JP2001512010A - Formulation for inducing glutathione-S-transferase function for gene therapy and protein therapy of pancreatic fibrosis - Google Patents

Abstract

  (57) [Summary] The present invention relates to a DNA whose expression product contains a common peptide sequence of CFTR, in particular, residues 430 to 660 thereof, and a DNA sequence having a glutathione transport function. The DNA molecule is useful for construction of a vector useful for gene therapy of cystic fibrosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Pancreatic fibrosis, sometimes referred to as cystic fibrosis, is described in the English expression "Cystic Fibro.
sis Transmembrane Conductance Regulator (pancreatic cystic fibrosis transmembrane conductance regulator) is abbreviated as being caused by mutations in the gene encoding a protein called CFTR.

The nuclear sequence encoding this CFTR protein with 1480 amino acid residues is described in Riordan J. R et al., "Identification of Pancreatic Cystic Gene: Cloning and Characterization of Complementary DNA" (1989). It was cloned and identified by Science 245: 1066-1072. The sequence coding for the CFTR protein is also Rior
dan himself is one of the co-inventors of PCT application WO / 1022796, published on March 7, 1981, in FIG. It is reproduced in 1. This PCT application and others that follow, in fact, describe the cloning of a coding sequence and its expression in a number of organisms.

[0003] According to the assumptions that appear repeatedly in the scientific literature, normal CFTR proteins are:
It has a function called “chlorine channel” or “chlorine ion channel”. In other words, in other words, transport of chloride ions (Cl ⁻ ) out of the epithelial cells and out of the epithelial cells under the action of its phosphorylation by protein kinase or protein kinase C which is dependent on cyclic AMP (cAMP) It seems to adjust. (Riordan, JR et al. (1989) Science 245: 1066-1073; Frizzell, R.A.
et al. (1986) Science 233: 558-560; Welsh, MJ and Liedtke, CM (198
6) Nature 322: 467: Li, M. et al. (1988) Nature 331: 358-360; Hwang, TC
et al. (1989) Science 244: 1351-1353)

[0004] Ko et al., (1997) Biochemistry 36: 505, which refers to previous authors on their own.
At 37 ° C, the CFTR protein also translocates from the endoplasmic reticulum to the Golgi apparatus and from there into the plasma membrane, as evoked by 3-5064.

[0005] In patients with pancreatic fibrosis, a number of mutations in CFTR have been observed. Epidemiological studies performed on patient populations show that in about 70% of cases CF (Cyst in English)
ic Fibrosis) or a deletion of the three nucleotides encoding for the phenylalanine residue at position 508 of the amino acid sequence of CFTR. The site of this mutation is more particularly the inclusion of the first fixed domain of ATP in the CFTR and, according to Ko et al., Cited previously, from phenylalanine residue 433 (F ₄₃₃ ) to serine 589 residue. (S ₅₈₉ ), which is localized in a so-called NBF1 (English expression “Nucleotide Binding Fold 1”, abbreviation of “nucleotide folding region”) region that extends to (S ₅₈₉ ). This mutation often results in the blockade of the mutated CFTR (called ΔF508) in the organelles (Golgi, endoplasmic reticulum) and then before the post-translational mutation can be achieved. It is thought to be responsible for impaired folding of the NBF1 domain in CF-deficient cells of patients, a disorder that will cause its destruction. And, in support of the diagnosis of pancreatic fibrosis, the so-called “sweat” test, which measures the secretion of chlorine in biological fluids from the subject patient, is trying to detect just the “chlorine channel” function. It is missing.

[0006] The correlations often made between the clinical phenomena observed in a large number of patients and the mutations observed in the gene coding for CFTR are then established in CF-deficient cells, especially in patients. A gene therapy-based method aimed at transferring the DNA encoding the "perfect" CFTR into cells in a manner that also allows their expression in epithelial cells of the human respiratory tract It is the basis of methods that have already been proposed for the treatment of pancreatic fibrosis.

[0007] Thus, studies using complexes of DNA encoding CFTR and cationic lipids have been performed by the intratracheal route (Yoshim) in order to gain its direct acceptance in the lung.
ura et al, Nucleic Acid Research, 20: 3233-3240, 1992) or alternatively by aerosol (Stribling et al., Proc, Natl. Acad. Sci, 89: 11277-11281.
1992). Similarly, it was also confirmed that administration of a gene encoding CFTR complexed with cationic lipids to a model mouse suffering from pancreatic fibrosis has an effect of repairing chloride channel dysfunction (Hyde et al.
al., Nature 362: 250-255, 1993).

For example, it has been proposed in the international applications WO 95/13365 and WO 96/13598 to induce the expression of the complete exogenous CFTR gene in CF cells of the patient to be treated. It is to be noted here that the gene is, for example, a vector, such as a genetically modified adenovirus or retrovirus, which is preferably defective but still capable of infecting these cells and transferring the CFTR gene thereto. The CFTR gene is then pre-incorporated under the control of a suitable regulator, where it can express itself under the control of said regulator.

[0009] These methods have proven to be at least inadequate, if not generally ineffective. As the authors of PCT application WO 95/25796 point out, this type of viral vector harbors, ie, packages, foreign or exogenous genes of a limited size of at most 4.5 kb. Can not. However, the DNA encoding for native CFTR reaches this critical size, thus regaining its arbitrary expression in infected cells under the control of the corresponding viral promoter.

This problem is accompanied by another problem which is apparently larger, namely, that the expression product is poorly soluble in the cytoplasmic environment of the cell in question. Ultimately, this also adds to the promising immunogenic nature of the high molecular weight expression products that are optionally formed.

