HK1018938A - Compositions useful for transferring therapeutically active substances into a target cell, and their use in gene therapy - Google Patents

Description

Composition for transferring therapeutically active substances into target cells and use thereof in gene therapy

The present invention relates to a composition which can be used for transferring therapeutically active substances into target cells, in particular vertebrate cells, more particularly mammalian cells. More particularly, the invention relates to the use of such compositions to prepare a carrier with which a therapeutically active substance, particularly a polynucleotide, can be transferred to a target cell.

Genetic diseases are in particular due to abnormal expression of specific genes or expression of non-functional mutant polypeptides. Cystic fibrosis, for example, is considered the most common lethal genetic disease (1/2000) with an average life expectancy of 20 to 30 years and is characterized by a large thickening of mucosal secretions, chronic lung disease, and exocrine adeno-pancreatic insufficiency. The disease is associated with a disturbance in electrolyte transport, particularly chloride ions across epithelial cell membranes, as a result of mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene (Rommens, Science 245 (1989)), 1059-. The most suitable treatment for this type of disease appears to be the transfer of the gene encoding a functional CFTR protein into the target cells to correct the observed cellular abnormalities. For this method, also called gene therapy, several authors of the paper considered that airway epithelial cells could be selected as target cells, in particular due to the accessibility of the airway epithelium, in particular by means of intrapulmonary transport, such as instillation into the lungs. It has been demonstrated that expression levels in target cells of about 5% to 7% of normal CFTR gene expression are necessary to restore the observed electrolyte transport. Similarly, some publications describe techniques for eliminating tumors or delaying the possibility of further progression of cancer by transferring genes into cancer target cells. Several approaches have been considered, in particular the transfer of immunostimulatory genes (immunotherapy) which induce or activate an immune response mediated by anti-tumor cells, and the mentioned genes which can be used for administration may be genes encoding cytokines, the transfer of toxin-producing cytotoxic genes into cells which express the genes, for example the tk gene of herpes simplex virus type 1 (HSV-1); or a metastasis anti-tumorigenic gene-i.e., a tumor suppressor gene, such as a retinoblastoma gene or a p53 gene; or a polynucleotide capable of inhibiting the activity of an oncogene, such as an antisense molecule or a ribozyme capable of degrading an oncogene-specific messenger RNA.

During the last 30 years, a number of methods have been established for introducing different heterologous genes into cells, in particular mammalian cells. These different methods can be divided into two categories. The first category relates to physical methods such as microinjection, electroporation or particle bombardment, which, while effective, are largely limited to in vitro applications, and are cumbersome and cumbersome to implement. The second category relates to molecular and cellular biology techniques, combining the genes to be transferred with biological or synthetic vectors that facilitate the transfer of said substances.

The most effective vectors currently are viral vectors, in particular adenoviral or retroviral vectors. These techniques have been developed on the basis of the natural properties of these viruses, which allow them to cross the cell membrane, preventing the genetic material from being degraded and allowing their genome to penetrate into the nucleus. These viruses have been the subject of a great deal of research, and some of them have been experimentally used as gene vectors for humans and are focused on treatments such as vaccines, immunotherapy or therapies in order to compensate for genetic defects. However, this approach using viruses is subject to a number of limitations, in particular due to the limited clonality of the viral genome, the risk of transmission of the infectious virions already produced in the host organism and environment, the risk of artificial mutations due to insertion in the host cells, and the fact that, in the case of retroviral vectors, strong immune and inflammatory responses are induced during in vivo Therapy, thus in fact limiting the number of conceivable methods of administration (McCoy, Human Gene Therapy (Human Gene Therapy)6(1995), 1553-. These many drawbacks, particularly in the case of viral vectors for use in humans, have led many organizations to develop alternative ways of designing for polynucleotide transfer.

There are many non-viral methods currently available. For example, cationic lipids such as DOTMA (Felgner, PNAS 84(1987), 7413-; receptors that mimic the viral system are used (see Cotten, Current Opinion in Biotechnology 4(1993), 705-); and the use of polyamidoamines (Haensler, bioconjugate chemistry 4(1993), 372-379). Although promising, these techniques suffer from limitations, particularly in their low levels of efficacy in vivo, which in fact limits their use in gene therapy. Furthermore, some of these techniques are limited to in vitro applications, in particular due to the toxicity of the molecules used (Polybrene-1, 5-dimethyl-1, 5-diaza-undecamethylene polymethine bromide-as an example), or due to inflammatory reactions caused by the introduction of these compounds (in the case of cationic lipids, for example Scheule, human Gene therapy 8(1997),689-707), or are difficult to control, for example in the case of receptors, in which a large amount of nucleic acid material enters the vesicles during endocytosis and thus no longer plays a role in therapy. Finally, these techniques are quite sensitive to environmental factors and require long and delicate efforts to adapt them for use in target cells or the chosen mode of administration, more specifically to transfer them from an in vitro model to an in vivo model.

Wolff (science 2478(1990),1465-1468) describes an attractive and convenient system for introducing polynucleotides into muscle cells, which involves merely injecting purified polynucleotides into target cells via the intramuscular route, without binding to any other compound capable of promoting the entry of the polynucleotide into the target cell. The results obtained recently by intratracheal injection (Meyer, Gene therapy 2(1995), 450-. Nevertheless, the expression of genes introduced into tissues is still so limited that this technique cannot be effectively used for gene therapy, in particular for the treatment of diseases associated with lung diseases. Some studies have proposed alternative methods to facilitate the entry of such polynucleotides into cells. For example, patent application WO 95/26718 relates to a method for introducing genetic material into a cell, which method comprises contacting the cell with a gene vaccine promoter and a nucleic acid molecule. According to a particular embodiment thereof, the gene vaccine facilitator may be present in DMSO. Nevertheless, no experimental data are provided in said patent application and the experiments carried out by Aubin (Somatic Cell mol. Genet.)14(1988), 155-. More specifically, Aubin describes a two-step process. The first step, which is also necessary, consists in contacting the DNA to be transfected with the electrostatic bridge molecule Polybrene, thus adsorbing the DNA to the cell surface, and the second step consists in contacting the DNA already adsorbed to the cell surface with DMSO, thus facilitating the entry of the DNA into the cells. These studies have not led to detailed protocols for in vivo applications, i.e., methods aimed at efficiently transferring polynucleotides, and more specifically polynucleotides that do not contain any transfection-facilitating compounds, into different cell types and increasing the expression level of nucleic acids in the target cells. Furthermore, Oudhiri (PNAS 94(1997), 1651) -1656) demonstrated that the firefly enzyme gene was not expressed in lung cells following intratracheal injection without any plasmid capable of facilitating entry of the plasmid into the cells.

