The present invention relates to novel cationic polymers which can be used for forming complexes of cationic polymers and of therapeutically active substances comprising at least one negative charge and the corresponding complexes, useful in particular for the transfer of a therapeutically active substance, in particular a nucleic acid, into a target cell.
Genetic diseases can be explained in particular by a dysfunction in the expression of specific genes or by the expression of mutated polypeptides which are nonfunctional in at least one cell type. The therapeutic solution which appears to be most appropriate for this type of condition is to transfer into specific target cells extracted and then reintroduced into the human body, or directly into the affected organs, the genetic information capable of correcting the defect observed. This may be for example the gene encoding the CFTR protein in the case of cystic fibrosis or the gene encoding dystrophin in the case of Duchenne's myopathy. In the context of this approach, also called gene therapy, the genetic information is introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body (ex viva method), or directly in vivo into the appropriate tissue. Many publications also describe the use of a gene therapy protocol in order to obtain in the target cells the expression of a protein of therapeutic value by introducing the corresponding genetic information. The therapeutic value may for example lie in the possibility of eliminating a tumor, or failing this to slow down its progression, by transferring into the target cancer cells immunostimulatory genes (immunotherapy) which are capable of inducing or of activating a cell-mediated immune response toward the tumor, or the administration of genes encoding cytokines, of cytotoxic genes conferring toxicity on the cells expressing them, for example the tk gene of the Herpes Simplex virus type 1 (HSV-1), or of antioncogenes, such as for example the gene associated with retinoblastoma or p53, or of polynucleotides capable of inhibiting the activity of an oncogene, such as for example the antisense molecules or the ribozymes capable of degrading the messenger RNAs specific for the oncogenes.
During the past 30 years, several studies have described techniques relating to the transfer of this genetic information into cells, in particular mammalian cells. These different techniques may be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are largely limited to applications in vitro and whose use is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology for which the genetic material to be transferred is combined with a vector of a biological or synthetic nature which promotes the introduction of said material.
Currently, the most efficient vectors are viral, in particular adenoviral or retroviral, vectors. The techniques developed are based on the natural properties which these viruses possess for crossing the cell membranes, for escaping degradation of their genetic material and for causing their genome to penetrate into the cell nucleus. These viruses have already been the subject of many studies and some of them are already used experimentally as vectors for genes in humans for the purpose, for example, of a vaccination, an immunotherapy or a therapy intended to make up for a genetic deficiency. However, this viral approach has some limitations, in particular linked to the risks of dissemination in the host organism and in the environment of the infectious viral particles produced, to the risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and to the induction of immune and inflammatory responses in vivo during the therapeutic treatment. Accordingly, alternative, nonviral systems for transferring polynucleotides have also been developed.
There may be mentioned for example coprecipitation with calcium phosphate, the use of cationic lipids such as DOTMA: N-[1-(2,3-dioleyl-oxyl)propyl]-N,N,N-trimethylammonium (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS: dioctadecylamido-glycylspermine (Behr et al., 1989, PNAS, 86, 6982-6986 or Transfectam™), DMRIE: 1,2-dimiristyloxypropyl-3-dimethylhydroxyethylammonium and DORIE: 1,2-diooleyl-oxypropyl-3-dimethylhydroxyethylammonium (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL: 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (Gao and Huang, 1991, BBRC, 179, 280-285), DOTAP™ (McLachlan et al., 1995, Gene Therapy, 2,674-622) or Lipofectamine™; or the use of polymers coupled to ligands recognized by a membrane receptor (for a review see Cotten and Wagner, 1993, Current Opinion in Biotechnology, 4, 705-710).
However, one of the major problems encountered when it is desired to transfer genes into target cells lies in the difficulty of causing the penetration of the nucleic acids because in particular of their polyanionic nature which prevents their passage across the cell membranes. The use of cationic polymers which can combine with the nucleic acids by electrostatic bonds makes it possible to solve this problem, at least partially. Thus, the document WO 95/24221 describes the use of dendritic polymers, document WO 96/02655 the use of polyethyleneimine, or of polypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316 the use of conjugates of polylysine.
The applicant company has now defined novel cationic polymers possessing particularly advantageous properties for the transfer into cells of therapeutically active substances comprising negative charges, in particular nucleic acids. Furthermore, these polymers have the advantage of being easily accessible, in particular by chemical synthesis, and inexpensive. They have a very low toxicity to cells, which constitutes a considerable advantage in the field of gene therapy.
