FR2766841A1 - GENE AND PROTEIN THERAPY PRODUCTS FOR MUCOVISCIDOSIS AND INDUCERS OF GLUTATHION CARRIER FUNCTION - Google Patents

Abstract

The invention concerns a DNA molecule containing a DNA sequence whereof the expression product has a common peptide sequence with part of that of CFTR, in particular the one extending from the 430<th> to the 660<th> residue of the latter and having a glutathione carrier function. Said DNA molecule is useful for constructing vectors useful in gene therapy for treating cystic fibrosis.

Description

Cystic fibrosis, also sometimes called cystic fibrosis, is reputed to be caused by mutations in the gene encoding the protein known as CFTR, according to the abbreviation of the English expression Cystic Fibrosis
Transmembrane Conductance Regulator.

The nucleic sequence encoding this CFTR protein which comprises 1480 amino acid residues, was cloned and identified by
Riordan JR et al., Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA (1989) Science 245: 1066-1072.

The coding sequence for the CFTR protein is also reproduced in FIG. 1 of PCT application WO / 9102796 made public on March 7, 1981 and of which Riordan himself is also one of the co-inventors.

This PCT application - and others which have followed - effectively describe the cloning of the coding sequence and its expression in a number of organisms.

According to a recurring hypothesis in the scientific literature, the normal CFTR protein would have a function called the chlorine channel or chloride ion channel, in other words would regulate transfers of anions, more particularly chloride ions (Cl-), out of the epithelial cells, outwards, under the effect of its phosphorylation by a protein kinase dependent on cyclic VAMP (cAMP) or protein kinase C (Riordan, JR et al. (1989) Science 245: 1066-1073; Frizzell, RA et al.

(1986) Science 233: 558-560; Welsh, M.J. and Liedtke, C.M. (1986) Nature 322: 467; Li, M. et al. (1988) Nature 331: 358-360; Hwang, T-C. et al.

(1989) Science 244: 1351-1353).

At 37 ° C, the CFTR protein would also pass from the endoplasmic reticulum to the Golgi apparatus, and from there into the plasma membrane as recalled by Ko et al., (1997) Biochemistry 36: 5053-5064, which refer themselves to previous authors.

Many CFTR mutations have been observed in patients with cystic fibrosis.

Epidemiological studies carried out in affected populations revealed in approximately 70% of cases a CF mutation (abbreviation of the English expression Cystic Fibrosis) or deletion of the 3 nucleotides encoding the phenylalanine residue at position 508 of the amino acid sequence of the CFTR. The site of this mutation would be located more particularly in the region called NBF1 (abbreviation of the English expression Nucleotide Binding Fold I (or
Folded region of nucleotides)) which would contain the first ATP binding domain in CFTR and which, according to Ko et al. already mentioned would extend from the residue Phenylalamine 433 (P433) to the residue Serine 589 (S589). This mutation is often held responsible for a defolding defect of the NBF1 domain in the then CF-defective cells of the patients, a defect which would cause a blockage of the CFTR thus mutated (called AF508) in the intracellular organelles (golgi, endoplasmic reticulum). , then its destruction before it could complete its post-translational maturation. And it is indeed the deficiency of the chloride ion channel function that the so-called sweat test aims to detect, which measures the secretion of chlorine in a biological fluid obtained from the patient concerned, in support of a diagnosis of the cystic fibrosis.

The correlation often made between clinical phenomena observed in many patients and the mutations observed in the gene encoding CFTR, is at the origin of one of the routes already proposed for the treatment of cystic fibrosis, in this case the a route based on gene therapy, for the purpose of transferring DNA encoding an intact CFTR into cells in a form which then allows its expression even in CF-defective cells, in particular in the epithelial cells of the airways of patients.

Thus tests using a complex of DNA encoding CFTR and cationic lipids have been administered to mice intratracheally in order to obtain their direct admission into the lungs (Yoshimura et al. Nucleic Acid Research, 20: 3233-3240,1992) or alternatively by aerosol, Stribling et al., Proc. Natl. Acad. Sci. 89: 1127711281, 1992). It was also found that the administration of the gene encoding CFTR complexed with cationic lipids to a model mouse suffering from cystic fibrosis, had the effect of correcting the defect in the function of the chloride ion channel (Hyde et al., Nature 362 : 250-255, 1993).

It has also been proposed, for example in international applications WO 95113365 and WO 96/13598 to induce the expression in the CF cells of the patients to be treated, of an entire CFTR gene supplied from the outside, this gene having at the previously been incorporated under the control of appropriate regulatory elements into vectors, for example genetically modified adenoviruses or retroviruses, preferably defective but still capable of infecting these cells, of transferring the CFTR gene therein, the latter then being capable of expressing itself under the control of the aforesaid regulatory elements.

These methods generally prove to be if not ineffective, at least insufficient. But as the requesters note
PCT WO 95/25796, viral vectors of this type can only harbor or package foreign or exogenous genes of restricted sizes, at most 4.5 Kb. Now, the DNA encoding natural CFTR reaches this size limit, which makes its expression random in the infected cells, under the control of the corresponding viral promoters.

To this difficulty is added another apparently major one, namely the low solubility of the expression product in the cytoplasmic medium of the cells in question. Finally, there is also the likely immunogenic character of the high molecular weight expression products that may be formed.

Also, the authors of application WO 95/25796 have proposed to replace the entire gene with a gene truncated by a significant part of the C-terminal region of the CFTR, with the proviso, however, that the deletion of this C region -terminal is not accompanied by the loss of the biological properties of the chloride ion channel type, characteristic of natural human CFTR, or even of functionally analogous properties of the entire recombinant CFTR.