[0011] Similarly, the authors of application WO 95/25796 have proposed replacing the entire gene with a gene in which a significant portion of the C-terminal region of CFTR has been truncated. Here, however, this deletion of the C-terminal region does not involve the loss of the biological properties of the chloride channel type, which is a characteristic of natural human CFTR, and also those properties that are functionally similar to all recombinant CFTR. Is a condition.

According to the authors of the application, the recombinant truncated proteins which can still fulfill their function must usually include:-about amino acid residues 76 to 360 (N of the CFTR protein) MSD (meaning "Membrane Spanning Domain" or "MSB"
Abbreviation for "domain extending through the membrane")-an MBD1 domain extending between about residues 360 to 708 of all CFTR (of the English expression "Nucleotide Binding Domain-1" or "nucleotide binding domain-1"). Abbreviation) or NBF1 domain; and finally: at rest, cAMP-dependent protein kinase or protein kinase C
A regulatory domain R extending from about CFTR amino acid residues 590 to 830, which is considered to ensure the closure and opening of chloride channels under the action of phosphorylation by R.

[0013] In particular, a recombinant protein having 884 N-terminal amino acids of CFTR further fulfills functions required for chloride ion channels and functions required for regulators of this channel.

However, assuming that better expression could actually be obtained with such a CFTR in which its C-terminal region has been deleted, inevitably more important excision of the expression product that may be obtained. Is expected to result in a sustained immunogenicity that makes its use in gene therapy trials more than accidental.

[0015] Similarly, this proposal for gene therapy, which would utilize only the truncated gene of CFTR that meets the conditions described in patent WO 95/25796, would further increase the efficacy of the treatment by making the functional CFTR protein more healthy You can see that this is the only function of the chloride channel that is restored to healthy subjects.

However, even though this impaired chloride transport actually constitutes the clinical manifestation often observed in patients with pancreatic fibrosis, the other manifestations that are also observed are But to ignore the fact that they remained unexplained.

The present invention is exactly as the inventors have demonstrated that CFTR itself plays a particularly important role in the active transport of glutathione (GSH), so that pancreatic fibrosis also plays a glutathione transporter function Due to the fact that most of the treatment of pancreatic fibrosis is done through different therapeutic approaches, because it could be linked in a significant way to mutations or other causes that would interfere with the ability of CFTR. It comes from confirmation.

Accordingly, the present invention relates to a DNA fragment whose sequence, including the sequence derived from the gene encoding CFTR, is selected from those encoding the active transport function of glutathione. The invention particularly relates to fragments whose size in gene therapy for the treatment of pancreatic fibrosis has been reduced even further than the size of DNA fragments already mentioned in the technical or scientific literature.

Indeed, what led the inventor to the invention on which the invention itself is based is:
-On the one hand, the discovery of the special properties of fusion polypeptides containing a part of the sequence of CFTR, generated in the frame of a study, with the aim of studying the three-dimensional structure of the sequence coding for the NBF-1 domain And, on the other hand, the so-called MRP (English expression "Multi-drug Resistance Associat
Observations on the sustained clinical improvement of symptoms associated with pancreatic fibrosis in patients who are also cancer patients receiving chemotherapy with overexpression of "ed Protein" (multidrug resistance-related protein).

For the study of the three-dimensional structure of the sequence encoding the NBF1 domain, with particular aim to study the effect of the mutation ΔF508, which is presumed to be responsible for the impaired folding of CFTR in the NBF1 domain. Previously involved in obtaining large amounts of pure, soluble and stable protein (about tens of mg). Such amounts are not obtainable by purification of the native protein, and we therefore have the ability to clone NBF- in a live expression system (E-Coli) in the form of a fusion protein with GST (glutathione-S-transferase). 1 (by its original definition). This selection was initially aimed at exploiting the specific recognition ability of glutathione S-transferase by glutathione to purify the required protein (GST-NBF1) from whole bacterial proteins. For this purpose, NB derived from the CFTR gene
Sequence containing the nucleotide sequence coding for F1 sequence (SEQ R ₄₅₀ -I _{5 86),} of the inserted sequences in bacteria transformed and transformed bacteria E. CoIi, expression in the bacteria capable Plasmid, for example, a pGEX-KT type plasmid. Culturing the thus transformed E. coli strain actually resulted in overproduction of the NBF-1 fusion protein and glutathione-S-transferase (GST). The yield obtained is 5 per liter of culture.
mg. The resulting soluble protein was sequenced. This protein was recognized by the anti-NBF1 antibody and also immobilized ATP. Cleavage with thrombin allowed the preparation of a free NBF1 domain exhibiting the same properties. However, it is not possible to separate the GST and NBF1 domains on an affinity column implanted with glutathione (GSH). That is, although completely separated on the electrophoresis gel, the two proteins NBF-1 and GST co-eluted on the GSH immobilized column.

The activity of the fusion protein was confirmed by fluorescence: in fact, ATP
The affinity for the fluorescent derivative (TNP-ATP) was observed.

However, many purification studies of the NBF1 domain separated from GST by thrombin splitting remained fruitless. In addition, GST-NBF1
The protein could be selectively and quantitatively resolved, but it was then revealed that the NBF1 domain alone was unstable. This domain had a strong tendency to form aggregates.

These results have led the inventors to re-consider and evaluate the definition of the NBF1 domain, as described by Riordan. Indeed, the definition of a domain is characterized by a unique fold that codes for its function and its stability is such that its stability is independent of the complete protein to which this domain belongs (here CFTR). Must support. To ensure this fold, the limits chosen for NBF1 must include the entire secondary structure (helix and sheet) elements necessary for stabilization of the domain in its native conformation.