The present invention relates first of all to a cationic polymer of formula I:
in which n is a whole number varying from 0 to 5 and p is a whole number varying from 2 to 20,000, more particularly p varies from 10 to 18,000 and advantageously from 200 to 1000,
characterized in that:
at least 10%, advantageously from 30 to 80%, preferentially 70%, of the free NH2 functions are substituted with identical or different hydrophilic R groups;
said cationic polymer may in addition comprise at least one targeting element combined covalently or not with the free NH2 functions and/or with said hydrophilic R groups provided that said cationic polymer contains at least 20%, preferably at least 30%, of free NH2 functions.
The invention relates more particularly to a cationic polymer defined by the following formula II:
Advantageously, said cationic polymer is defined by the formula III:
The polymers of formula II and III exhibit the characteristics as defined above for the polymer of more general formula I.
According to the present invention, “hydrophilic group” is understood to mean a group comprising at least one hydrophilic function. It may be for example a hydrophilic function chosen from the amine, hydroxyl, amide and ester functions.
These hydrophilic functions may be directly combined with the free NH2 functions of the polymer through an N—C bond, or indirectly via an arm. In the latter case, the invention relates, for example, to a cationic polymer for which R is chosen from the groups:
where R′ designates a group containing at least one hydrophilic function and n′ is a whole number varying from 1 to 5.
According to an advantageous embodiment, the hydrophilic group R or R′ consists of a polymer exhibiting hydrophilic properties, such as for example polyethylene glycol (PEG) or its derivatives, for example a methoxy-PEG, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polylactic acid, polyglycolic acid and cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose. According to the invention, the molecular weight of such polymers preferably varies from 300 to 5000, more particularly from 1000 to 4000, and is advantageously 2000. The preferred polymer for the use of such a variant of the invention is polyethylene glycol (PEG) and more particularly PEG 2000.
In general, when n and/or n′ are greater than 5, the solubility of the cationic polymer thus formed or its capacity to form stable interactions with a negatively charged molecule may be disrupted. However, it is within the capability of persons skilled in the art to analyze the properties of such structures and to determine through experiments the most favorable conditions for the use of such polymers, in particular for the formation of complex with nucleic acids and for the transfection of cells.
Representative examples of cationic polymers according to the invention are glycolilated polyallylamines, in particular a polymer of formula III (n=1) where:
or alternatively the ethoxylated polyallylamines, in particular a polymer of formula IV (n=1) where R is —(CH2)2—OH
and about 50 to about 80%, preferably from 50 to about 70%, of free NH2 functions are substituted with R. According to an even more particular embodiment, p=592.
Advantageously, the invention relates to a cationic polymer as defined by formulae I, II, III or IV and which comprises, in addition, at least one targeting element. Such targeting elements can make it possible to direct the transfer of an active substance toward certain cell types or certain specific tissues (tumor cells, pulmonary epithelium cells, hematopoietic cell, muscle cell and the like). They can also make it possible to direct the transfer of an active substance toward certain preferred intracellular compartments such as the nucleus, the mitochondria and the like. They may be in addition elements which facilitate penetration into the cell or the lysis of the endosomes. Such targeting elements are widely described in the literature. They may be for example whole or part of lectins, peptides, in particular the peptide JTS-1 (see patent application wo 94/40958), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands capable of reacting with an antiligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. In particular, they may be galactosyl residues which make it possible to target the receptor for the asialoglycoproteins at the surface of the hepatic cells, ligands which can react with receptors such as the receptors for growth factors, receptors for cytokines, lectins, or adhesion proteins; they may also be an antibody fragment such as the Fab fragment, a fusogenic peptide INF-7 derived from the influenza virus hemagglutinin HA-2 subunit (Plank et al., 1994, J. Biol. Chem. 269, 12918-12924), a nuclear localization signal derived from the SV40 virus T antigen or from the Epstein-Barr virus EBNA-1 protein.
This targeting element is attached covalently or noncovalently to the polymer of formula I, or II, or III, or IV. In the case where the cationic polymer is combined with an active substance comprising negative charges to form a complex according to the present invention, it is possible for said targeting element to be attached to said active substance.
Taken in isolation, the cationic polymer according to the invention contains monomers carrying free NH2 functions which are capable of becoming NH3 + under appropriate pH conditions. Under these conditions, said cationic polymer is capable of forming a complex with at least one substance, in particular a therapeutically active substance, comprising negative charges.
The invention therefore also relates to a complex comprising at least one cationic polymer according to the present invention combined with at least one therapeutically active substance, comprising at least one negative charge.
Advantageously, such a complex is characterized in that said therapeutically active substance is chosen from nucleic acids and proteins.