According to the authors of the application, a recombinant truncated protein still capable of exercising its functions must normally comprise: - the MSD sequence (abbreviation of the English expression Membrane
Spanning Domain or Domain extending across the membrane)
which extends approximately between amino acid residues 76 to 360
(from the N-terminal residue of the CFTR protein), - the MBD1 domain (English abbreviation for the expression Nucleotide
Binding Domain-1 or binding nucleotide domain-1) which
extends approximately from residue 360 to residue 708 of CFTR
integer, and finally - the regulatory domain R which extends approximately from the residues
amino acids 590 to 830 of the CFTR, this domain being known to ensure
at rest, the closure of the chloride ion channel and its opening under
the effect of phosphorylation by the cAMP-dependent protein kinase
or protein kinase C.

In particular, a recombinant protein comprising the 884 N-terminal amino acids of CFTR would still exercise the required functions of chloride ion channel and of regulator of the opening and closing of this channel.

But assuming that one can effectively obtain a better expression with such CFTRs amputated of their C terminal regions, one cannot fail to note the still large size of the expression products capable of being obtained, and consequently of anticipate the persistence of an immunogenic character making its use more than random in gene therapy trials.

It will also be noted that this proposal for gene therapy, which would only use the truncated CFTR gene meeting the conditions described in patent WO 95/25796, further links the efficacy of the treatment to the reestablishment of the sole function of the channel. Chloride ions exerted in healthy subjects by a functional CFTR protein.

This was to omit, however, that if the lack of transport of chloride ions is indeed a clinical manifestation often observed in patients with cystic fibrosis, other manifestations also observed remain unexplained to this day.

The invention stems precisely from the observation that the treatment of cystic fibrosis would largely require a different therapeutic approach, since, as has been demonstrated by the inventors, CFTR would also play a particularly important role in the active transport of glutathione (GSH), so cystic fibrosis could also be significantly linked to mutations or other causes that interfere with the ability of CFTR to perform glutathione transporter function.

The invention therefore relates to DNA fragments, the sequences of which, including sequences derived from the gene encoding CFTR, are chosen from those which encode a function of active transport of glutathione. And the invention is particularly interested in fragments of smaller sizes than those of DNA fragments, the use of which in gene therapy for the treatment of cystic fibrosis has already been mentioned in the technical or scientific literature.

It is in fact thanks to: - on the one hand, the discovery of particular properties of a polypeptide
fusion containing part of the CFTR sequence, which had been
produced as part of studies aimed at studying the structure
of the sequence encoding the NBF-1 domain, and - on the other hand, observations relating to a lasting clinical improvement
symptoms related to cystic fibrosis in a patient as well
cancer patient treated with chemotherapy, which was accompanied by
overexpression of the so-called MRP protein (abbreviation of
Multidrug Resistance Associated Protein
multiple drug resistance)), which the inventors were led to discover, which is the very basis of the invention.

The study of the three-dimensional structure of the sequence encoding the NBF1 domain - in particular for the purpose of studying the effect of the aF5o8 mutation presumed to be responsible for the deflection of CFTR in the NBF1 domain - implied the prior obtaining of large amounts of pure, soluble and stable protein (a few tens of mg). Since such quantities are not accessible by purification of the natural protein, the inventors therefore undertook to produce NBF1 (in its initial definition) by cloning in a living expression system (E-colt), in the form of a protein. fusion with GST (Glutathione-S-Transferase).

This choice was initially intended to use the capacity for specific recognition of glutathione S-transferase by glutathione to purify the desired protein (GST-NBF1) from all the bacterial proteins. To this end, a sequence derived from the gene for
CFTR (sequence R450-1586) and containing the nucleotide sequence encoding the NBF1 sequence was inserted into a plasmid allowing the transformation of E.coli bacteria and the expression therein of the insertion sequence in the transformed bacteria , for example a plasmid of the pGEX-KT type. The cultivation of the strain of E. coli thus transformed effectively led to an overproduction of a fusion protein of NBF1 and of glutathione-S-transferase (GST). The yields obtained were 5 mg / l of culture. The soluble protein obtained was sequenced. It was recognized by anti-NBF1 antibodies and also binds ATP. Cleavage with thrombin made it possible to prepare free NBF1 domains which exhibited the same characteristics.

However, it is not possible to separate the GST and the NBF1 domain on an affinity column grafted with glutathione (GSH): although perfectly separated on an electrophoresis gel, the two proteins NBF1 and GST co-eluted on a column. of immobilized GSH.

The activity of the fusion protein was verified by fluorescence: an affinity for a fluorescent derivative of ATP (the TNP
ATP) has indeed been observed.

However, multiple attempts to purify the domain
NBF1, isolated from GST, by thrombin cleavage were unsuccessful.

Furthermore, the GST-NBF1 protein could be selectively and quantitatively cleaved, but the NBF1 domain alone was then found to be unstable. It had a strong tendency to form aggregates.

These results led the inventors to reconsider or re-evaluate the definition of the NBF1 domain, as it had been described by
Riordan. Indeed, the definition of a domain must correspond to a stable entity, characterized by its own folding encoding its function, and whose stability is independent of the complete protein (here CFTR) to which this domain belongs. To ensure this folding, the limits chosen for NBF1 must contain all of the secondary structural elements (helices and leaflets) necessary for the stabilization of the domain in its native conformation.