However, CFTR is a transporter that forms one of a larger family of membrane proteins (A11,96) found in both eukaryotes and prokaryotes.
It belongs to the family of BC (ATP binding cassette). These proteins are generally
It is an active transporter that hydrolyzes ATP to provide the necessary chemical potential for transport. In eukaryotes, these proteins transport molecules (ions, vitamins, peptides, sugars, drugs, etc.) across all membranes. Their global organization exhibits common properties: the region (TM) and the nucleotide anchoring domain that participate in the selection of the chemical entity to be transported. These genes are often derived from fusions between two “half genes”. Then the comprehensive topology is (T
M1)-(NBF-1)-(TM2) -NBF2). And in fact, CFT
R belongs more particularly to the subfamily of CFTR / MRPs and has about 50% sequence similarity to the human MRP protein (multidrug resistance associated protein). This likewise confers resistance to the cadmium ion (Tom. 96) on the yeast Saccharomyces cerevisiae.
It is also very similar to the YCF1 protein (yeast cadmium resistance factor 1) provided to visiae. To a lesser extent, this is also true for the yeast Saccharomyces
STE6 (of subfamily MDR / TAP), which transports the "factor a" pheromone in cerevisiae (Tom 94), and human MDR (multidrug resistance protein) as well as A
It is also close to bovine ATPase-F1, the only structural protein known within the family of BC proteins (Abr 94).

Similarly, the NTR1 domain of CFTR has its N
The BF domain was chosen by the inventors because the structure of bovine ATPase is known. From this selection, which is discussed in two recent publications (Ann, 97a, Ann. 97b), a method developed in (Pat. 96) that allows, among other things, the bovine ATP N, constructed by homology with the structure of Fase-F1
A three-dimensional structural model for BF1 is provided: 1. Understanding the phenotype associated with the mutation ΔF508, unlike the previous models (Hyd 90, Mim 91); Consensus sequence LS conserved at a high level among ABC proteins
2. Understand the main role of GGQ; The NBF1 domain (hereafter referred to as “reviewed”) which has been considered to extend from the amino acid residue at about position 430 of CFTR to about residue 650 (only about 70 residues protrude on domain R in its initial determination). NBF1 ")
Redetermining the limits of

This last point comes directly from the hypothesis of a comparable fold for the bovine ATPase-F1 and the NBF region of CFTR. The main argument is that the family of ABC transporters forms a homogenous group of components from one phylogenetic branch, distinct from other proteins that bind ATP. Within this frame these proteins must exhibit an equivalent fold within the conserved region (typically within the "basic brick" NBF). It is then justifiable to model one structure of these proteins from the known structure of another protein.

[0027] Thus, the nucleotides of bovine ATPase-F1 (Abraha) according to the method described by Annaeau et al. (FEBS Letters, (1997) 407: 303-308).
ms JP, Jeslie AGW, Lutter R., Walker JF (1994) Nature 370; 621-6
Each structure models built for NBF-1 domain of CFTR by homology to known structures of anchoring domain of _{28) L 453 -S 456, N} 505 -S 511, N 53 8 ~G 542, D 567 ~ Β with six parallel strands D ₅₇₂ , R _{600 to} V ₆₀₃
The structure comprises a hydrophobic center. These lamellas alternate with six α helices and are organized on either side of the mid-plane of the central β structure, as shown in the paper of FEB Letters, Vol. 407, p303-308, 1997. . A loop on the surface of the domain connects the helix to the sheet.

In fact, the expression product of the fragment corresponding to the gene encoding for CFTR (a fragment that itself forms part of the present invention) has been shown to be stable, indicating that the new NBF1 Domain (that is, reviewed N
Within the BF1 domain), a fusion DNA encoding E. coli in the polyhistidine system, ie, for the “reviewed NBF1” (which contains 44 amino acids of CFTR).
A fragment encoding a polypeptide extending from residue 8 to residue 650) and a tail containing only six histidine residues (where it is "
(Which can be considered "produced alone"). The resulting production rate is significantly higher than on the preceding fragment,
For the polyhistidine system, it has reached about 200 mg per liter of culture (from initial testing before any optimization). This purified fragment “Reviewed NBF1” was purified in large quantities in the form of a soluble and stable protein.

CFT that can be actually superimposed at the glutathione fixation site in GST
The possibility of a glutathione anchoring site within NBF1 of R
A protein that exhibits an anchoring site and acts as a catalyst in glutathione nucleophilic attack
Known structure of GST (glutathione-S-transferase)
Is proved by comparing the models of Potentially involved in glutathione fixation
The conferring NBF1 residue forms a crack at the end of the involved strand in the beta structure
(See FEBS letter paper). More precisely, these residues are in the loop at the N-terminus of sheets 1-4 of the structure of NBF1.
It is positioned. These loops consist of residues I₄₄₈−Q₄₅₂(IERSQ), S ₄₇₈ -K₄₈₁(SEGK), M₄₉₈-I₅₀₆(MPGTIKENI), A₅₆₆-L₅₆₈ (ADLVL), A₅₉₆-T₅₉₉(ANKT).

[0030] The new definition of NBF1 and the model of the three-dimensional structure that was constructed suggest that the CFTF of CFTR was suggested by the unexpected fact that NBF1 is retained on a glutathione column even after resolution of the fusion protein GST-NF1. Enabled the inventor to propose a new function for In fact, by comparing this model with the known three-dimensional structure of GST, a protein that exhibits glutathione fixation sites and acts as a catalyst for glutathione nucleophilic attack (Lim, 94), potential glutathione fixation sites can be determined. The existence of is proved.

The two ABC proteins closest to CFTR (human MRP and yeast YCF1
Functional information about) seems to reinforce this hypothesis. Indeed, it has now been demonstrated that MRP exports drugs via glutathione in the form of direct adducts of "glutathione drugs" or by simultaneous or sequential immobilization of glutathione and drug (Zarn. 95). Similarly, YCF1 transports cadmium ions in the form of a glutathione double adduct. Thus, it is believed that CFTR can be a "glutathion pump" such as MRP and YCF1.