The technical problem underlying the present invention is therefore to provide a method for efficiently transferring therapeutically active substances into target cells.

This technical problem is solved by the embodiments characterized in the claims, i.e. the applicant has now developed a composition for transferring a therapeutically active substance, preferably a polynucleotide, into target cells. It is envisaged that the composition will be used in particular in vivo gene therapy procedures, particularly since the components of the composition are not harmful.

The invention therefore relates firstly to a composition characterized in that it consists of at least one therapeutically active substance and at least one polar compound chosen from a group of specific aprotic polar compounds.

According to the invention, an "aprotic polar compound" is understood to mean a compound which does not contain any positively charged hydrogen atoms (aprotic). The properties of such compounds are described extensively in the literature (see, for example, Vollhardt and Schore,1994, trade De ChimieOrganique [ organic chemistry paper ], second edition, De Boeck university). Such compounds can be obtained in particular from natural or synthetic sources, or by chemical modification of a compound which does not have the above-mentioned properties per se. The person skilled in the art has the knowledge required to identify such compounds. The aprotic polar compounds to be used in the process and applications of the invention described below are commercially available from various sources or prepared as described in the prior art. There are a large number of polar compounds that are contemplated for use in the present invention.

According to a first aspect of the invention, the aprotic polar compounds mentioned as being preferred are defined as:

(a) formula I:

wherein R is₁And R₂Is aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl, or alkoxy of 1 to 8 carbon atoms, which may be the same or different, may be linear or branched, and may be optionally substituted, provided that when R is₁When it is a group of 1 or 2 carbon atoms, R₂Is a radical of at least 3 carbon atoms, R₂Is a radical of 1 or 2 carbon atomsWhen R is₁Is a radical of at least 3 carbon atoms, R₁And R₂Possibly linked to form a cyclized molecule;

(b) formula II:

wherein R is₃And R₄Are identical or different and are aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl or alkoxy having 1 to 8 carbon atoms, which are optionally repeated, may be linear or branched and may be optionally substituted, R₃And R₄Possibly linked to form a cyclized molecule;

(c) formula III:

wherein R is₃And R₄Are identical or different aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl or alkoxy radicals having 1 to 8 carbon atoms. These radicals being optionally repeating, may be linear or branched and may be optionally substituted, R₅Is (CH)₂)_xAt each [ R ]₅-S]_nAre independent of each other in the repeating unit, wherein X =1 to 6, and R₅Optionally substituted, n =1 to 50, R₃And R₄Possibly linked to form a cyclized molecule, or

(d) Dimethylformamide, dimethylacetamide, tetramethylurea and a derivative of any of them.

Said R₁,R₂,R₃And/or R₄The radical preferably consists of 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and particularly preferably 2 to 3 carbon atoms.

The meaning of alkenyl is understood to include one or more double chains in the carbon chain.

According to the invention, R of these polar compounds₁,R₂,R₃And/orR₅The groups may be substituted. In particular, such a substitution may consist of a marker molecule (see US 4711955), a cellular target molecule (ligand) or an anchoring molecule, which enables, for example, the distribution of the compound or of the complex containing it to be observed after in vivo or in vitro administration. These have been described in numerous scientific publications as factors that allow targeting of specific cell types, facilitate penetration into cells, lysis of endosomes or even intracellular trafficking to the nucleus. These factors may consist of all or part of the following: sugars, glycols, peptides (e.g., GRP, gastrin releasing peptide), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies (or fragments thereof), specific membrane receptor ligands, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or combinations of said compounds, e.g., targeting galactose residues of the hepatic Cell surface asialoglycoprotein receptor, INF-7 fusogenic peptides derived from the HA-2 subunit of influenza hemagglutinin for membrane fusion (Plank, J.Biol.Chem., 269(1994),12918-12924), nuclear signaling sequences derived from SV40 viral T-antigen (Lanford, Cell (Cell)37(1984),801-813) or from EBNA-1 protein of Epstein Barr virus (Ambinder, J.Virol., 65(1991), 1466-1478).

According to the invention, the aprotic polar compound is in particular selected from di-n-propyl-sulfoxide (DPSO), dimethylsulfone, sulfolane, cyclobutanesulfoxide (TEMSO), 1-methyl-2-pyrrolidone, methyl-di-methylsulfoxide, methyl-di-ethylsulfoxide and derivatives thereof. According to an advantageous form of the invention, the aprotic polar compound is selected from di-n-propyl-sulfoxide (DPSO) and derivatives thereof. As described in the accompanying examples, it was found that when DNA supplemented with DPSO was administered, DPSO significantly promoted the uptake of DNA by cells. Enantiomers of such molecules are also included within the scope of the invention.

For the purposes of the present invention, the term "derivative" of any of the compounds mentioned above is intended to mean that the chemical structure of the molecule is based on the compounds described above and can be used to transfer a substance into a target cell. The ability of the cells to promote uptake of the substance during transfection of the substance with such derivatives may even be enhanced compared to naturally occurring aprotic polar compounds as well as the compounds mentioned above. Methods for preparing such derivatives are well known to those skilled in the art and are described, for example, in the book by Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., N.Y.N.Y. (10010), No. 175 street 5, and Organic Synthesis (Organic Synthesis), Wiley, N.Y., USA). The derivatives can be subjected to transfection assays according to the prior art or, for example, as described in the accompanying examples. Moreover, suitable derivatives and analogues can be designed according to methods known in the art, for example with the aid of a computer. In particular, DPSO and other related aprotic polar compounds can be used in their three-dimensional and/or crystal structures to design compounds with the ability to promote DNA uptake (Rose, biochemistry (Biocholistry) 35(1996), 12933-.