However, CFTR belongs to the ABC transporter family (ATP
Binding Cassette) which form one of the largest families of membrane proteins (AI 1.96), found in both eukaryotes and prokaryotes. These proteins are generally active transporters which hydrolyze ATP to provide the chemical potential necessary for transport. In eukaryotes they transport molecules (ions, vitamins, peptides, sugars, drugs, etc.) across all membranes. Their global organization presents common characteristics: regions (TM) which participate in the selection of the chemical entity to be transported, and nucleotide binding domains. These genes are often the result of the fusion between two half genes. The global topology is then: (TM1) - (NBF1) - (TM2) -NBF2). And in fact, CFTR belongs more particularly to the CFTR / MRP subfamily, and has a sequence similarity of approximately 50% with the human MRP protein (Multi-drug
Resistance associated Protein). It is also very close to the YCFî protein (Yeast Cadmium resistance Factor 1) which gives the yeast Saccharomyces cerevisiae resistance to cadmium ions (Tom.96). To a lesser extent it is also close to
STE6 (from the MDR / TAP subfamily) and which transports the factor a pheromone in the yeast Saccharomyces cerevisiae (Tom.93), and
Human MDR (Multi-drug Resistance protein), or even beef ATPase-F1, the only structural protein known in the ABC protein family (Abr. 94).

And it is because the structure of the bovine ATPase is known that the inventors have chosen its NBF domains as a reference structure for the re-evaluation also of the NBF1 domain of the
CFTR. It follows from this choice, discussed in two recent publications (Ann.97a, Ann.97b), a 3D structure model for NBF1, built by homology with the structure of bovine ATPase-F1, method developed in (Pat .96), allowing, among other things: 1. to account for the phenotype linked to the Au508 mutation, unlike
previous models (Hyd.90, Mim.91); 2.report the key role of the LSGGQ consensus sequence, very
preserved in ABC proteins; 3. to redefine the limits of the NBF1 domain - hereinafter NBF1 revisited
which is now considered to extend approximately from
430th amino acid residue up to approximately residue 650
(encroaching by about 70 residues on the R domain, in its definition
initial) of the CFTR sequence.

This last point follows directly from the hypothesis of a comparable folding for the NBF regions of bovine ATPase-F1 and CFTR. The main argument is that the ABC transporter family forms a homogeneous group that is quite distinct from other ATP binding proteins, the members of which are derived from divergent evolution. In this context, these proteins must exhibit a comparable folding in their conserved regions (typically, in the NBF building blocks).

It is then lawful to model the structure of one of these proteins on the basis of the known structure of another.

The structure model thus constructed for the NBF1 domain of
CFTR, by homology with the known structure of the nucleotide binding domains of bovine ATPase-F1 (Abrahams JP, Jeslie AGW,
Lutter R., Walker JF (1994) Nature 370: 621-628), according to the method described by Annereau et al. (FEBS Letters, (1997) 407: 303-308), comprises a hydrophobic core consisting of an ss sheet with 6 parallel strands, respectively: L453-S456, N505-S511, N538-G542, D567-D572, R600-V603 )
These sheets alternate with 6 a-helices are organized on either side of the mean plane of the central ss sheet, as shown in the article by FEBS
Letters, Vol. 407, pp.:303-308, 1997. Loops located on the surface of the domain connect the helices to the leaflets.

And in fact the expression product of the fragment corresponding to the gene encoding CFTR (fragment which itself forms part of the invention) has been shown to be stable, which is confirmed by the results of the cloning in E. coli of the new NBF1 domain (or revisited NBF1 domain), in E.coli in the polyhistidine system, that is to say of a fusion DNA encoding the revisited NBF1 domain (it is the fragment encoding the polypeptide extending from 448th to 650th amino acid residues of CFTR) and a tail comprising only 6 histidine residues (it can then be considered that it is produced alone). The production rates obtained are noticeably greater than on the preceding fragment, reaching for the polyhistidine system approximately 200 mg per liter of culture (from the first test, before any optimization). This revisited NBF1 purified fragment was purified in massive amounts as a soluble and stable protein.

The potential existence of a glutathione binding site in
NBF1 of CFTR practically superimposable on the glutathione binding site in GST is demonstrated by comparison of this model with the known structure of GST (Glutathione-S-Transferase), a protein which has a glutathione binding site and which catalyzes reactions of nucleophilic attacks of glutathione. The NBF1 residues potentially involved in glutathione binding are located in a region forming a crevice at the ends of strands involved in a Q sheet (see the article by FEBS letter). These residues are located more precisely in loops, at the N-terminal ends of sheets 1 to 4 of the structure of NBF1. These loops contain residues 1448
Q452 (IERGQ), S478-K481 (SEGK), M498-l506 (MPGTIKENI), A566-L568 (ADLVL), A596-T599 (ANKT).

The new definition of NBF1 and the 3D structural model constructed have enabled the inventors to propose a new function for CFTR, suggested by the unexpected fact that NBF1 is retained on a glutathione column, even after cleavage of the GST NF1 fusion protein. Indeed, the comparison of this model with the known 3D structure of GST, a protein which has a glutathione binding site and which catalyzes the reactions of nucleophilic attacks of glutathione (Lim. 94), highlights the presence of a potential glutathione binding site.

Functional information about the two proteins
ABCs closest to CFTR (human MRP and YCF1 from yeast) seem to support this hypothesis. Indeed, it is now established that
MRP exports drugs via glutathione, either as direct glutathione-drug adducts or by simultaneous or sequential binding of glutathione and the drug (Zarn.95). Likewise, YCF1 transports cadmium ions in the form of double adducts of glutathione. Thus, it appears that CFTR can be a glutathione pump, like MRP and YCF1.

