For rectal administration, the composition may be in the form of suppositories prepared with binders melting at rectal temperature, for example cocoa butter or polyethylene glycols. For a composition for parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions are used which contain pharmacologically compatible dispersing agents and / or wetting agents.

The present invention also relates to a complex comprising: i) a copolymer according to the invention; ii) at least one anionic or potentially anionic active substance.

The copolymer according to the invention, as mentioned above, is cationic or potentially cationic, thus an electrostatic type bond is created between the copolymer of the invention (i) and the active substance (ii). In the context of the present invention, the term “potentially cationic” means a substance which, after reaction with a bronsted acid or a halide, produces a cationic compound.

In the context of the present invention, the term “potentially anionic” is understood to mean a substance which, after reaction with a base, produces an anionic compound.

Preferably, in the context of the invention, the active substance is a nucleic acid. The nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or hybrid sequences (DNA / RNA). They may be sequences of natural or artificial origin, and in particular genomic DNA, complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA. (RRNA), or synthetic or semi-synthetic sequences. The nucleic acid can vary in size from oligonucleotide to chromosome. These nucleic acids can be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of libraries, by chemical synthesis, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by screening of libraries. They can in particular be obtained by a chemical modification at the level of the sugar part of the nucleobase part or of the internucleotide backbone. Among the advantageous modifications in the sugar moieties, mention may in particular be made of the modifications occurring in the 2 'position of the ribose, such as the 2'-deoxy, 2'-fiuoro, 2'-amino, 2'-thio, or 2' modifications. -0-alkyl, in particular 2'-0-methyl, instead of the normal 2'-OH group on the ribonucleotides, or the presence of a methylene bridge between the 2 'and 4' positions of the ribose (LNA). Regarding the nucleobases, it is possible to use modified bases, such as in particular 5-bromouridine, 5-iodouridine, N ³ -methyl-uridine, 2,6-diaminopurine (DAP), 5-methyl-2'-deoxyCytidine, 5- (1-propynyl) -2'-deoxy-Uridine (pdU), 5- (1-propynyl) -2'- deoxyCytidine (pdC), or bases conjugated with cholesterol. Finally, advantageous modifications of the internucleotide backbone include the replacement of phosphodiester groups of this backbone by phosphorothioate, methylphosphonate, phosphorodiamidate groups, or the use of a backbone composed of N- (2-aminoethyl) -glycine units linked by peptide bonds (PNA, Peptide Nucleic Acid). The different modifications (base, sugar, backbone) can of course be combined to give modified nucleic acids of morpholino type (bases fixed on a morpholine ring and linked by phosphorodiamidate groups) or PNA (bases fixed on units of N- (2-aminoethyl ) -glycine linked by peptide bonds). They can moreover be incorporated into vectors, such as plasmid vectors.

Preferably, the nucleic acid is selected from the group consisting of RNA, complementary DNA (cDNA), genomic DNA, plasmid DNA, antisense DNA, messenger RNA. , antisense RNA, interfering RNA, ribozymes, transfer RNA, ribosomal RNA, or DNA encoding these types of RNA.

Regarding deoxyribonucleic acids, they can be single or double stranded. These nucleic acids can comprise a sequence of genes chosen from a) marker genes, b) genes for therapeutic purposes and c) genes for vaccination, and the elements allowing its expression.

For the purposes of the invention, the term "gene for therapeutic purposes" is understood to mean, in particular, any gene encoding a protein product having a therapeutic effect. The protein product thus encoded can be a protein, a peptide, etc. This protein product can be homologous to the target cell (that is to say a product which is normally expressed in the target cell when the latter shows no pathology). In this case, the expression of a protein makes it possible, for example, to overcome insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or even to overexpress said protein. The therapeutic gene can also encode a mutant of a cellular protein, having increased stability, altered activity, etc. The protein product can also be heterologous with respect to the target cell. In this case, an expressed protein can for example complete or provide a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response. The therapeutic gene can also code for a protein secreted in the body.

The therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular RNAs. Such sequences can, for example, be transcribed in the target cell into RNA complementary to cellular mRNAs and thus block their translation into protein. It can also be synthetic oligonucleotides, optionally modified. Antisenses also include sequences encoding ribozymes, which are capable of selectively destroying target RNAs. The therapeutic gene can also be a gene encoding an siRNA or a shRNA. Advantageously, the genes for therapeutic purposes are chosen from the genes encoding:

- dystrophin and minidystrophin (myopathies);

- the protein CFTR cystic fibrosis transmembrane conductance regulator associated with cystic fibrosis;

- alphal -antitrypsin;

- Erythropoietin (EPO);

- cytokines such as interleukins and TNF tumor necrosis factor;

- growth factors such as TGFbeta and PDGF;

- lysosomal enzymes such as beta-glucosidase;

- adenosinedesaminase (ADA);

- sense and antisense RNAs;

- interfering RNAs;

- ribozymes;

- low density lipoprotein receptors, deficient in hypercholesterolemia;

- coagulation factors such as factors VII, VIII and IX;

- phenylalanine hydroxylase;

- tyrosine hydroxylase;

- Herpes simplex virus thymidine kinase;

- proteins of the major histocompatibility complex, preferably HLA-B7;

- cytosine deaminase;

As indicated above, the nucleic acid may also contain one or more genes for vaccine purposes capable of generating an immune response in humans or animals. In this particular embodiment, the invention therefore allows the production either of vaccines or of immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, parasites, bacteria, viruses or cancers. . They may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus, the pseudorabies virus, or else specific for tumors. Thus, the genes for vaccine purposes are advantageously chosen from the genes coding for:

- Viral antigens, advantageously the nucleoprotein of the influenza virus or the NEF or GAG protein of the HIV virus;

- antigens associated with tumors, advantageously the MARTI antigen for melanoma cancers, mucins or the PSA antigen for prostate cancer;

- bacterial antigens;

- parasitic antigens.

In another advantageous embodiment, the marker gene is chosen from the genes of:

- firefly luciferase;

- green jellyfish Aequora Victoria protein;

- beta-galactosidase from E. Coli;

- chloramphenicol acetyl transferase;

- resistance to an antibiotic, preferably resistance to neomycin or hygromycin.

To obtain an optimum effect of the complexes of the invention, the respective proportions of the copolymer according to the invention and of the nucleic acid are preferably determined so that the mass ratio (pg / pg) between the copolymer and the nucleic acid or between 1 and 10, preferably between 3 and 6.

This ratio can of course be adapted by a person skilled in the art depending on the copolymer used, the possible presence of an adjuvant, the nucleic acid, the target cell and the mode of administration used.

The complexes according to the invention can be prepared by mixing a solution of the nucleic acid concerned and a solution of the copolymer according to the invention.

Advantageously, these solutions are prepared from physiological serum or from a buffer, such as hepes buffer, or from a cytocompatible medium.

The present invention further relates to a pharmaceutical composition comprising a complex according to the invention and optionally a pharmaceutically acceptable excipient. The pharmaceutical composition according to the invention can be used as it is or in combination with other compounds. Thus, in a particular embodiment, the composition according to the present invention additionally comprises an adjuvant capable of combining with the copolymer / nucleic acid complex according to the invention and of improving the transfecting power. Indeed, the transfecting power of the compositions of the invention can be further improved in the presence of certain adjuvants (lipids, proteins, lipopolyamines, synthetic polymers for example), capable of combining with the copolymer / nucleic acid complex according to the invention.

Advantageously, the adjuvants used in the compositions according to the invention are cationic lipids (comprising one or more cationic charges in their polar part) or neutral lipids.

As regards the cationic lipids, it may more particularly be lipopolyamine. Other adjuvants which are particularly advantageous for the preparation of the compositions of the invention are represented by neutral lipids.

The use of neutral lipids is particularly advantageous when the charge ratio R (amines / phosphates) is low. In a particularly advantageous manner, use is made of natural or synthetic lipids, zwitterionic or devoid of ionic charge under physiological conditions. These various lipids can be obtained either by synthesis or by extraction from organs (for example: the brain) or from eggs, by standard techniques well known to those skilled in the art.

In a particularly advantageous embodiment, the compositions of the present invention comprise a targeting element making it possible to direct the transfer of the nucleic acid. This targeting element can be an extracellular targeting element, making it possible to direct the transfer of the nucleic acid to certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells, etc.). It can also be an intracellular targeting element, making it possible to direct the transfer of the nucleic acid to certain privileged cellular compartments (mitochondria, nucleus, etc.). Among the targeting elements which can be used in the context of the invention, mention may be made of sugars, peptides, oligonucleotides, lipids or proteins. Advantageously, they are sugars, peptides or proteins such as antibodies or fragments of antibodies, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc. In particular, they may be ligands of growth factor receptors, cytokine receptors, cellular lectin receptors or adhesion protein receptors. We can also cite the transferrin, HDL and LDL receptor. The targeting element can also be a sugar making it possible to target the asialoglycoprotein receptors, or else an antibody Fab fragment making it possible to target the receptor for the Fc fragment of immunoglobulins.