According to another embodiment of the invention, the aprotic polar compound is selected from Dimethylformamide (DMF), dimethylacetamide, Tetramethylurea (TMU) and derivatives thereof.

The active substances of the compositions of the present invention include, but are not limited to, peptides, proteins, polynucleotides, antibodies, small organic compound ligands, hormones, peptidomimetics, Pentose Nucleic Acids (PNAs), and the like, and preferred substances are those capable of inducing and/or mediating a physiological response in a subject. According to the invention, the active substance of the preferred composition is a polynucleotide, which, when added to an aprotic polar compound, results in an increased capacity of the polynucleotide to transfect into cells.

"Polynucleotide" is understood as a naturally isolated or synthetic, linear or circular, double-or single-stranded DNA and/or RNA fragment, the term being used to denote the exact sequence of a nucleic acid, labeled or unlabeled (see, for example, US 4711955 or EP 302175), modified or unmodified (see, for example, US 5525711) and to define a fragment or a region of a nucleic acid without limiting the size of the nucleic acid. Polynucleotides are understood to mean, in particular, cDNA, chromosomal DNA, plasmid DNA, polynucleotides which do not contain any compounds which promote the entry of polynucleotides into cells; polynucleotides which bind to at least one polypeptide, in particular a polypeptide of viral origin, more particularly a polypeptide of adenoviral or retroviral origin, or to a synthetic polypeptide; a polynucleotide that binds to a ligand; polynucleotides associated with at least one cationic amphiphile, in particular a lipid; polynucleotides, messenger RNAs, bound to at least one cationic or neutral polymer; antisense RNA; a ribozyme; transferring RNA; ribosomal RNA; or a DNA encoding such RNA.

According to a particular embodiment of the invention, the polynucleotide comprises a gene of interest and an agent enabling the expression of the gene of interest. In this embodiment, the polynucleotide is advantageously in the form of a plasmid, and the factor enabling expression to occur is the sum of the factors enabling transcription of the DNA fragment into RNA (antisense RNA or mRNA) and translation of the mRNA into polypeptide. These factors are in particular promoter sequences and/or regulatory sequences which function in the cell in question and which, where appropriate, enable the polypeptide to be secreted or expressed on the surface of the target cell. For example, RSV, MPSV, SV40, CMV or 7.5k viral promoters, or vaccinia viral promoters, or promoters encoding creatine kinase, actin and lung surfactant genes may be mentioned. It is also possible to select promoter sequences which are specific for a given cell type or which can be activated under certain conditions. The literature provides a great deal of information about such promoters. Furthermore, the polynucleotide may comprise at least two identical or different sequences having transcription promoter activity and/or at least two identical or different DNA coding sequences, located consecutively or at a distance apart, in the same or opposite orientation to each other, as long as the function of the transcription promoter and the transcription of said sequences are not affected. Similarly, "neutral" nucleic acid sequences or introns that have no effect on transcription and are cleaved prior to translation may be introduced in the construction of such nucleic acids. Sequences of this nature and their use are described in the literature. The polynucleotide may contain sequences required for intracellular trafficking, replication and/or integration. Such sequences are well known to the skilled person. Alternatively, the polynucleotide according to the present invention may be a polynucleotide which is modified so as not to be integrated into the genome of the target cell, or a polynucleotide stabilized with a substance such as spermine.

The compositions of the invention may also include a selectable marker gene, either linked to the polynucleotide or as a separate nucleic acid molecule, e.g., on a recombinant plasmid. This embodiment is particularly useful for ex vivo treatment of tissues, cells and organs. After introduction of the exogenous DNA, the engineered cells can be cultured on an enrichment medium for 1-2 days and then transferred to a selection medium. The selectable marker in the recombinant plasmid confers resistance to the selector and allows those cells which have stably integrated the plasmid into their chromosomes to be selected for growth to form foci which can then be cloned and developed into cell lines. Many selection systems are available, including but not limited to separately existing tk^-,hgprt^-Or aprt^-Herpes simplex virus thymidine kinase in cells (Wigler, cell 11(1977),233), hypoxanthine guanine phosphoribosyl transferase (Szybalska, proceedings of the american academy of sciences (proc. natl. acad. sci. usa)48(1962),2026), and adenine phosphoribosyl transferase (Lowy, cell 22(1980), 817). Antimetabolite resistance may alternatively be selected from dihydrofolate reductase dhfr (Wigler, proceedings of the american academy of sciences, usa 77(1980), 3567; O' Hare, proceedings of the american academy of sciences, usa 78(1981),1527) conferring resistance to mycophenolic acid, gpt (Mulligan, proceedings of the american academy of sciences, usa 78(1981), 2072); neo (Colberre-Garapin, J. Mol. biol., 150(1981),1) conferring aminoglycoside G-418 resistance; conferring hygromycin resistance (Santerre, Gene 30(1984), 147); or a puromycin resistant hygro (pat, puromycin N-acetyltransferase). Other selectable genes are also described, for example trpB enables cells to use indole instead of tryptophan; hisD enables cells to utilize histidinolReplacement of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85(1988), 8047); and ODC (Ornithine decarboxylase) conferring resistance to an ornithine decarboxylase inhibitor, i.e., 2- (difluoromethyl) -DL-ornithine, (DFMO) resistance (McConlogue, 1987; see: Current communications in Molecular Biology, Cold spring harbor laboratory eds.).