However, the human MRP and MDR proteins are involved in the phenomena of multi-resistance to anti-tumor drugs (Dea.95).

These proteins are able to actively transport these drugs, thus participating in the cell's detoxification mechanisms. Their overexpression is induced by the drugs themselves, which is the main cause of failure of cancer treatment with chemotherapy.

However, it was recently shown that the overexpression of CFTR could also be triggered by anti-tumor drugs, and that it confers the multi-resistance phenotype (Ll.95).

From this also follows the hypothesis formulated by the inventors that the function of the transporter of glutathione could be important and that mutations capable of disturbing it would also explain a number of symptoms of cystic fibrosis. The role of CFTR as a glutathione pump (in addition to its known role as a chlorine channel) also allows a more comprehensive understanding of cystic fibrosis (Sto. 97).

A deficit in the transport function of glutathione would also better account for chronic inflammatory reactions and / or the very high number of polymorphonuclear cells that are often found in the airways of patients with cystic fibrosis, since it is known that glutathione plays a central role in the control of the inflammatory reaction by protecting the cells from the aggression of free radicals by the neutrophils and, moreover, that it intervenes in the protection vis-à-vis oxidants such as free radicals or hyperoxides.

Likewise, it is the organs in which glutathione is normally secreted and in which it exerts a detoxifying function (lungs, intestines, colon) which are generally seriously affected by cystic fibrosis.

A certain number of clinical observations agree with the hypothesis which is the basis of the invention. The level of GSH is indeed lowered in the serum and the bronchial mucus of the subjects suffering from cystic fibrosis. The organs where CFTR is best expressed are those where GSH plays an important role and is actively transported: pancreas, lung, liver, kidney. Detoxifying liver function appears to be impaired, a function in which GSH may play a central role. This is reflected, for example, by a particular sensitivity of patients to a heavy metal such as mercury. Oxidative stress phenomena have also been observed for a long time in the lungs. N-acetyl cysteine, a drug used with benefit for patients, is deacetylated by entering the cell, providing cysteine (a semi-essential amino acid in humans).

And in fact the validity of the hypothesis is already being confirmed in the lasting improvement which has been observed in terms of cystic fibrosis in a patient, aged 29, carrying cystic fibrosis (with clinical signs since birth ). The treated patient had exon 12 of CFTR on one allele and, G673X, located in exon 13 on the other allele. This patient presented, since his birth in 1968, all the clinical signs associated with the disease: positive sweat tests, repeated sinusitis, repeated bronchitis, polyps of the nasal cavity etc ... In 1989 he presented an infection with Pseudomonas aeruginosa , classic in cystic fibrosis and usually almost definitive in these patients. In addition, in April 1993, left thigh fibrosarcoma was diagnosed. The patient underwent surgical and radiotherapy treatment and then chemotherapy from July to October 1993, followed by a new treatment in January 1994.

More particularly, the anti-tumor treatment was as follows:
- epirubicin, 110 mg for two days (D1 and D2)
- ifosfamide, 3.3 g for five days (D1 to D5);
- Filgrastime, 300, ug per day for eight days (D8 to Jet);
under cover:
- granisetron, for the prevention of nausea;
- Methylprednisolone;
- Mesna.

Three months later, the patient again received six cycles of epirubicin and ifosfamide.

Cancer treatment has been effective. Also concerning the clinical picture of cystic fibrosis, following chemotherapy treatment, the following improvements were observed:
- the patient's respiratory condition has improved considerably
is lying;
- his infection with Pseudomonas aeruginosa has disappeared:
- his respiratory parameters reach about 75% of
theoretical values,
- he recovered a respiratory state roughly equivalent to that
which he presented at the beginning of his adolescence,
- he declares himself cured of cystic fibrosis.

A sweat test taken at his home in early 1997 was still very positive, which means that the chloride ion channel function was not restored in this patient. This observation, linked to the fact that the patient declares himself cured of cystic fibrosis, leads to the conclusion that another essential function has been restored by the chemotherapy treatment. In view of the foregoing, it can be assumed that it is in this case the transport function of glutathione which has been considerably increased.

As has already been indicated, the invention is therefore more particularly aimed at DNA fragments corresponding to parts of the gene coding for CFTR, these parts also coding for a polypeptide possessing a function of active transport of glutathione. In what follows, the DNA sequences are generally defined by reference to the peptide sequences which correspond to them, these peptide sequences being each time, for the convenience of language, generally designated by the expression product of expression of the sequences of DNA of the invention. These DNA sequences can of course be reconstituted by the reader, in particular on the basis of Figure 1 which, for the most part, is a reproduction of the sequence of the gene coding for CFTR which was published in the article by Riordan et al.

already mentioned or also in PCT application WO 91/02796.

In the definitions of the DNA sequences - or of their expression product - which follow, reference will frequently be made to regions downstream or upstream of the DNA sequence or of its expression product to which the invention to be related to. It will immediately appear that a region upstream of a given DNA sequence within the gene encoding the CFTR represented in FIG. 1 or of the corresponding expression product is located upstream of the 5 'end of this DNA sequence or the N-terminus of the amino acid sequence of the corresponding expression product. Conversely, the downstream regions are those which are located beyond the 3 'end of this DNA sequence or the C'-terminus of the amino acid sequence of the corresponding expression product. .