The compositions according to the invention can be formulated with a view to topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for direct injection into the desired organ, or for topical administration (on the skin and / or mucosa). They may in particular be sterile, isotonic solutions or dry decompositions, in particular lyophilized ones, which, by adding sterilized water or physiological serum as the case may be, allow the constitution of injectable solutions. The doses of nucleic acid used for the injection as well as the number of administrations can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or else. the duration of the treatment sought.

The complexes and compositions according to the invention can be used for the transfer of nucleic acids into cells in vivo, in vitro or ex vivo. In particular, the copolymers according to the invention can be used for very efficiently transferring nucleic acids into numerous cell types, and in particular in certain cell types which are usually difficult to transfect.

The present invention therefore also relates to a method for transfecting in vitro or ex vivo a nucleic acid in cells, said method comprising bringing said cells into contact with at least one complex according to the present invention or a pharmaceutical composition according to the invention.

The cells can be of prokaryotic or eukaryotic origin, in particular animal, plant or human.

Advantageously, the cells are mammalian cells, advantageously chosen from:

- hematopoietic stem cells; - cells of the immune system, such as dendritic cells, macrophages and lymphocytes;

- liver cells;

- skeletal muscle cells;

- skin cells, such as fibroblasts, keratinocytes, dendritic cells and melanocytes;

- cells of blood vessels and microvessels, such as endothelial cells and smooth muscle cells;

- epithelial cells of the airways;

- cells of the central nervous system;

- cancer cells;

- connective tissue cells, such as tenocytes.

According to an advantageous embodiment of the invention, the in vitro or ex vivo transfection method is characterized in that a complex or a pharmaceutical composition according to the invention is brought together in a medium containing cells to be transfected. , under conditions such that there are:

- passage of the complex from the medium into the cytoplasm of the cells,

- release of the nucleic acid involves in the aforesaid complex in the cytosol and / or the nucleus of the cells,

- transcription and expression of nucleic acid in transfected cells,

- expression of the protein corresponding to the transfected gene.

The present invention further relates to the use of a copolymer according to the invention for the preparation of a pharmaceutical composition intended for the intracellular delivery of anionic molecules, advantageously of nucleic acid in particular as defined above.

It further relates to a complex according to the invention for its use as a medicament intended for the intracellular delivery of nucleic acid, advantageously intended for the treatment of tumors, more advantageously of leiomyomeuterin or of a malignant tumor such as carcinoma of the ovaries, of the breast or endometrium, for the preparation of a vaccine or for the treatment or prevention of congenital or acquired metabolic deficiency or diseases associated with deficient gene expression, more preferably for the treatment of diseases related to disorders of a gene unique such as severe combined immune deficiency syndromes, cystic fibrosis, hemophilia, sickle cell anemia, beta thalassemia and muscular dystrophy.

The invention will be better understood on reading the examples of the present invention which follow.

The examples of the present application implement a cell viability assay. This MTT assay evaluates cell viability in a 3- (4,5) -dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) bromide assay. The cells are seeded at a rate of ^2.5 × 10 4 cells per well in a culture medium (100 μL) in a 96-well plate. The culture medium is MEM (minimum essential medium) supplemented with 10% decomplemented fetal calf serum (FBS) containing 100 U / ml of penicillin, 100 mg / ml of streptomycin, 1% of non-essential amino acid and 1 % of GlutaMAX. The cells are seeded at a rate of 2 × 10 ⁴ cells per ml and are maintained at 37 ° C. in a humid oven in an atmosphere at 5% CO 2.

Two days later, the culture medium is removed and the polymers whose cytotoxicity is to be evaluated are added (100 µL of solution) to each well, and the cells are incubated at 37 ° C. After 4 hours, the medium is removed and the cells are cultured for 48 hours at 37 ° C. in MEM culture medium. 48 hours later, MTT is added (5 mg / mL in PBS) at the rate of 10 μL in each well and the whole is incubated for 4 hours at 37 ° C. MTT is converted into an insoluble dye by living cells and it is solubilized using acidified isopropanol. Absorbance is measured at 570 nm. The percentage of cytotoxicity is (A ₀ - A) / A ₀ x100 where A is the absorbance of the treated cells and A ₀ that of the control cells cultured under the same conditions

Example 1: Preparation of a copolymer according to the invention

Step ^1: Synthesis of IPEI

In a 500 mL three-necked flask equipped with a condenser, 10 g of poly (2-ethyl-2-oxazoline) with a molar mass of 25,000 g. mol ¹ (0.2 mmol) are dissolved in 115 mL of distilled water. To this mixture is added 12N hydrochloric acid (75 ml; 2.44 mol), and the hydrolysis is carried out for 48 hours, at reflux under nitrogen.