Incidentally, human MRP and MDR proteins are involved in the phenomenon of multiple resistance to antitumor drugs (Dean et al., Current Opinion in Genetic & Developmen).
t S, 779-785, 1995). These proteins have the ability to actively transport these drugs, and thus participate in the detoxification mechanism of the cell. Their overexpression is induced by the drug itself, which is a major cause of failure of cancer treatment with chemotherapy. However, in recent years it has been shown that CFTR overexpression could also be induced by the drug itself, and that this overexpression conferred a multiple resistance phenotype (LI. 95).

[0033] From this as well, the inventors hypothesized that the function of glutathione transporter could be important and that the mutations that could result in it would explain many symptoms of pancreatic fibrosis. It was reached. "Glutathione pump"
The role of CFTR as well (in addition to its known role of chloride channels) also allows for a more comprehensive understanding of pancreatic fibrosis (Sto. 97).

Glutathione plays a central role in controlling inflammatory responses by protecting cells from free radical attack by polynuclear neutrophils, and furthermore, it protects against oxidants such as free radicals or peroxides. Glutathione transport dysfunction, as well as known intervening interventions, should better account for the extremely high number of granulocytes and / or chronic inflammatory responses often found in the airways of patients with pancreatic fibrosis right.

Similarly, suffering from generally severe pancreatic fibrosis are those organs (pulmonary, intestinal, colon) in which glutathione is normally secreted and in which it performs a detoxifying function.

[0036] Several clinical observations are consistent with the hypothesis underlying the present invention. GSH rates are indeed reduced in bronchial mucus and serum of pancreatic fibrosis patients. The organs in which CFTR is best expressed are those in which GSH plays an important role and are actively transported,
That is, the pancreas, lung, liver, and kidney. The detoxification function of the liver, a function in which GSH may play a central role, appears to be unusual. This manifests itself, for example, in the patient's particular sensitivity to heavy metals, such as mercury. Oxidative stress phenomena have also been observed long ago at the lung level. N-acetylcysteine, a drug that has been utilized in an advantageous manner for patients, is acetyl-depleted by penetrating into cells, thus providing cysteine (an amino acid that is anti-essential in humans).

And, in fact, the truth of the hypothesis is now already (with chronic signs from birth) 2
This can be supported by the sustained improvement seen in pancreatic fibrosis in a 9-year-old patient with pancreatic fibrosis. Treated patients have CFTR on one allele
Exon 12 and G6 in exon 13 on another allele
73X. The patient has had all the chronic signs associated with the disease since birth in 1968: positive sweat tests, recurrent sinusitis, recurrent bronchitis, nasal polyps and the like. In 1989, this patient was identified as Pseudomonas aerugino, who is classic in pancreatic fibrosis and is usually virtually final in these patients.
showed sa infection. Further, in April 1993, a diagnosis of fibrosarcoma of the left thigh was made. The patient underwent radiation therapy and surgery, followed by chemotherapy from July to October 1993 and then new treatment in January 1994.

More specifically, the antitumor treatment was as follows: granisetron for nausea prevention methylprednisolone as mesna epirubicin. 110 mg for 2 days (Days 1 and 2)-Ifosfamide. 3.3 g for 5 days (1st to 5th days)-Fill glass time 8 days. 300 μg daily (Days 8 to 15)

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

The anti-cancer treatment was effective. Similarly, the following improvements were observed in the clinical tables of pancreatic fibrosis after chemotherapy treatment:-the patient's respiratory status improved significantly;-the patient's infection with Pseudomonas aeruginosa disappeared:-the patient Respiratory parameters reached about 75% of theory; the patient returned to a respiratory state approximately equivalent to that presented at the beginning of his adolescence. -The patient has declared that pancreatic fibrosis has healed.

The perspiration test performed on patients in the beginning of 1997 has always been found to be very positive, meaning that the function of chloride channels has not been restored in this patient. are doing. This confirmation, coupled with the fact that the patient has declared himself cured of pancreatic fibrosis, can conclude that other basic functions have been restored by chemotherapy. From the above, it can be inferred that what significantly increased here is the transport function of glutathione.

As already indicated, the present invention thus more particularly relates to polypeptides having a DNA fragment corresponding to a part of the gene encoding CFTR, which part also has an active transport function for glutathione. For encoding DNA fragments. In the following, DNA sequences are generally defined on the basis of their corresponding peptide sequences, and these peptide sequences are in each case generally referred to by the expression "expression product" of the DNA sequences of the invention for the sake of language. Is done. These DNA sequences are, of course, notably essentially the same as those already mentioned in Riordan
et al., as well as the CFT published in PCT application WO 91/02796.
R, which is a reproduction of the sequence of the gene coding for R. It can be reconstructed by the reader based on 1.

In the following definitions of the DNA sequence or its expression product, the DN to which the present invention relates
Frequent reference is made to regions "downstream" or "upstream" of the A sequence or its expression product. FIG. A given DNA sequence within the gene encoding the CFTR represented in No. 1 or the "upstream" region of the corresponding expression product
It will be readily apparent that the sequence is located at the 5 'end of the sequence or N-terminal upstream of the amino acid sequence of the corresponding expression product. Conversely, the “downstream” region is
This is a region located beyond the 3 'end of the NA sequence or beyond the C-terminus of the amino acid sequence of the corresponding expression product.

Evidently, the one letter designation (in the amino acid symbolic identification system utilizing a unique letter for each amino acid) followed by the index “n” is counted from the first amino acid residue of CFTR. The attached FIG. This refers to the "n-th" amino acid derived by considering 1.