“Nucleic acid” is understood to mean a synthetic or isolated natural, linear or circular, double-stranded or single-stranded, DNA and/or RNA fragment designating a precise succession of nucleotides, modified or otherwise, which make it possible to define a fragment or a region of a nucleic acid with no size limitation. According to a preferred embodiment, the therapeutically active substance is a nucleic acid chosen from the group consisting of a cDNA; a genomic DNA; a plasmid DNA; a messenger RNA; an antisense RNA; a ribozyme; a transfer RNA; a ribosomal RNA; or a DNA encoding such RNAs; a polynucleotide free of any compound facilitating its introduction into cells; a nucleic acid combined with at least one polypeptide, in particular a polypeptide of viral origin, and more particularly of adenoviral or retroviral origin, or a synthetic polypeptide; a nucleic acid combined with a ligand; a nucleic acid combined with amphiphilic agents, in particular cationic lipids; a nucleic acid combined with cationic polymers different from the polymers according to the present invention or with neutral or anionic polymers.
According to a variant of the invention, said therapeutically active substance contained in said complex is a nucleic acid which comprises a gene of interest and elements for expressing said gene of interest. Such a gene of interest may for example encode the whole or part of a ribozyme or of an antisense nucleic acid. According to another embodiment of the invention, said gene of interest encodes the whole or part of a polypeptide, in particular of a marker polypeptide (luciferase, β-galactosidase, product conferring resistance to an antibiotic, and the like) or of a polypeptide exhibiting a therapeutic or prophylactic activity (therapeutically active polypeptide), and more particularly an immunogenic activity of the cellular or humoral type. The term polypeptide extends with no restriction as to its size or its degree of glycosylation. It is also possible to mention, by way of examples of genes of interest, the genes encoding an enzyme, an enzyme inhibitor, a hormone, a cytokine, a membrane receptor, a structural polypeptide, a polypeptide forming a membrane channel, a transport polypeptide, an adhesion molecule, a ligand, a factor for regulating transcription, translation or replication, for stabilizing the transcripts, a coagulation factor, a polypeptide with antitumor effect, a polypeptide capable of slowing down the development of a bacterial, viral or parasitic infection, a toxin, or an antibody, such as for example the gene encoding the CFTR protein, dystrophin, factor VIII or IX, E6/E7 of HPV, MUC1, BRCA1, β-interferon, γ-interferon, interleukin (IL)2, IL-4, IL-6, IL-7, IL-12, tumor necrosis factor (TNF) type alpha, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), the Herpes Simplex virus type 1 (HSV-1) tk gene, the gene associated with restinoblastoma or p53 or the whole or part of immunoglobulins, such as the F(ab)2, Fab′, or Fab fragments or the anti-idiotypes (U.S. Pat. No. 4,699,880). Of course this list is not limiting, and the use of other genes may be envisaged.
In the case where the nucleic acid comprises the whole or part of a gene of interest encoding the whole or part of a polypeptide, it should be specified that said nucleic acid comprises, in addition, the elements necessary to ensure the expression of said DNA after transfer into a target cell, in particular promoter sequences and/or regulatory sequences which are efficient in said cell, and optionally the sequences required to allow the excretion or the expression, at the surface of the target cells, of said polypeptide. By way of example, there may be mentioned the promoters such as the promoters of the viruses RSV (Rous Sarcoma Virus), MPSV, SV40 (Simian Virus), CMV (Cytomegalovirus) or of the vaccinia virus, the promoters of the gene encoding muscle creatinine kinase, for actin, or for the pulmonary surfactant. It is in addition possible to choose a promoter sequence specific for a given cell type, or which can be activated under defined conditions. The literature provides a large amount of information relating to such promoter sequences. Moreover, said nucleic acid may comprise at least two identical or different sequences exhibiting a transcriptional promoter activity and/or at least two identical or different DNA coding sequences situated contiguously, distantly, in the same direction or in the opposite direction, relative to each other, provided that the function of the transcriptional promoter or the transcription of said sequences is not affected. Likewise, in this type of nucleic acid construct, it is possible to introduce “neutral” nucleic sequences or introns which do not hamper the transcription and are spliced before the translation step. Such sequences and their uses are described in the literature (WO 94/29471). Said nucleic acid may also contain sequences required for intracellular transport, for replication and/or for integration, for secretion, for transcription or translation. Such sequences are well known to persons skilled in the art. Moreover, the nucleic acids which can be used according to the present invention may also be nucleic acids modified such that it is not possible for them to become integrated into the genome of the target cell or nucleic acids stabilized with the aid of agents, such as for example spermine, which as they are do not have any effect on the transfection efficiency.