Also obviously, the indication of a letter (in the symbolic identification system of amino acids implementing a unique letter for each of them) followed by an index n refers to the nth amino acid which follows. from the examination of Figure 1 attached, counting from the first amino acid residue of CFTR.

In general, the longest DNA molecule according to the invention contains a DNA sequence whose expression product has a peptide sequence in common with part of that of CFTR, this part being essentially free from at least the Nterminal region of CFTR which normally extends upstream of the MSD region of the latter and which comprises, in particular, the 360 Nterminal amino acids of CFTR, or, alternatively, any DNA sequence resulting from the modification of the preceding one by deletion, substitution or addition of nucleotides, with the proviso that the capacity of the corresponding expression product to exert a function of transporter of glutathione is preserved at least in part.

By compound transporter of glutathione, intracellular and confers a phenotype of resistance to multiple drugs); Biophysical Journal (1995) 69: 883-895.

It is also possible to have recourse to the measurement of the proportions of mRNA corresponding to the CFTR in the epithelial cells previously taken from a test mammal suffering from cystic fibrosis, such as the mouse, to which the DNA molecule had previously been administered. in accordance with the invention to be tested, under the control of regulatory elements allowing transcription and translation of its DNA sequence in epithelial cells, in particular under the conditions described by Yoshimura et al., in the article identified upper. The demonstration of an expression of the glutathione transporter function is then assessed by the increase in the amounts of mRNA induced in the treated mice compared to what is observed in untreated control cystic fibrosis mice, for example after quantification of mRNAs against an internal RT reference
PCR involving the level of ss2-microglobulin mRNA, according to the method of Lechapt-Zalcman E. et al. described in Resp. J. 1997, M.

MDR1-PgP 170 expression in human Bronchus.

As a variant, it is also possible to use, to define the DNA molecules of the invention, the test which has already been mentioned above. Within the scope of the invention, any DNA molecule meeting the conditions indicated above and which is characterized by a conservation of the affinity of the corresponding expression product for glutathione, this affinity being able to be demonstrated by the implementation of the test consisting in cleaving the expression product of the DNA molecule in question with the aid of thrombin in a C-terminal fragment and in an N-terminal fragment, in collecting the N-terminal fragment and in placing it in contact with an affinity column onto which glutathione is grafted, the affinity of said expression product for glutathione then resulting from its capacities, similar to those of glutathione-S-transferase, to be fixed on this column d activity and to be elected.

Preferably, however, this DNA molecule is shorter, in particular characterized in that its expression product is also essentially free at its C-terminus side of the amino acid sequence of the region of CFTR which s 'normally extends downstream of the R region of the latter.

It is even sufficient that the amino acid sequence of the expression product of the aforesaid DNA sequence itself comprises, on the one hand and on the side of its C-terminal end, the sub-sequence of approximately 80 residues d. 'amino acids (which were previously considered to belong to the R region and now are part of the revisited NBF1 region) which in CFTR extends downstream from its 570th amino acid residue and, on the other hand and in the direction of its N-terminal end, the NBF1 region of CFTR, or even only a part thereof, so that the capacity of the expression product of the aforesaid DNA sequence to exercise said product is retained. glutathione transporter function.

In general, the DNA sequence according to the invention therefore essentially comprises the region of the sequence encoding all or part of the revisited NBF1 region of CFTR which extends from its C-terminal end to 'beyond the presumed glutathione binding site (naturally in the reverse reading direction of the amino acid sequence of this revisited NBF1, i.e. from its C-terminus to its N-terminus.

A preferred DNA molecule is one whose expression product itself has an amino acid sequence which extends between approximately the 430th and the 660th amino acid residue of CFTR.

Among the preferred DNA molecules according to the invention, mention will be made of those which are characterized in that the amino acid sequences of their respective expression products themselves contain, on the one hand and on the side of their C- end terminal, the sequence
NLQPDFSSKLMGCDS (N635 to S649) of CFTR and, on the other hand and in the direction of their N-terminal end, one or more of the following peptide sites:
- ANKT (A596àT599),
- ADLVL (A566 to L570),
- MPGTIKENI (M498 to 1506),
- SEGK (S478 to K481),
- IERGQ (1448 to Q4s2), from the CFTR.

In particular, the DNA molecule according to the invention can be chosen from those having a DNA sequence characterized in that the amino acid sequence of its expression product includes, on the one hand and near its end C-terminal, the sequence
NLQPDFSSKLMGCDS (N635 to S649) and, on the other hand and near its N-terminal end:
- either the ANKT peptide site (A596 to T599),
- either the ADLVL peptide site (A566 to L570),
- either the MPGTIKENI peptide site (M498 to 1506),
- either the SEGK peptide site (S478 to K481),
- or the peptide site IERGQ (1448 to Q452), of CFTR.

However, the amino acid sequence of the expression product of the aforesaid DNA sequence may also include, downstream of the NLQPDFSSKLMGCDS site (N635 to S649), a segment of 10 to 20 amino acid residues whose sequence corresponds to that which follows this same site also in the CFTR or again, upstream of the aforesaid site near its N-terminal end, a segment of 10 to 20 amino acid residues which precedes this same site also in the CFTR, or both segments at the same time.

These segments can also be replaced by binding oligonucleotides (linkers) containing restriction sites facilitating their incorporation into a vector intended to transfer them into appropriate competent cells.

In general, the preferred DNAs of the invention are those whose expression products are soluble in water or in physiological serum, in particular in an amount of at least 0.5, preferably at least. 1 mg / ml of water, in particular in the form of physiological solution.