Generally speaking, the longest DNA molecules according to the invention contain a DNA sequence whose expression product has a peptide sequence in common with a part of the sequence of CFTR, this part comprising the basic In addition, usually extending upstream of the MSD region of CFTR and especially CFTR
Not contain at least the N-terminal region of the CFTR containing the 360 N-terminal amino acids of the above, or otherwise, as a variant, at least partially conserved the ability of the corresponding expressed material to perform glutathione transporter function And any DNA sequence resulting from modification of the preceding sequence by deletion, substitution or addition of nucleotides.

“Glutathione transport factor compounds”, according to the present invention, refer to compounds belonging to the already reported family of ABC transporters that perform their transport function via glutathione. Similarly, any fragment or analog of such a transporter that is the result of one or more mutations that preserve the transport properties via glutathione is also meant.

Glutathione is an adduct of glutathione with glutathione itself or other compounds. It can be a natural adduct such as leukotriene, or a detoxifying adduct with heavy metals, strong oxidants (free radicals, peroxides ...), or a drug.

In particular, the ability of the expression product of the DNA sequence according to the invention to perform glutathione transporter function is described in "Overexpression of the cystic fibrosis transmembrane regula".
tor in NIH3T3 cells lowers membrane potential and inatracellular pH and
The paper entitled "confers a multidrug resistance phenotype" (overexpression of transmembrane regulators of cystic fibrosis in NIH3T3 cells lowers membrane potential and intracellular pH and confer a multidrug resistance phenotype) ( Biophysical Journal (1995) 69: 883-8
By performing experimental techniques already used to assess the same type of induction under the action of anti-tumor drugs, such as described and utilized by Wey LY et al. It can be assessed by their ability to induce CFTR production in epithelial cells in vitro.

Similarly, under the control of regulators that allow the transcription and translation of that DNA sequence in epithelial cells, especially under the conditions described by Yoshimura et al, in the aforementioned article,
Method for determining the proportion of mRNA corresponding to CFTR in epithelial cells previously collected from the body of an experimental mammal suffering from pancreatic fibrosis, such as a mouse to which a DNA molecule according to the invention to be tested has been previously administered Can also be used.

TgH obtained from homologous recombination at the level of exon 10 of the Cftr gene
The (Une) type of mouse (neomycin resistance gene insertion) is one interesting model for this study. Heterozygous animals do not show any modification of the phenotype. Homozygous mutants show abnormalities similar to human pancreatic fibrosis in young mice (Koller BH et al. Science, 248, 1227-1229).
These mice are suitable models because they suffer from intestinal obstruction and die even before they reach the age of maturity, thus highlighting a study model of inflammatory exacerbations at the level of this organ.

Expression vectors available in this test and which can be administered by systemic routes include, for example, the adenovirus of the Av1CF2 type (Genetic Therapy Inc.), in which the coding sequence to be studied is replaced by a sequence coding for the entire CFTR.
Gaitherbourg, MD. USA).

At this time, the proof of the expression of the glutathione transporter function is described in, for example, Resp. J. 1997 “M. MDR1-PgP170 expression in human Bronchus” (F. MDR-1 in human bronchi).
-PgP170 expression) untreated after quantification of mRNA in relation to an internal standard for RT-PCR utilizing β2-microglobulin mRNA rate according to the method of Lechapt-Zalcman E. et al. Assessed by the increase in the amount of mRNA induced in the treated mice compared to that found in control pancreatic fibrosis mice. Moreover, since the amount of MDR protein is high in intestinal epithelium, transfection of the above-described experimental mammal with the CFTR fragment under study should induce absorption of the exacerbated inflammatory condition at the intestinal level.

As variants, the tests already questioned above can also be used for the purpose of defining the DNA molecules according to the invention. Included within the framework of the present invention are any DNA molecules which fulfill the above-mentioned conditions and are characterized by a conservation of the affinity of the corresponding expression product for glutathione, wherein the affinity of the DNA molecule in question with thrombin Can be substantiated by performing a test consisting of contacting the glutathione-implanted affinity column with the affinity column on which glutathione is to be collected, splitting the expression product into a C-terminal fragment and an N-terminal fragment, and The affinity of the expression product for glutathione derives from its ability to immobilize on and elute from this activity column, similar to that of glutathione-S-transferase.

Preferably, however, the DNA molecule is shorter, in particular a CFTR whose expression product likewise essentially extends on its C-terminal side, usually downstream of the R region of this CFTR. Is characterized in that it does not include the amino acid sequence of the region of

Similarly, the amino acid sequence of the expression product of said DNA sequence is itself, on the one hand, about 80 amino acid residues extending at its C-terminal side and downstream of its 570th amino acid residue in the CFTR. A subsequence of the glutathione transporter, which in the direction of its N-terminus contains a subsequence of the group (which was previously considered to belong to the R region and is now part of the "reviewed NBF1" region) Said DN that performs the function
It is sufficient to include the CFTR and thus only a portion of the NBF1 region, so that the ability of the expression product of the A sequence is retained.

Thus, generally speaking, the DNA sequence according to the invention basically consists of its C
Extending from the end to beyond the putative fixation site of glutathione (naturally, the direction of reverse reading of the amino acid sequence of this "reviewed NBF1", that is, the direction from its C-terminal to its N-terminal) "reviewed NBF1" of CFTR The coding region for all or part of the region.

A preferred DNA molecule is a DNA molecule whose expression product itself has an amino acid sequence extending between amino acid residues 430 and 660 of CFTR.