Suitable vectors and plasmids for use in the compositions used in the in vitro or in vivo gene therapy according to the invention are described in the literature and are well known to the person skilled in the art; see, for example: giordano, Natural Medicine (Nature Medicine)2(1996), 534-539; schaper, cycling research (circ. Res.)79(1996), 911-; anderson, science 256(1992), 808-; isner, Lancet (Lancet)348(1996), 370-; muhlhauser, cycling Studies, 77(1995), 1077-; wang, Natural drug 2(1996), 714-716; WO 94/29469; WO97/00957 or Schaper, modern Biotechnology review 7(1996),635-640, and the references cited therein. Preferably, the vector is a gene transfer or targeting vector. The above polynucleotides, plasmids and recombinant vectors can be constructed by methods known to those skilled in the art; see, for example, Sambrook, in Molecular Cloning A laboratory Manual, N.Y., Cold spring harbor laboratory (1989) and Ausubel in methods in modern Molecular biology (Current protocols in Molecular biology, Green Pulsells Associates and Wiley Interscience, N.Y. (1989)).

In the present invention, the polynucleotide may be homologous or heterologous to the target cell. It may be advantageous to select polynucleotides encoding all or part of a polypeptide, in particular a polypeptide having therapeutic or prophylactic activity, more particularly a cellular or humoral type of immunological activity. The term polypeptide is to be understood as being without limitation as to the size of the polypeptide and the extent to which it is modified (e.g., glycosylated). For example, mention may be made of genes encoding enzymes, hormones, cytokines, membrane receptors, structural polypeptides, polypeptides forming membrane channels, transport polypeptides, adhesion molecules, ligands, factors regulating transcription, translation, replication or transcription stability, orGenes coding for antibodies, such as, for example, genes coding for the CFTR protein, dystrophin, factor VIII or factor IX, HPV E6/E7, MUC1, BRAC1, interferons, interleukins (IL-2, IL-4, IL-6, IL-7 and IL-12), Tumor Necrosis Factor (TNF) alpha or GM-CSF (granulocyte macrophage colony stimulating factor), or tk gene of herpes simplex virus type 1 (HSV-1), retinoblastoma gene or p53 and genes coding for all or part of immunoglobulins, such as F (ab)₂Fab' and Fab fragments, or genes encoding anti-idiotype immunoglobulins (U.S. Pat. No. 4,699,880). It will be appreciated that the genes mentioned are not limiting to the invention and that other genes may be used.

According to the present invention, it may be desirable to have a composition containing the largest possible concentration of polynucleotide so that, when desired, the smallest possible amount of the composition of the invention can be administered. The skilled person has sufficient knowledge to be able to adjust the concentration of the polynucleotide in the solution medium according to its ability to dissolve the polynucleotide. According to a preferred embodiment, the aprotic polar compound is dissolved in an aqueous solution, that is to say the compound is diluted in an aqueous solution, the latter being, where appropriate, saline or a buffer. The skilled person has sufficient knowledge to select the most suitable aqueous solution for the target cell type. More specifically, the aprotic polar compound is dissolved in water or a buffer, e.g., 20mM Hepes, at a pH of 7.5. The volume of the aprotic polar compound may be from 0.1 to 100% of the total volume of the composition, preferably from 5 to 50% by volume, most preferably from 15 to 20%.

The present invention further relates to a pharmaceutical composition comprising the above-mentioned composition of the invention and optionally a pharmaceutically acceptable carrier or excipient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions and the like. Compositions containing such carriers can be prepared by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in a suitable dosage. Can go in different waysAdministration of suitable compositions is, for example, intravenous, intratracheal, intrapulmonary, intraperitoneal, subcutaneous, intramuscular, topical or intradermal. The dosage regimen employed should be determined by the clinician and other clinical factors. As is well known in the medical arts, the dosage administered to each patient depends on many factors, including the size, body surface area, age, the nature of the drug used, sex, time and route of administration, health, and other drugs used concurrently. Generally, a dosage regimen using the present pharmaceutical composition is from 1. mu.g to 10mg of the pharmaceutically active compound per day. If the dosage regimen is a continuous infusion, the biologically active compound should be administered in an amount ranging from 1 μ g to 10mg per minute per kilogram of body weight. The progress of the disease can be monitored by periodic assessment. The dosage will vary, but the preferred dosage for intravenous administration of the polynucleotide is, for example, that the DNA should be from about 10⁶To 10¹⁶A DNA molecule of mussel. The compositions of the present invention may be administered topically or systemically. Parenteral administration, such as intravenous administration, is common; the DNA may also be administered directly to the target site, for example, by catheter administration into an artery. Furthermore, the compositions of the present invention may be incorporated into microcapsules or microspheres prior to injection. One can use in vivo transgenic methods for which a variety of devices have been designed, such as dual balloons or other catheters, or by direct injection into the target tissue as described above. Alternatively, cells in tissue can be isolated ex vivo from the body, transfected with the above-mentioned compositions and reinjected.

In another embodiment the invention relates to a vaccine comprising any one of the compositions mentioned above. In this embodiment, the preferred therapeutically active substance is an antigen or a polynucleotide encoding an antigen capable of eliciting protective immunity to a disease in a human or animal susceptible to the disease. The vaccines of the present invention may be prepared according to methods well known in the art. For example: the immunogenicity of antigens for vaccination can be improved by designing Th-1-or Th-2-directed immune responses by co-transfection of cDNAs encoding secreted cytokines or chemokines or membrane molecules. The vaccine may be injected, for example, either intradermally, subcutaneously, intramuscularly, or, particularly if used as an anti-tumor vaccination, into the tumor growth site or into the lymphatic or lymph node drainage zone at the tumor growth site.