The invention naturally also relates to any recombinant DNA molecule, in which the DNA sequence as defined above is found to be recombined with exogenous nucleic acid elements, in particular recombinant molecules coding for a fusion protein nevertheless exhibiting the essential biological properties, as defined above, of the product of expression of the DNA sequence characteristic of the molecules according to the invention.

Another category of DNA molecules in accordance with the invention are those in which the exogenous nucleic acid elements constitute an expression vector, comprising appropriate regulatory elements, said nucleic acid sequence therein. being in the state of insertion sequence placed under the control of these regulatory elements, the latter then authorizing the expression of said nucleic acid sequence in a competent cellular organism.

They are in particular expression vectors capable of transforming competent cellular organisms for the purpose of ensuring the expression of the DNA sequence characteristic of the invention, the expression product then being able, if appropriate, be recovered from the cells in culture, or even from the culture medium. It goes without saying that the invention cannot be limited to vectors modified by the DNA sequence according to the invention which can only be used for the purposes of gene therapy. It also extends its effects to any other form of vectors capable of being used for the production of the corresponding expression product in cultured cells, in particular for the purpose of producing the recombinant expression product which could itself be directly used, as discussed below.

Reference is made to the descriptions of international applications
WO 91/02796 already cited, WO 91/10374, WO 92/05273 and WO 93/17040 with regard to examples of expression vectors which have already been described for the expression of the entire CFTR gene, and upon request WO 95/25796 already mentioned in which are described vectors which can be used for the expression of a CFTR of which the C-terminal region had been truncated.

These various vectors can naturally also be used for the expression of the DNA sequences in accordance with the present invention. The descriptions of these patent applications should be regarded as forming part of the description of the present application as regards the variants of expression vectors which can be used for the production of the products of expression of the DNA molecules according to the invention. .

Of particular interest are vectors which allow transformation of human cells, in particular epithelial cells.

Particularly preferred expression vectors are vectors derived from viruses of the adenovirus or retrovirus type or also from viruses related to these (AAV virus), the use of which has already been envisaged for carrying out gene therapies applied to the treatment of Cystic fibrosis. We will also mention international requests
WO 95/13365 and WO 96/13598 which describe vectors and more generally techniques applicable to the expression of the gene encoding CFTR, for application in gene therapy in the form of transcription and expression cassettes comprising the sequence encoding CFTR, recombined with regulatory sequences functional in-vivo in mammalian cells. Again these techniques are applicable to the expression of the DNA sequences according to the invention (instead of the DNA sequences encoding the entire CFTR) so that the descriptions of these applications should also be considered as making part of the description of the present application, as regards the description of techniques and vectors which can be used for this purpose.

Everything indicates that the expression induced in-vivo of the DNA molecules according to the invention, which are all intended to encode a polypeptide having a function of glutathione transporter, may remedy an insufficiency in this regard of an endogenous CFTR. due to the mutations it would carry and to which at least some of the clinical signs of cystic fibrosis seem to have to be attributed and, more generally, to stimulate endogenous expression of CFTR and, in so doing, even to partly restore the channel function with chloride ions, since this would not be completely annihilated by other mutations affecting this endogenous CFTR.

The therapeutic effect expected from a gene therapy using DNA sequences corresponding to the above-indicated definitions can be all the greater the smaller the products of expression of these DNA sequences, the more soluble, stable, and of its own reduced immunogenicity, or even non-existent in practice.

In general, the invention also relates to any pharmaceutical composition combining the DNA molecules in accordance with the invention, where appropriate already in vectorized form, with pharmaceutical vehicles promoting their penetration into human cells and their expression in them. this.

These are all types of cells capable of being targeted by molecules used directly or indirectly (for example when it is a question of polypeptides generated in situ as in the case of gene therapy) in an anti-gene therapy. -cystic fibrosis. These are for example epithelial cells, in particular in local treatments, for example at the pulmonary level, or any other cells, for example hepatic cells, which are capable of intervening in the beneficial effect of the treatment, in particularly when it results from treatment by the general route. This is of very particular interest in the context of the invention, taking into account the preferred mode of action from which it takes advantage, namely the improvement of the function of glutathione transporter.

The invention therefore also relates to all forms of pharmaceutical compositions suitable for the treatment of cystic fibrosis, allowing the targeting of the selected cells and containing, associated with an appropriate pharmaceutically acceptable vehicle, a DNA cassette comprising the DNA molecule according to the invention. , under the control of regulatory elements capable of controlling the transcription and translation of its coding sequence in mammalian cells, preferably human.

A first type of preferred compositions are pharmaceutical compositions suitable for systemic administration for the reasons mentioned above. Other forms of compositions are suitable for administration via the respiratory tract, the preferred compositions according to the invention then being those which can be put in the form of aerosols, nebulisates or the like allowing absorption of the composition. in particular by inhalation, and bringing these compositions into direct contact with the targeted lung cells.

Examples of pharmaceutical vehicles, in particular of the cationic lipid type, particularly suitable for this mode of administration and for similar purposes (in connection with gene therapies using full-length CFTR) have already been described, for example in international applications. WO 93/12240, WO 93/12756 or WO 93/24640. It goes without saying that they are also applicable to the constitution of pharmaceutical compositions using the DNA molecule according to the invention.