Preferred DNA molecules according to the invention include the amino acid sequence of the respective expression product itself, while the sequence N of CFTR on its C-terminal side
LQPDFSSKLMGCDS comprises (N ₆₃₅ ~S _649), on the other hand of CFTR in the direction of the N-terminus _{- ANKT (A 596 ~T 599)} , - ADLYL (A 566 ~L 570), - MPGTIKENI (M 498 ~I 506) _{, - SEGK (S 478 ~K 481} ), - IERGQ (I 448 ~Q 452). A DNA molecule comprising one or more of the following peptide sites:

In particular, a DNA molecule according to the invention is characterized in that the amino acid sequence of its expression product contains, on the one hand, the sequence NLQPDFSSKLMGCDS (N ₆₃₅ -S ₆₄₉ ) near its C-terminus and, on the other hand, near its N-terminus -Peptide site of CFTR: ANKT (A _{596 to} T ₅₉₉ ),-Peptide site: ADLYL (A _{566 to} L ₅₇₀ ),-Peptide site: MPGTIKENI (M _{498 to} I ₅₀₆ ),-Peptide site: SEGK (S ₄₇₈₎ ~K _481), - a peptide _{site: IERGQ (I 448 ~Q 452)} . Can be selected from those having a DNA sequence characterized by including

Accordingly, the amino acid sequence of the expression product of the DNA sequence may further comprise the site NLQP
Downstream of DFSSKLMGCDS (N ₆₃₅ ~S _649), or to entailment segments of 10-20 amino acid residues having a sequence corresponding to the subsequent sequence even in CFTR on the same site, further their N-terminal nearby Upstream of said site, the CFTR may also include a segment of 10-20 amino acid residues preceding this same site, or these two segments may be included simultaneously. This segment may also be replaced by a binding oligonucleotide (linker) that contains a restriction site that facilitates its incorporation into a vector intended for transfer into suitable competent cells.

In general, preferred DNAs according to the invention are such that their expression products are soluble in water or physiological serum, in particular in the form of physiological solutes, at a rate of at least 0.5 mg, preferably at least 1 mg per ml of water. DNA.

The invention is, of course, characterized by any recombinant DNA molecule, in particular a molecule according to the invention, in which the DNA sequence as described above has been recombined with an exogenous nucleic acid element. It relates to a recombinant molecule encoding a fusion protein that still exhibits the basic biological properties as described above of the expression product of a DNA sequence.

Another category of DNA molecules according to the present invention is that exogenous nucleic acid elements are comprised of expression vectors containing appropriate regulatory elements, wherein said nucleic acid sequences are placed under the control of these regulatory elements. DNA molecules that are found in the inserted insertion sequence, where these regulatory elements allow expression of the nucleic acid sequence in competent cell organisms.

What is at stake here is, in particular, an expression vector capable of transforming a competent cell organism for the purpose of ensuring the expression of the DNA sequence, which is a feature of the present invention. Can optionally be recovered from the cells in culture and even from the medium. Of course, the invention is not restricted to a vector modified by a DNA sequence according to the invention which can only be used for gene therapy purposes. The invention can likewise be used for the production of the corresponding expression product in cells in culture, in particular for the purpose of producing a recombinant expression product which can itself be directly used as described below. It extends its potency to all other forms of vectors.

For examples of expression vectors already described for expression of the whole CFTR gene, see WO 91/02796, WO 91/10374, WO 92 /
No. 05273 and WO 93/17040, and also the previously mentioned application WO 95/25796 which describes vectors which can be used for the expression of CFTR with truncated C-terminal regions. Please refer to. These different vectors are, of course, likewise DNA according to the invention.
It can also be used for expression of sequences. The description of these patent applications should not be regarded as forming part of the description of the present application with respect to the variants of the expression vectors that can be used for the production of the expression product of the DNA molecule according to the invention. No.

Vectors which allow the transformation of human cells, especially epithelial cells, are particularly advantageous. Particularly preferred expression vectors are viruses of the adenovirus or retrovirus type, and even closely related viruses (AAV virus), whose use has already been considered for the implementation of gene therapy applied to the treatment of pancreatic fibrosis. ). It is also worth mentioning that in-vivo in mammalian cells
A vector available for the expression of a gene encoding CFTR, intended for application in gene therapy in the form of a transcription and expression set comprising a sequence encoding CFTR, which is recombined with a functional regulatory sequence at International patents WO95 / 13365 and WO96 / 13598 specifically describe the technology.
Again, these techniques are based on (DNA encoding for all CFTRs).
(Instead of the sequence) can be used for the expression of the DNA sequence according to the invention, and therefore the description of these applications likewise relates to the description of the technology and the vectors available for that purpose. Must be considered part of the description of

The in-vivo-induced expression of a DNA molecule according to the invention, which is all responsible for encoding one polypeptide with glutathione transporter function, is associated with endogenous CFTR and pancreatic A deficiency in this regard of this CFTR due to a mutation that is believed to be at least part of the clinical manifestation of fibrosis can be corrected for, and more generally the endogenous CFTR Stimulation of sexual expression and, while doing so, can even partially restore chloride channel function, unless other mutations affecting this endogenous CFTR completely abolish the function of the chloride channel. Everything is written.

The expected therapeutic effect of gene therapy utilizing DNA sequences that meet the above definition is that the expression products of these DNA sequences are small, soluble, stable, and have their unique immunogenicity reduced. And thus can be so large that they do not actually exist.

In general, the present invention also provides a "vectorized" form, optionally together with a pharmaceutical vehicle that already favors its entry into and expression in human cells. The invention also relates to all pharmaceutical compositions which bind a DNA molecule according to the invention.