Another embodiment of the invention relates to a diagnostic composition comprising any of the above compositions and optionally suitable detection means. In this embodiment, therapeutically active substances carrying a label are preferred. There are many different labels and methods of labeling known to those of ordinary skill in the art. Labels useful in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Additionally or alternatively, the therapeutically active substance comprises a marker gene encoding a protein that can be detected directly or indirectly, such as β -lactosidase, green fluorescent protein or luciferase; see also the examples which follow. The diagnostic compositions of the present invention may be advantageously used for targeting, e.g., emitting light to a selected area, and for tracking objects within a subject. In addition, animal models of disease states, methods for localizing and tracking pathogens or disease processes in animals, and methods for screening putative therapeutic compounds for compounds effective in inhibiting a disease or pathogen can be accomplished using the compositions described above. The detection and localization of light emitted by mammals is described in the prior art, such as 97/18841 and the references cited therein.

The invention also relates to the use of a composition according to the invention for transferring at least one therapeutically active substance, in particular a polynucleotide, into target cells in vitro, ex vivo or in vivo, in particular in vivo.

These introduction or transfer methods are known per se. By "transferred" is meant that the therapeutically active substance is transferred into the cell and is localized within or at the surface of said cell or within the membrane of the cell at the end of the procedure. If the active substance is a nucleic acid, it is called "transfection". Transfection may be identified by a variety of suitable methods, for example by measuring the expression of the gene or by measuring the concentration of the expressed protein.

According to the invention, "target cells" are to be understood as meaning prokaryotic cells, yeast cells and also eukaryotic cells, plant cells, human or animal cells, in particular mammalian cells. In addition, cancer cells should also be mentioned. In vivo, the invention may be applied to the stroma or lumen of a tissue, such as the lung, trachea, skin, muscle, brain, liver, heart, spleen, bone marrow, thymus, bladder, lymph, blood vessels, pancreas, stomach, kidney, ovary, testis, rectum, peripheral or central nervous system, eye, lymphatic organ, cartilage and endothelium. According to an advantageous alternative of the invention, the target cells are muscle cells, hematopoietic stem cells or respiratory tract cells, more particularly tracheal or pulmonary cells, advantageously respiratory tract epithelial cells.

The invention also relates to a method for transferring a therapeutically active substance into target cells by contacting said cells with a composition according to the invention. Advantageously, this method comprises an additional step of heating the composition prior to contact with the cells.

It is to be understood according to the present invention that by "composition" is meant a therapeutically active substance and a polar compound contained therein that are associated or bound together in such a way that the active substance cannot be considered to be free of an enhancer. For example, in the particular case where the active substance is plasmid DNA, the applicants have demonstrated that the physical properties of the DNA are altered in the presence of DPSO. Thus, one skilled in the art can easily imagine that a composition consisting of the active substance and the polar compound can be obtained in vitro, but also in vivo, by injecting the compound separately into the target organ or tissue, followed by injection of the second compound while the first compound is still present, to form the claimed composition in situ. Such separate modes of administration should be considered as equivalent implementations of the present invention.

As mentioned above, the composition according to the invention can be used as a medicament for diagnostic, therapeutic, prophylactic or vaccination purposes. For this reason, the present invention also relates to the use of the composition of the invention as a medicament for therapeutic, prophylactic or vaccination purposes.

In particular, the compositions of the invention may be used to carry out a method of treatment by transferring at least one therapeutically active substance, in particular a polynucleotide, into a target cell. Preferred target cells are mammalian cells. In particular, the mammalian cells are lung cells, muscle cells or hematopoietic stem cells.

The compositions of the invention may be administered by intramuscular, intratracheal, intranasal, intracerebral, intrapleural, intratumoral, epidermal, intravenous or intraarterial routes, using syringes or any other similar means, including systems adapted for use in the respiratory tract or mucosa, e.g. inhalation, instillation, aerosolization. Administration may be mentioned by using emulsion forms, by oral means or any other means known to the skilled person and applicable to the present invention.

According to the present invention, and from the point of view of in vivo gene therapy, the proposed mode of administration can be repeated several times for the patient, and any compound used does not elicit any significant immune response. The mode of administration may be a single dose or multiple doses, repeated one or more times over a time interval. The route of administration and the dosage will depend on various factors, such as the individual or disease being treated and the polynucleotide to be transferred.

More particularly, the invention relates to the use of a composition according to the invention for the preparation of a medicament for diagnostic, therapeutic, prophylactic or vaccination purposes for the human or animal body by gene therapy. The first possibility is that the drug may be administered directly into the body (e.g. intramuscularly, or to the lungs with an aerosol, etc.). It is also possible to use ex vivo methods in which cells (bone marrow stem cells, peripheral blood lymphocytes, muscle cells, etc.) are first extracted from a patient, transfected according to the invention, and then administered to the patient.

Finally, the invention relates to a cell, in particular a prokaryotic cell, or a yeast cell or a eukaryotic cell, in particular an animal cell, in particular a mammalian cell, more particularly a cancer cell, transfected with a composition as defined above. According to a preferred embodiment of the invention, the cells are respiratory tract cells, more particularly tracheal cells or lung cells, advantageously respiratory tract epithelial cells. For respiratory administration, it is advantageous to administer a bronchodilator compound (e.g., theophylline) in advance. The applicant has demonstrated that oral administration of 0.3mg of theophylline (dilantane) to mice 30 minutes prior to incubation of the composition with DPSO results in a slight increase in expression levels compared to mice not pretreated with this.

The specification and examples of the invention disclose and describe and incorporate the various embodiments. Literature pertaining to any of the methods, uses and compounds used in the present invention may be available from public libraries, or from electronic devices, for example. For example, a public database "Medline" on the Internet may be used, such as the one found at the website http:// w.ncbi.nlm.nih.gov/PubMedMedline.html. Further databases and web addresses, such as http:// www.ncbi.nlm.nih.gov/, http:// www.infobiogen.fr/, http:// www.fmi.ch/biology/research ___ tools. html, http:// www.tigr.org/, are well known to the skilled person and information in this respect can be obtained via the web address http:// www.lycos.com, for example. An overview of patent information in the biotechnology field and a survey of relevant patent information sources for retrospective retrieval and updating of information is given in the Berks article TIBTECH 12(1994), 352-.