Advantageously, a specific ratio between the number of positive charges of said cationic polymer and the number of negative charges of said therapeutically active substance will be chosen. Without wishing to be limited by a specific ratio, quantities of the different charges will be preferably chosen such that the ratio between the positive charges of the cationic polymer and the negative charges of the active substance is between 1 and 30, in particular between 1.5 and 10, and preferably between 2.5 and 5. The calculation to arrive at such a ratio will take into consideration the negative charges carried by the active substance and the quantity of cationic polymer necessary to satisfy the ratio indicated above will be adjusted. When the polymer also contains one or more targeting elements, the quantities and the concentrations of targeting elements are adjusted as a function of their respective molar mass and of the number of their positive and/or negative charges.
The invention also relates to a method of preparing the cationic polymer/therapeutically active substance complexes according to the invention, characterized in that one or more polymers according to the invention are brought into contact with one or more therapeutically active substances comprising at least one negative charge and in that said complex is recovered. The recovery of said complex may be accompanied by a purification step. Several purification techniques may be envisaged, such as those based on the use of a density gradient or of a specific or nonspecific affinity column. These purification techniques are well known to persons skilled in the art who possess the necessary expertise for their use.
The invention also relates to a method for transferring a therapeutically active substance, in particular a nucleic acid, into a target cell in vitro, ex vivo or in vivo, characterized in that at least one complex according to the invention is brought into contact with target cells. According to one embodiment of the method according to the invention, cells cultured on an appropriate medium are brought into contact with a suspension comprising at least one cationic polymer/substance comprising negative charges complex as described in the present invention. After incubating for a certain period, the cells are washed and recovered. The transfection may be verified by any appropriate means, and in particular by measuring the expression of the gene carried by the nucleic acid forming a complex with the cationic polymer or by measuring the concentration of the polypeptide expressed. The method of transfection is well known per se and designates the introduction of a nucleic acid of interest into a cell for the purpose of expressing said nucleic acid.
“Target cells” according to the invention are understood to mean prokaryotic cells, yeast cells and eukaryotic cells, in particular animal cells, and in particular mammalian cells, especially human and/or cancer cells. In vivo, the complexes according to the invention may be administered into the interstitial or luminal space of tissues such as the lungs, the trachea, the skin, the muscle, the brain, the liver, the heart, the spleen, the bone marrow, the thymus, the bladder, the lymph, the blood, the pancreas, the stomach, the kidney, the ovaries, the testicles, the rectum, the peripheral or central nervous system, the eyes, the lymphoid organs, the cartilages and the endothelium.
The expression of a gene after transfection into cells may be analyzed by conventional techniques such as for example the detection of messenger RNAs by Northern blotting or digestion with S1 nuclease and/or the detection of proteins by Western blot immuno-precipitation or a functional test. The latter test is particularly suitable when the gene encodes a protein marker such as for example luciferase or β-galactosidase.
The invention also relates to the use of complexes as described above for the preparation of a medicament for the treatment of the human or animal body, in particular by gene therapy. According to a first possibility, the medicament may be administered directly in vivo or by an ex vivo approach which consists in removing the cells from the patient, in transfecting them in vitro according to the invention and in readministering them to said patient.
The invention relates to a pharmaceutical composition characterized in that it comprises at least one complex according to the present invention.
According to one variant of the invention, said pharmaceutical composition comprises at least one adjuvant capable of enhancing the capacity for transfection of said complex into a target cell in vitro, ex vivo or in vivo. More particularly, said adjuvant is chosen from the group consisting of a lysosomotropic agent such as for example chloroquine, a protic polar compound chosen in particular from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof, and an aprotic polar compound chosen in particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof.
Depending on the mode of administration chosen, it is also possible to add to the composition of the invention a pharmaceutically acceptable carrier allowing administration to humans or to animals. The use of such carriers is described in the literature.
A complex or a pharmaceutical composition according to the invention may be administered in vivo in particular in a form which is injectable, in particular by the intramuscular route. It is also possible to envisage injection by the intratracheal, intranasal, epidermal, intravenous, intraarterial, intratumoral, intrapleural, or intracerebral route using a syringe or any other equivalent means. According to another embodiment, it is possible to use systems appropriate for the treatment of the airways or of the mucous membranes such as inhalation, instillation or aerosolization, by the topical route, by oral administration or any other means perfectly known to persons skilled in the art and applicable to the present invention. The administration may take place in a single dose or in a dose repeated once or several times after a certain time interval. The route of administration and the dosage which are most appropriate vary according to different parameters such as for example the individual or the disease to be treated, or alternatively the nucleic acid to be transfected or the target organ/tissue.
Finally, the invention relates to a cell transfected with a complex or a pharmaceutical composition as defined above, particularly a prokaryotic cell, a yeast or eukaryotic cell, in particular an animal cell, in particular a mammalian cell, and more particularly a cancer cell.
The present invention is illustrated by the following Examples 1 to 5, with reference to FIGS. 1 to 9.