What is at issue here is whether directly or indirectly in anti-pancreatic fibrosis therapy (
Any type of cell that can be targeted by the molecule used (for example, for polypeptides produced in situ, as in gene therapy). For example, epithelial cells, especially in local treatment, for example at the pulmonary level, or also hepatocytes, which may intervene in the beneficial effects of treatment, for example, especially when consequent to treatment by the systemic route All other cells. This systemic route of treatment is particularly advantageous within the framework of the present invention, taking into account the mode of action utilized by the present invention, i.e., improved glutathione transporter function.

Thus, the present invention also provides for the targeting of selected cells under the control of a regulator capable of controlling the transcription and translation of its coding sequence in mammalian, preferably human, cells. It also relates to any form of a pharmaceutical composition suitable for the treatment of pancreatic fibrosis, comprising a DNA cassette comprising a DNA molecule according to the present invention in association with a suitable pharmaceutically acceptable vehicle.

A first type of preferred composition is a pharmaceutical composition adapted for administration by the systemic route for the reasons mentioned above. Other composition forms are adapted for administration via the respiratory tract, where the preferred compositions according to the invention are particularly suitable for the absorption of the composition by inhalation and the targeted lung cells and the composition of these compositions. A composition that can be in the form of an aerosol, spray, or the like that allows direct contact.

Examples of pharmaceutical vehicles, especially of the cationic lipid type, which are particularly suitable for this mode of administration and for similar purposes (associated with gene therapy utilizing whole CFTR) have already been described, for example, in the international applications WO 93/12240, WO 93/12756. Or WO93 /
24640. Of course, they are equally applicable to the construction of pharmaceutical compositions utilizing DNA molecules according to the present invention.

Finally, the invention also relates to the polypeptide itself, characterized by an amino acid sequence as defined above in relation to the DNA sequence which is characteristic of the DNA molecule according to the invention. These polypeptides are particularly useful for transforming cells in culture with a suitable vector containing a DNA sequence encoding these polypeptides under the control of appropriate regulatory elements and expressing the polypeptides obtained from these cell cultures. Is the polypeptide produced in the cells in culture after recovery.

As an example, we have already mentioned by way of example a number of patent applications already published on culture systems whose use has already been described in connection with the production of recombinant CFTR. The same system can advantageously be used for the production of a polypeptide as defined in the present application.

Similarly, the present invention relates to pharmaceutical compositions which bind to said polypeptides a suitable pharmaceutical vehicle of the same type as the ones considered above, especially for pharmaceutical compositions based on DNA molecules.

The present invention is not limited to the sole use of a DNA molecule as defined above, especially with respect to peptide sequences shared with the gene of CFTR. The present invention also relates to a completely different DNA molecule, wherein the expression product which is structurally different from the expression product of a DNA molecule as defined above will also exhibit glutathione transporter function. As well as compositions that are substituted for or added to. In particular, human M that also supports such genetic information
DNA containing the NBF-1 region of a protein of the RP or MDR protein type can be used for this purpose. One example is Cole SPC et al., Sc
There is a DNA sequence encoding human MRP-1 described by ience 258: 1650-1654 (1992). Similarly, replacement of the human MRP protein or a region useful for this purpose with the expression product of the DNA molecule according to the invention as defined above (
Or, similarly, added thereto).

Finally, the present invention relates to a method wherein the active ingredient is a DNA molecule according to the invention or, more generally, a D-encoding polypeptide encoding glutathione ion transporter activity.
It is not limited to the composition constituted by the NA molecule nor the corresponding expression product. The invention likewise relates to compositions in which the active ingredient is associated with others, such as, for example, completely different DNA molecules or optionally their expression products. For example, it has the aim of restoring deficient chloride channel function in treated patients, possibly in a much more prominent manner than a single DNA molecule according to the invention allows, the international application WO 95/95. 25796
Is the truncated DNA or the total DNA encoding for CFTR, or the corresponding expression product. In this way, it is then possible in this manner to ensure, under the most advantageous conditions, simultaneous recovery of glutathione transporter function and chloride channel function in the patient concerned.

Similarly, the present invention relates to compositions of the type described above, such as DN according to the invention.
A molecule, in particular a DNA molecule derived from the gene encoding CFTR, is intended in particular to increase the effect of the glutathione transporter function of the composition according to the invention,
D encoding a glutathione-S-transferase such as that supported by the human GSTM1 gene (Zhong S et al., Biochem. J. 291: 41-50 (1993)).
It also relates to compositions that will be associated with the NA sequence.

As it refers to peptide-based compositions, the present invention likewise extends its effects to those containing glutathione-S-transferase for the same purpose.

Finally, the clinical indications of the present invention are not limited to the treatment of pancreatic fibrosis.
The pharmaceutical compositions of the present invention are also applicable to other diseases that are frequently found in CF heterozygous patients, such as asthma and polyarthritis, and even more generally those caused by glutathione transporter dysfunction. Can also be applied to the treatment of

Finally, the DNA molecule according to the invention induces glutathione transporter function in animals, such as mice, for the purpose of creating an animal model that expresses this function, and it is this glutathione transport of its expression product. Whether to stimulate factor function even more or, conversely, to evaluate how these compounds interfere with the glutathione transporter function of these expression products, these animal models It can be used to study the effect of a third compound administered.