The compositions, uses, methods of the invention are useful for treating a variety of diseases whose treatment and/or diagnosis involves or depends on the transfer of a therapeutic substance in a cell. Although treatment of animals is also included in the methods and uses described herein, it is desirable to use the pharmaceutical compositions, methods and uses of the present invention in humans.

The following examples 1 to 7 illustrate the present invention with reference to fig. 1 to 5.

FIG. 1: map of plasmid pTG11033

FIG. 2: the level of expression of the gene encoding luciferase in the lung (RLU/mg protein) following injection of the composition. The composition consists of 50. mu.g DNA in 20mM Hepes buffer at pH7.5 and 15% (v/v) DESO or DPSO

FIG. 3: expression level of luciferase-encoding gene (RLU/mg protein) in lung after injection of composition. The composition contained 50. mu.g of DNA dissolved in 20mM Hepes buffer at pH7.5 and, where appropriate, 10 or 15% (v/v) of a different polar compound. The values shown are calculated from the average of each observation after injecting mice with the same composition.

FIG. 4: expression level of luciferase-encoding gene (RLU/mg protein) in lung after injection of composition. The composition contained 50mg of DNA dissolved in 20mM Hepes buffer at pH7.5 and, where appropriate, 10 or 15% (v/v) TMU or DPSO. The values shown are calculated from the average of each result observed after injection of n mice with the same composition. SEM: mean standard error (see: Burke, Scientific data management 9(1997), 32-38).

FIG. 5: plasmid DNA melting curve +/-DPSO. 50. mu.g/ml plasmid DNA in 20mM HEPES pH7.5 (pTG11033) or 50. mu.g/ml plasmid DNA in 20mM HEPES pH7.5 containing 5%, 10%, 15% or 20% DPSO, respectively, were heated (4 ℃/min). The absorbance was recorded and normalized to the initial absorbance at 35 ℃.

The invention and many of its advantages will be better understood from the following illustrative examples.

Example 1: preparation of a composition containing a polar Compound as DESO or DPSO

The selected polynucleotide, plasmid pTG11033 (FIG. 1), included the gene encoding luciferase under the control of the CMV promoter, intron 1 of the HMG gene and the SV40polyA termination signal. Precipitation of 250. mu.l of plasmid DN from a 1mg/ml solution of purified plasmid (purified by cesium chloride gradient centrifugation)A. After centrifugation, the nucleic acid pellet was washed with 70% ethanol and dried, and dissolved in a given volume of DESO or DPSO and 20mM Hepes buffer pH7.5 to prepare the composition shown in Table I below:

	15％DESO(v/v)	15％DPSO(v/v)
			50. mu.g of DNA/50. mu.l	37.5. mu.l of DESO20mM Hepes, pH7.5 in sufficient quantity to 250. mu.l	37.5. mu.l DPSO,20mM Hepes, pH7.5, in sufficient quantity to 250. mu.l

Example 2: administering the composition by intratracheal injection

Female mice (B6/SJLF1, Iffa Credo) of 8 weeks of age were anesthetized by intraperitoneal injection (saline, IMALENE 1000, xylazine/ROMPUN). After disinfecting the skin with 70% alcohol, the trachea was exposed by incision, and 50. mu.l of the composition of example 1 was injected intratracheally with a syringe. At least three different mice were injected with each composition. The expression levels were compared with those obtained after injection with 50. mu.g of DNA dissolved in 50. mu.l of 20mM Hepes pH7.5 without addition of any polar compound. Non-injected mice are also included in the method as controls.

Example 3: the luciferase activity in the injected mouse tissues was measured.

Mice were sacrificed two days after injection. The lung and trachea are treated separately. Tissues were frozen in liquid nitrogen and stored at-80 ℃. To measure luciferase activity, tissues were mechanically ground in a mortar placed in dry ice. To the tissue fragments obtained from the lung or trachea, 500. mu.l or 200. mu.l of lysis buffer (Promega) was added, respectively, and the resulting solution was subjected to three-step freeze/thaw treatment. Cell debris was removed by centrifugation and luciferase activity (RLU/min, relative light units per minute) was measured by adding 100. mu.l of reagent to measure luminescence from 20. mu.l of supernatant according to the instructions of the supplier (Promega). The measured luciferase activity was normalized to the amount of protein with the help of a standard scale established for commercially available luciferase (Promega). Aliquots of the supernatant were then subjected to color comparison using bicinchoninic acid (BCA) colorimetry (Smith, anal. biochem.150(1985),76-85) to determine the total amount of protein. Thus, the activity of luciferase can be expressed as RLU per mg of protein (protein extracted from tissue). For a given composition, the activity studied corresponds to the mean value obtained from three injected mice.

Example 4: results obtained

Figures 2 and 4 illustrate the results obtained with extracts obtained from the lungs. These results demonstrate that when lung tissue was injected with polynucleotide alone (50 μ g), no luciferase gene expression was observed, whereas the addition of DPSO to the injected composition promoted expression.

These results also demonstrate that DPSO is itself a compound effective in promoting the ability of polynucleotides to transfect into cells following injection of the composition into the trachea, because of the significantly increased expression compared to polynucleotide injection alone.

Example 5: other aprotic polar compounds to be tested

In the same way, we demonstrate that other aprotic polar compounds can be used according to the invention. To this end, various compositions were prepared according to the methods described in examples 1 and 4. In particular, these compositions comprise: 50. mu.g of plasmid pTG 11033/50. mu.l and variable percentages (10%/15%) of the following: DPSO, cyclobutanesulphoxide (TEMSO), (see Johnson and Keiser,1973, Organic Synthesis discol 5(1973),791), DMSO, tetramethylurea or sulfolane, and the resulting composition was injected into 8 to 12 week-old mice (C57BL/6, Iffo Credo) according to the method described in example 2 above.

The results obtained (FIGS. 3 and 4) demonstrate that the invention can be carried out with a variety of aprotic polar compounds. It was found that intratracheal injection of a test composition comprising a polynucleotide and one of the aprotic polar compounds as listed above significantly improved transfection of lung cells under the same conditions, especially with respect to concentration, compared to injection of the polynucleotide alone.