Sodium hydroxide (NaOH) pellets are then introduced into the reaction medium to increase the pH of the solution to pH = 12 (pH paper control) allowing total precipitation of the desired polymer. The reaction medium is filtered through a sintered glass of porosity 4, then washed with distilled water until the washing water reaches a pH = 7. The white precipitate is recovered and dried in a freeze-dryer. The polymer is thus obtained in the form of a white powder. Its structure is determined by NMR ( ¹ H, ¹³ C), in solution in D ₂ 0 containing 5 μL of TFA, and by size exclusion chromatography (SEC).

Yield: 4.232 g (96.3%), ¹ H NMR (300 MHz, at 10 mg in 0.7 ml D ₂ 0 + 5mPTA): 5 (ppm) = 3.50 (s, 4H, NHCFbCFb) . ¹³ C NMR (75 MHz, D ₂ 0 + 5plTAF): d (ppm) = 48.97 (s, 2C, NHÇHsÇHs); 0 (s,)

^2nd step: synthesis of the copolymer of formula II.

50mg of IPEI 11kDa and 85mg of boc-his (1-boc) -OSu (compound of formula IV) were dissolved in 2mL of DMSO. 150 μL of diisopropylamine were then added. The reaction is heated for 48 hours at 65 ° C., then the reaction is left under stirring for 24 hours at room temperature. The process is then distinguished according to whether one wishes to obtain the deprotected product (R = H) or the protected product (R = Boc).

To obtain the deprotected product (R = H), after 48 h, 9 ml of 50% TFA were added dropwise at 0 ° C. and the reaction is left to stir for 24 h at room temperature. After evaporation, the product is, firstly, purified by carrying out two liquid-liquid extractions (H ₂ 0 / DCM) and then in a second step, by dialysis for at least 48 hours using 100OMWCo sausages. After lyophilization, the product is recovered in the form of a white powder.

To obtain the protected product (R = Boc), after evaporation, the product thus obtained is the copolymer of the invention, called protected copolymer. It is, firstly, purified by carrying out two liquid-liquid extractions (H ₂ 0 / DCM) and then secondly, by dialysis for at least 48 hours using 100OMWCo strands. After lyophilization, the product is recovered in the form of a white powder.

Example 2: Toxicity of the copolymer of the invention compared to that of the linear polyethylenimine polymer

The deprotected polymer according to the invention or the IPEI was placed in incubation at different concentrations with HeLa cells (2.5 × 10 ⁴ cells / ml) for 24 hours at 37 ° C. Cellular viability then was measured using the MTT test described above. The results are reported in the table below.

[Table 1] according to the invention deprotected

Whatever the concentration of the polymer (according to the invention or the IPEI), the cytotoxicity of the IPEI turns out to be always greater than that of the polymer according to the invention, the cell viability resulting from the incubation of the IPEI being lower. .

The cytotoxicity of the deprotected copolymer according to the invention and of the IPEI was evaluated in HeLa cells by incubating increasing levels of the copolymers. The tests were carried out according to the MTT protocol described above. The IC50 results are given in Table II below, at a concentration of 27 pg / mL.

[Table2]

It should be noted that the toxicity to kill half of the cells (IC50%) of the IPEI polymer is high. With IPEI there are fewer complete viruses than with the copolymers of the invention, which results in lower efficiency for gene therapy.

Example 3: Preparation of the copolymer complex of Example 1 (protected or unprotected) with pCMV-EGFP

112.5 µl of the copolymer of Example 1 (protected or unprotected) was mixed with 112.5 µl of Hepes buffer (10 mM) pH 7.4. The resulting mixture was added to 75 µg of pCMV-EGPF in 975 ml of Hepes buffer (10 mM) pH 7.4. The solution obtained is stirred vigorously for 4 seconds and then left for 30 min at room temperature. The solution was made up to 4 ml with the following culture medium (OptiMEM): Human cervical epithelioid carcinoma cells (HeLa cells; CRL1772, C2C12, Rockville, MD, USA) are grown in medium. MEM (Minimum Essential Medium) supplemented with 10% decomplemented fetal calf serum (FBS) containing 100 U / ml of penicillin, 100 mg / ml of streptomycin, 1% of non-essential amino acid and 1% of GlutaMAX. The cells are seeded at a rate of 2 × 10 ⁴ cells per ml and are maintained at 37 ° C. in a humid oven in an atmosphere at 5% CO 2.