Finally, the invention also relates to the polypeptides themselves which are characterized by amino acid sequences as defined above in relation to the DNA sequences characteristic of the DNA molecules according to the invention. These polypeptides are in particular those which have been produced in cultured cells, after transformation of the latter with suitable vectors containing DNA sequences encoding these polypeptides under the control of appropriate regulatory elements, and recovery of the polypeptides. expression obtained from these cell cultures.

Reference has already been made above and by way of example to a certain number of patent applications already published relating to culture systems the use of which has already been described in relation to the production of recombinant CFTR. The same systems can be used with advantage for the production of the polypeptides defined in the present application.

Likewise, the invention relates to pharmaceutical compositions combining the above-mentioned polypeptides with suitable pharmaceutical vehicles, in particular of the same type as those envisaged above for pharmaceutical compositions based on DNA molecules.

The invention is not limited to the sole use of a DNA molecule as defined above, in particular with regard to the peptide sequence which it has in common with the gene for
CFTR. It also relates to compositions in which this DNA molecule would be substituted - or also added - a distinct DNA molecule whose expression product, structurally different from that of the DNA molecule, as it was. defined above, would also have a glutathione transporter function. In particular, DNAs containing an NBF-1 region of proteins of the human MRP protein type, which also carries such genetic information, could be used for this purpose. By way of example, mention will be made of the DNA sequence encoding human MRP-I described by Cole SPC et al., Science 258: 16501654 (1992). Likewise, the human MRP protein or the region useful for this purpose can be substituted - or also added - to the product of expression of the DNA molecule in accordance with the invention, as it has been further defined. high.

The invention is ultimately not limited, nor to compositions of which the active principle consists only of a DNA molecule in accordance with the invention or more generally of a DNA molecule encoding a polypeptide endowed with an activity of glutathione ion transporter, or also corresponding expression products. It also relates to compositions in which this active principle is also associated with others, for example with a separate DNA molecule or, as the case may be, with its expression product. It is, for example, the whole DNA encoding the CFTR or the truncated DNA of international application WO 95/25796, or the corresponding expression product, for the purpose of restoring a deficient chloride ion channel function. in the patient treated perhaps more markedly than the single DNA molecule according to the invention would allow. It is then possible in this way to ensure, under the most favorable conditions, the simultaneous restorations of the glutathione transporter function and of the chloride ion channel function in the patients concerned.

Likewise, the invention relates to compositions of the above-mentioned type, for example those in which the DNA molecule according to the invention, in particular that derived from the gene encoding CFTR, would be associated with a DNA sequence encoding for glutathione-stransferase, such as that carried by the human GSTM1 gene (Zhong S. et al., Biochem. J. 291: 41-50 (1993)), in particular for the purpose of potentiating the glutathione transporter function of the composition according to the invention.

With regard to compositions based on peptides, the invention therefore also extends its effects to those which would contain, for the same purposes, glutathione-S-transferase.

Finally, the clinical indications of the invention are not limited to the treatment of cystic fibrosis. The pharmaceutical compositions of the invention are also applicable to other conditions frequently observed in CF-heterozygous patients, for example asthma and polyarthritis and, more generally still to the treatment of conditions attributable to a deficit of the function. of glutathione transporter.

Finally, the DNA molecules according to the invention can be used to induce a glutathione transporter function in animals, for example mice, for the purpose of creating animal models expressing this function and for studying the effect of administered third compounds. to these animal models, whether to further stimulate this glutathione transporter function of their expression products or, on the contrary, to assess to what extent these compounds interfere with the glutathione transporter functions of these expression products .

Claims (31)