The best results were observed with compositions containing DPSO or TMU. For each of these polar compounds, it was demonstrated that the expression level of the measured gene encoding luciferase could be increased by increasing the percentage of the polar compound at the amount of DNA tested (50. mu.g) (FIG. 4).

Example 7: recording of plasmid DNA melting curves

Plasmid DNA melting curves were recorded on a thermostated UV/visible CARY/VARIAN spectrophotometer (software version: 3.04) using two quartz colorimetric rings (L =1cm) and a Teflon final plug. Total volume of sample: 1ml (50. mu.g/ml plasmid DNA (pTG11033) was dissolved in 20mM HEPES pH7.5 containing varying percentages (0,5,10, 20/%) of di-n-propyl-sulfoxide (DPSO) with a temperature gradient of 40 ℃/min and experiments performed at 260nm, 35 ℃ to 100 ℃ (range: 0.9-1.2 OD; 50. mu.g-1 OD.) the melting curve of DNA (FIG. 5) shows that DNA alone ("naked" DNA) behaves differently than DNA in the presence of DPSO ("solvent complex") in that the change in absorbance in the presence of sulfoxide compound is reduced and occurs at higher temperatures.

Example 8: aerosol formulation of lipid-solvent complexes

The final form of delivery of the vector to the airway epithelium is in the form of an aerosol. However, if only DNA is used, the DNA will degrade rapidly during aerosolization. This can be avoided by complexing the DNA as a lipid complex (complexing the DNA with cationic amphiphiles such as those described in patent application n0FR97/15805, for example pTG 90). It has been observed in advance that lipid complexes alone cause no or only low levels of expression in the lung. According to the present invention, it was tested to combine the advantageous properties of solvent complexes (complexes of the invention) and lipid complexes. For this purpose, a lipo-solvent complex (DNA homocationic lipid and DPSO) was prepared; the mixture was aerosolized, collected in a cold trap tank, and then injected into the trachea of a mouse. Intratracheal injection of the lipid complex and pTG 90/DOPE alone did not result in luciferase expression, but in the presence of DPSO, the pTG 90/DOPE lipid solvent complex resulted in luciferase expression. Generally, the expression level of the lipid-solvent complex assay decreased after aerosolization, but 86% activity of the same complex was observed before aerosolization for the pTG/DOPE/DPSO lipid-solvent complex.

Example 9: synthesis of disulfoxide

9.1 methyl-bis-methylsulfoxide (bis (methylsulfinyl) methane BiMsum) (= CH₃-SO-CH₂-SO-CH₃)。

10.5ml of CH are added at room temperature₃SOCH₂SCH₃(12.5g,100 mmol; Aldrich 17.795) was diluted in 125ml of methanol. After stirring, the solution was stored at 0 ℃. 21.4 g NaIO were added dropwise over 90 minutes₄(100 mmol) in 280ml of water. By thin layer chromatography (TLC, solvent: CH)₂Cl₂/CH₃OH 95/5 with I₂,KMnO₄Detection, CH₃SOCH₂SCH₃，Rf=0.9；CH₃SOCH₂SOCH₃Rf =0.33) to control the reaction progress. The reaction mixture was filtered through Celite and 200ml of CH with 300ml of water₂Cl₂And finally washed with 200ml of methanol.

The enriched organic fractions were pooled and taken over Na₂SO₄Dried and evaporated.

Purifying the obtained product and passing the fraction containing the two enantiomers through the column three times550mlCH₂Cl₂ 250ml CH₂Cl₂/CH₃OH 97/3,1000ml CH₂Cl₂/CH₃OH 95/5, and 1000ml CH₂Cl₂/CH₃OH 90/10 silica gel column (ID4cm, 100g silica in CH)₂Cl₂Middle). Both enantiomers of bimsu eluted in fraction 95/5. Fractions were analyzed by high performance liquid chromatography (NH)₂A column, which can separate the two enantiomers). The enriched fractions of one or the other enantiomer were pooled and evaporated. By NH₂High performance liquid chromatography and¹h nuclear magnetic resonance (200 MH)₂,CDCl₃) And (6) carrying out analysis.

Enantiomeric purity R, S-BiMSuM/R, R-BiMSuM 87/13:4.5g

Enantiomeric purity R, S-BiMSuM/R, R-BiMSuM 05/95:0.6g

9.2 methyl-di-ethylsulfoxide (bis (ethanesulfinyl) methane, BiESuM) (= CH)₃CH₂-SO-CH₂-SO-CH₂CH₃)

4ml of CH are added at room temperature₃CH₂-SO-CH₂-S-CH₂CH₃(4.45g, 29 mmol, FluKa 02845) was diluted in 25ml methanol. After stirring, the solution was stored at 0 ℃. 7g of NaIO was added dropwise₄(33 mmoles) in 50ml of water. The reaction mixture was kept at 0 ℃ for 5 hours with stirring. By thin layer chromatography (TLC, solvent: CH2 Cl)₂/CH₃OH 90/10 with I₂,KMnO₄Detecting; CH (CH)₃CH₂SOCH₂SCH₂CH₃,Rf=0.95；CH₃CH₂SOCH₂SOCH₂CH₃Rf =0.7) controls the reaction progress. The reaction mixture was filtered through Celife, using 300ml of water, 200ml of CH₂Cl₂Then washed with 200ml of methanol. By CH₂Cl₂The liquid phase was then extracted 3 times with ethyl acetate. The organic fractions were pooled and taken over Na₂SO₄Dried and evaporated.