Example 4: Lentivirus production

60 ml of the copolymer of Example 1 (protected or unprotected) are added to 10 ml of a Flepes 10 mMphFI 7.4 buffer comprising 5 μg of DNA plasmid. The solution is stirred vigorously for 5 seconds and then stored for 30 min at room temperature (approximately 20 ° C.). The solution is then made up to 1.5 ml with OptiMEM to obtain the transfection solution. GibcoOpti-MEM I Serum Reduced Medium is a modification of Eagle's Minimum Essential Medium, buffered with FIEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, oilgo elements and growth factors.

Tri-transfection (pTRIP-GFP, 8.81 and VSV-G) was performed with amounts of plasmid in the mass ratio 2-2-1 pg / well respectively (5 pg / well).

The cell medium is removed and replaced with the transfection solution. The incubation lasts 16 hours, the medium is then changed and the cells are allowed to incubate in OptiMEM without transfection agent. The supernatant is collected after 20, 24, 40, 44 and 48 hours (after the start of the experiment, ie 4 hours, 8 hours, 24 hours, 28 hours and 32 hours after removing the transfection agent) and the viral titer is measured. The results for the deprotected polymer according to the invention are given in Table 3 below.

The viral titer is assayed by a qPCR method well known to those skilled in the art. The viral titer can also be measured by a protein assay to have the quantity of viral particles present, which are assemblages of 3 proteins (VP1, VP2, VP3) (MTT assay). [Table 3]

Example 5: Transfection of HeLa cells

Study of the transfection of HeLa cells of the plasmid pCMV-EGFP using the copolymer of the invention (protected or unprotected) at the relative mass concentration of 1/3 DNA / polymer. The procedure used is that of the MTT assay described above.

[Table 4]

The transfection efficiency tests were also carried out with the protected copolymers of the invention, with a DNA / copolymer mass ratio of 1/6 and with different proportion of histidine.

The results are shown in Table 6. [Table 6]

These results show that the polymers of the invention have excellent transfection efficiency. More particularly, these results show that the protected copolymers, despite steric hindrance of the free amine function on the histidine functions of the copolymer, unexpectedly exhibit very good transfection efficiency.

The use of protected product advantageously makes it possible to facilitate the synthesis process by avoiding a deprotection step.

Claims

1. Linear polyethyleneimine copolymer comprising monomeric units of formula (I) and monomeric units of formula (II) wherein :

R ¹ and R ² , identical or different, represent a hydrogen atom, an alkyl group, linear or branched, comprising from 1 to 3 carbon atoms, preferably from 1 to 2 carbon atoms, at least one of R ¹ or R ² is H;

R, identical or different, is selected from H or an amine protecting group, for example Boc, Fmoc; n is a number between 1 and 99% of the total monomers; and m is a number between 1 and 99% of the total monomers.

2. A copolymer according to claim 1, wherein R is an amine protecting group, eg Boc, Fmoc.

3. A copolymer according to claim 1, wherein at least one of R's is an amine protecting group, eg Boc, Fmoc.

4. Copolymer according to any one of claims 1 to 3, in which R ¹ and R ² represent Fl.

5. Process for preparing the copolymers according to any one of claims 1 to 4, comprising the reaction between a polyethylene imine compound of formula (I) and a compound of formula (IV) and optionally the partial or total deprotection of the amine functions. (I wherein R ¹ is as defined in claim 1 and R 'is an amine protecting group, for example Boc or Fmoc.

6. A copolymer according to any one of claims 1 to 4, as an active substance carrier.

7. A pharmaceutical composition comprising a copolymer according to any one of claims 1 to 4, an active substance and optionally a pharmaceutically acceptable excipient.

8. Complex comprising:

- a copolymer according to one of claims 1 to 4;

- at least one anionic or potentially anionic active substance.

9. The complex of claim 8, wherein the active substance is a nucleic acid.

10. The complex of claim 9, wherein the nucleic acid is selected from the group consisting of RNA, cDNA, genomic DNA, plasmid DNA, antisense DNA, messenger RNA, antisense RNA, interfering RNA, ribozymes, transfer RNA, ribosomal RNA, or DNA encoding these types of RNA.

11. Pharmaceutical composition comprising at least one complex according to one of claims 8 to 10.

12. Complex according to one of claims 8 to 10 for its use for the intracellular delivery of anionic or potentially anionic active substance.