1. DNA molecule containing: - either a DNA sequence whose expression product has a sequence peptide in common with part of that of CFTR, this part being essentially free from at least the N-terminal region of the CFTR which normally extends upstream of the MSD region of this last and which includes, in particular the 360 amino acids N terminals of the CFTR, this expression product having a function glutathione transporter, - either a DNA sequence resulting from the modification of the previous one by deletion, substitution or addition of nucleotides, provided that is at least partially preserved the capacity of the expression product corresponding to exert a glutathione transporter function. 2. DNA molecule according to claim 1, characterized in that the expression product of the aforesaid DNA sequence is also essentially free on the side of its C-terminal end, from the amino acid region of the region of CFTR which normally extends downstream of the R region of the latter, and which comprises in particular the 650 C-terminal amino acids of CFTR. 3. DNA molecule according to claim 1 or claim 2, characterized in that the amino acid sequence of the expression product of the aforesaid DNA sequence itself comprises, on the one hand and on the side of its C-terminus, the subsequence of approximately 80 or more amino acid residues which in the CFTR, extends downstream of its 570th amino acid residue and, on the other hand and on the side of its N-terminal end, a sufficient part of the region NBF1 of CFTR, or even of a part upstream thereof, so that the capacity of the expression product of the aforesaid DNA sequence to exercise said glutathione transporter function is retained. 4. DNA molecule containing: - either a DNA sequence whose expression product has a sequence peptide in common with part of that of CFTR, this part being essentially free from at least the N-terminal region of the CFTR which normally extends upstream of the MSD region of this last and which includes, in particular the 360 amino acids N terminals of the CFTR, this expression product exhibiting an affinity for glutathione - either a DNA sequence resulting from the modification of the previous one by deletion, substitution or addition of nucleotides, provided that is at least partially preserved the affinity of the expression product corresponding for glutathione. 5. DNA molecule according to claim 4, characterized in that the expression product of the aforesaid DNA sequence is also essentially free on the side of its C-terminal end, from the amino acid region of the region of the CFTR which extends downstream of the region R of the latter, and which comprises in particular the 650 amino acids C-terminals of the CFTR. 6. DNA molecule according to claim 4 or claim 5, characterized in that the amino acid sequence of the expression product of the aforesaid DNA sequence, itself comprises, on the one hand and on the side of its C-terminus, the subsequence of approximately 80 or more amino acid residues which in the CFTR, extends downstream of its 570th amino acid residue and, on the other hand and on the side of its N-terminal end, a sufficient part of the region NBF1 of CFTR, so that the affinity of said expression product for glutathione is preserved. 7. DNA molecule according to any one of claims 1 to 6, characterized in that the amino acid sequence of the expression polypeptide of the aforesaid DNA sequence itself contains, on the one hand and on the side from its C-terminal end, the sequence NLQPDFSSKLMGCDS (N635 to S649) of CFTR and, on the other hand and in the direction of its N-terminal end, one or more of the following peptide sites: - ANKT (A596 to T599), - ADLVL (A566 to Lus70), - MPGTIKENI (M498 to 1506), - SEGK (S478 to K481), - IERGQ (1448 to Q452), from the CFTR. 8. DNA molecule according to claim 7, characterized in that the amino acid sequence of the expression polypeptide of the aforesaid sequence includes, on the one hand and near its Cterminal end, the sequence NLQPDFSSKLMGCDS (N635 to S649 ) and, on the other hand and near its N-terminal end: - either the ANKT peptide site (A596 to T599), - either the ADLVL peptide site (A566 to L570), - either the MPGTIKENI peptide site (M498 to 1506), - either the SEGK peptide site (S478 to K481), - or the peptide site IERGQ (1448 to Q452), of CFTR. 9. DNA molecule according to any one of claims 1 to 6, characterized in that the essential part of the sequence is essentially delimited by the codons of nucleotides which code respectively for the 430th and the 660th amino acid residues of the CFTR. 10. DNA molecule according to claim 8 or claim 9, characterized in that the amino acid sequence of the expression product of the aforesaid DNA sequence also includes downstream of the site. NLQPDFSSKLMGCDS (N635 to S649) a segment of 10 to 20 amino acid residues whose sequence corresponds to that which follows this same site also in the CFTR or even upstream of the aforesaid site near its N-terminal end, a 10-20 amino acid residue segment that precedes this same site also in CFTR, or both segments at the same time. 11. A DNA molecule whose nucleotide sequence coincides with the DNA sequence, as defined in any one of claims 1 to 10. 12. DNA molecule according to any one of claims 1 to 11, characterized in that its expression product is soluble, in particular at a rate of at least 0.5 mg / ml of water, for example of solute. physiological, preferably at least 1 mg / ml physiological saline. 13. DNA molecule according to any one of claims 1 to 12, characterized in that it is formed of a DNA of recombination between the aforesaid DNA sequence and a distinct sequence, the whole encoding a molecule. fusion retaining the biological properties of the expression product of the first. 14. DNA molecule according to any one of claims 1 to 12, characterized in that it consists of an expression vector comprising appropriate regulatory elements, said nucleic acid sequence being found therein. state of insertion sequence placed under the control of these regulatory elements, the latter then authorizing the expression of said nucleic acid sequence in a competent cellular organism. 15. DNA molecule according to claim 14, characterized in that the competent cellular organism comprises human cells, in particular epithelial or hepatic cells. 16. DNA molecule according to claim 14 or claim 15, characterized in that the expression vector is a vector of the adenovirus or retrovirus type, or of viruses which are related to them and functionally analogous to them. 17. A DNA molecule according to any one of claims 1 to 16 for application in gene therapy. 18. Cellular host, characterized in that it contains the DNA molecule according to any one of claims 1 to 17 in a form allowing its expression within this cellular host. 19. A composition containing the DNA molecule according to any one of claims 1 to 17, associated with a vehicle allowing its penetration into human cells and its expression therein. 20. Composition according to claim 19, characterized in that the DNA molecule is associated with a cationic lipid vehicle promoting this expression. 21. A composition according to claim 19 or claim 20, characterized in that the aforesaid DNA molecule is associated with a pharmaceutically acceptable vehicle allowing its administration in vivo by the general route. 22. A composition according to claim 19 or claim 20, characterized in that the aforesaid DNA molecule is associated with a pharmaceutically acceptable vehicle allowing its administration in vivo by the systemic route via the respiratory tract. 23. Composition according to any one of claims 19 to 22, characterized in that it further comprises a DNA molecule encoding glutathione-S-transferase, and this in a form allowing its expression in vivo. 24. Use of the DNA molecule according to any one of claims 1 to 17 or of the composition according to any one of claims 19 to 23, for the production of a pharmaceutical composition suitable for the treatment of cystic fibrosis. 25. A polypeptide the sequence of which coincides with that of the product of expression of the DNA sequence as defined in any one of claims 1 to 14. 26. A composition containing the polypeptide of claim 25 in association with a pharmaceutically acceptable carrier. 27. A composition according to claim 26, characterized in that the pharmaceutically acceptable vehicle which allows administration by the systemic route. 28. A composition according to claim 26, characterized in that the pharmaceutically acceptable carrier is of a type which allows administration via the respiratory tract. 29. A composition according to claim 28, characterized in that it is releasable in the form of aerosols or other form of product which can be administered by inhalation. 30. Composition according to any one of claims 26 to 29, characterized in that it also contains giutathione-S-transferase. 31. Use of the composition of the polypeptide according to claim 25 or according to any one of claims 26 to 29 for the production of medicaments for the treatment of cystic fibrosis.