The product obtained was purified and the fractions containing the two enantiomers were subjected to silica gel column (ID 2cm,38 g silica in CH)₂Cl₂Middle) three times through the column, eluent is 100ml CH₂Cl₂,200ml CH₂Cl₂/CH₃OH 99/1,200ml CH₂Cl₂/CH₃OH 97/3,200mlCH₂CI₂/CH₃OH 95/05,200ml CH₂Cl₂/CH₃OH 92.5/7.5,200mlCH₂Cl₂/CH₃OH 90/10. The enantiomer of BiESuM was eluted in fraction 95/5. Fractions were analyzed by high performance liquid chromatography (silica gel column, which separates the two enantiomers). The enriched fractions of one or the other enantiomer were pooled and evaporated. Using silica gel high performance liquid chromatography and¹h nuclear magnetic resonance (200MH2, CDCl)₃) And (3) analysis: delta.3.89 (quintuple, 2H, -SO-CH)₂-SO-),3.1 (quartet, 4H, -CH₂-),1.40(t,6H,-CH₃)。

Fraction 1: enantiomeric purity R, S-BiESuM/R, R-BiESuM 84/16:1.9g

Fraction 2: enantiomeric purity R, S-BiESuM/R, R-BiESuM 16/84:0.075g

The specific embodiments described herein are merely illustrative of specific aspects of the invention and are not intended to limit the invention to that scope, and any functionally equivalent compositions and compounds are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are also intended to fall within the scope of the appended claims. Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth above as many apparent variations thereof are possible without departing from the spirit or scope thereof.

Claims (29)

1. A composition comprising a mixture of at least one therapeutically active substance and at least one aprotic polar compound selected from the group consisting of compounds defined as:

(a) formula I:

wherein R is₁And R₂Aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl, alkoxy which are identical or different and are 1 to 8 carbon atoms and may optionally be substituted,

with the proviso that when R₁When it is a group of 1 or 2 carbon atoms, R₂A group of at least 3 carbon atoms, R₂When it is a group of 1 or 2 carbon atoms, R₁A group of at least 3 carbon atoms, R₁And R₂Possibly linked to form a cyclic molecule;

(b) formula II:

wherein R is₃And R₄Aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl or alkoxy which are identical or different and are of 1 to 8 carbon atoms, which are optionally repeating, linear or branched and optionally substituted,

R₃and R₄Possibly linked to form a cyclic molecule;

(c) formula III:

wherein R is₃And R₄Aryl, alkyl, cycloalkyl, fluoroalkyl, alkenyl or alkoxy which are identical or different and are of 1 to 8 carbon atoms, which are optionally repeating, linear or branched and optionally substituted,

R₅is (CH)₂)_xAnd at each [ R ]₅-S]_nAre independent of each other in the repeating unit, wherein x =1 to 50

R₃And R₄Possibly linked to form a cyclic molecule; or

(d) Dimethylformamide, dimethylacetamide, tetramethylurea or derivatives thereof.

2. The composition of claim 1, wherein the aprotic polar compound is selected from the group consisting of di-n-propyl sulfoxide (DPSO), cyclobutanesulfoxide (TEMSO), dimethylsulfone, sulfolane, 1-methyl-2-pyrrolidone and derivatives thereof.

3. The composition of claims 1 and 2, wherein the therapeutically active substance is a polynucleotide.

4. The composition of claim 3, wherein said polynucleotide is a polynucleotide that does not contain any compounds that facilitate its entry into a target cell.

5. The composition of claim 3, wherein said polynucleotide is a polynucleotide associated with at least one viral polypeptide.

6. The composition of claim 3, wherein the polynucleotide is a polynucleotide associated with at least one cationic amphiphile, in particular a lipid.

7. The composition of claim 3, wherein the polynucleotide is a polynucleotide associated with at least one cationic or neutral polymer.

8. The composition of any one of claims 3 to 7, wherein said polynucleotide is an antisense polynucleotide.

9. The composition of any one of claims 3 to 7, wherein said polynucleotide is a ribozyme.

10. The composition of any one of claims 3 to 7, wherein the polynucleotide comprises a gene of interest and a factor enabling expression of the gene of interest.

11. The composition of claim 10, wherein the polynucleotide encodes all or part of a polypeptide.

12. The composition of claim 11, wherein said polypeptide exhibits therapeutic activity.

13. The composition of claim 11, wherein the polypeptide exhibits prophylactic activity, in particular immunogenic activity.

14. The composition of claim 11, wherein the polypeptide is an enzyme, hormone, cytokine, membrane receptor, antibody, factor that regulates transcription, translation or replication and is involved in transcriptional stability, structural polypeptide, polypeptide that forms a membrane channel, transport polypeptide, adhesion molecule or ligand.

15. A composition as claimed in any one of claims 1 to 14 wherein said aprotic compound is in aqueous solution.

16. A composition as claimed in any one of claims 1 to 15 wherein the volume of said polar compound is equivalent to 15-20% of the total volume of the composition.

17. A composition as claimed in any one of claims 1 to 16, wherein the aprotic polar compound is in aqueous solution.

18. A pharmaceutical composition comprising a composition as claimed in any one of claims 1 to 17 and optionally a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18 for use in transferring at least one therapeutically active substance into a target cell.

20. The pharmaceutical composition as claimed in claim 18 or 19, which is designed for administration by intramuscular route or by inhalation, intratracheal injection, instillation or aerosol method.

21. A vaccine comprising the composition of any one of claims 1 to 17.

22. A pharmaceutical composition as claimed in any one of claims 18 to 20 or a vaccine as claimed in claim 21 for use in gene therapy.

23. A diagnostic composition comprising a composition as claimed in any one of claims 1 to 17.

24. Use of a composition as claimed in any one of claims 1 to 17 for the preparation of a pharmaceutical composition for transferring at least one therapeutically active substance into target cells in vivo or in vitro.

25. The use of claim 24, wherein the target cell is a mammalian cell.

26. The use of claim 24 or 25, wherein the target cell is a muscle cell.

27. The use of claim 24 or 25, wherein the target cell is a respiratory cell.

28. A method for transferring a therapeutically active substance into target cells in vitro, wherein said cells are contacted with a composition according to any one of claims 1 to 17.

29. The method of claim 28, comprising the additional step of heating said composition prior to contacting said composition with said cells.