[Brief description of the drawings]

FIG. 1. Basically the papers of Riordan et al. Mentioned above and also PCT application WO 91/0
2A shows a reproduction of the sequence of the gene encoding for CFTR published at 2796.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 1/18 A61K 35:52) C07K 14/47 C12N 15/00 ZNAA C12N 9/10 A61K 37/02 / / (A61K 38/00 37/52 35:52) 35:52) (72) Inventor Barthe, Joel FR-75007, Paris, Avigne-Constant-Cochran 8 (72) Inventor Lalman, Jean-Eve France, F-91120 Pareso, Rue Anatole France 20 (72) Inventor Stven, Veronique France, F-75006 Paris, Rue de Ren, 108 Bis (72) Inventor Anlo, Jean-Philippe France Country, F-75015 Paris, Ryu Dar 4

Claims (31)

[Claims] 1. the expression product has a peptide sequence in common with a part of the peptide sequence of CFTR, which extends essentially upstream of the MSD region of CFTR and in particular the 360 N-terminals of CFTR N of CFTR containing amino acids
A DNA sequence that is at least free of the terminal region and is provided with a glutathione transporter function, or-at least partially retains the ability of the corresponding expression product to perform a glutathione transporter function A DNA sequence obtained as a result of modifying the above sequence by deletion, substitution or addition of nucleotides. 2. The expression product of the DNA sequence also contains a region R of CFTR.
Of CTR, which typically extends downstream of and contains the 650 C-terminal amino acids of CFTR,
2. The DNA molecule according to claim 1, wherein the amino acid region of the FTR region is basically not included on the C-terminal side. 3. The amino acid sequence of the expression product of said DNA sequence is itself, on the one hand, about 80 amino acid residues extending C-terminal to the 570th amino acid residue in the CFTR. A subsequence, on the other hand, on its N-terminal side, a sufficient part of the region NBF1 of CFTR, and thus the part upstream thereof, so that the expression product of the DNA sequence exhibiting the glutathione transporter function is retained. The DNA molecule according to claim 1, wherein the DNA molecule comprises a DNA molecule. 4. The expression product has a peptide sequence in common with a part of the peptide sequence of CFTR, which extends essentially upstream of the MSD region of CFTR and in particular the 360 N-terminals of CFTR A DNA sequence that does not contain at least the N-terminal region of CFTR containing amino acids and the expression product exhibits an affinity for glutathione, or that the affinity of the corresponding expression product for glutathione is at least partially retained. A DNA sequence obtained as a result of modifying said sequence by deletion, substitution or addition of nucleotides, provided that: 5. The expression product of the DNA sequence also contains a CFTR region R.
The DNA molecule according to claim 4, characterized in that the amino acid region of the region of CFTR that extends downstream of and specifically includes the 650 C-terminal amino acids of CFTR is essentially not included on its C-terminal side. 6. The amino acid sequence of the expression product of the DNA sequence itself, on the one hand, on the C-terminal side, approximately 80 amino acid residues extending downstream of the 570th amino acid residue in the CFTR Or, on the other hand, on the N-terminal side thereof, a sufficient part of the region NBF1 of CFTR such that the affinity of the expression product for the glutathione is retained. 6. The DNA molecule according to 5. 7. The amino acid sequence of the expressed polypeptide of said DNA sequence is itself, on the one hand, on the C-terminal side, the sequence NLQPDFSSKLMGCDS of CFTR.
(N ₆₃₅ -S ₆₄₉ ), on the other hand, in the N-terminal direction of CFTR,-ANKT (A _596- T ₅₉₉ ),-ADLYL (A _566- L ₅₇₀ ),-MPGTIKENI (M _498- I ₅₀₆ ), _{- SEGK (S 478 ~K 481)} , - IERGQ (I 448 ~Q 452), characterized in that it comprises one or more of the peptide portion of any one of claims 1 to 6 A DNA molecule as described. 8. The amino acid sequence of the expressed polypeptide of the sequence, on the one hand and entailment its C-terminal nearby sequence NLQPDFSSKLMGCDS the (N ₆₃₅ to S _649), it is near its N-terminus on the other, CFTR peptide sites: ANKT (A _{596 to} T ₅₉₉ ), Peptide site: ADLYL (A _{566 to} L ₅₇₀ ), Peptide site: MPGTIKENI (M _{498 to} I ₅₀₆ ), Peptide site: SEGK (S _{478 to} K ₄₈₁ ), Peptide site: IERGQ ( I _{448 to} Q ₄₅₂ ). 9. The method according to claim 1, wherein the main part of the sequence is essentially limited by nucleotide codons encoding amino acid residues 430 and 660 of CFTR, respectively. A DNA molecule according to claim 1. 10. The amino acid sequence of the expression product of the DNA sequence further comprises a site N
Downstream of LQPDFSSKLMGCDS (N ₆₃₅ -S ₆₄₉ ), it also contains a segment of 10 to 20 amino acid residues having a sequence corresponding to the sequence following this same site in CFTR, or its N-terminal. Close upstream of the site, CFT
10. As in R, a segment of 10 to 20 amino acid residues preceding the same site is included, or these two segments are included simultaneously. DNA molecule. 11. A DNA molecule having a nucleotide sequence corresponding to the DNA sequence according to any one of claims 1 to 10. 12. The expression product may be, for example, at least 0 in saline, for example.
. 12. The DNA molecule according to any one of claims 1 to 11, characterized in that it is soluble at a rate of 5 mg / ml, preferably at least 1 mg / ml in physiological saline. 13. The assembly formed of recombinant DNA between the DNA sequence and a completely different sequence, wherein the aggregate encoding the fusion molecule preserves the biological properties of the former expression product. The DNA according to any one of claims 1 to 12,
molecule. 14. An expression vector comprising appropriate regulatory elements, wherein said nucleic acid sequence is found in an inserted sequence placed under the control of these regulatory elements,
13. The DNA molecule according to any one of claims 1 to 12, wherein these regulators enable the expression of the nucleic acid sequence in a competent cell organism. 15. DNA molecule according to claim 14, characterized in that the competent cell organism comprises a human cell, in particular an epithelial cell or a hepatocyte. 16. The expression vector according to claim 14, wherein the expression vector is an adenovirus or retrovirus type vector or a virus type vector closely related to and functionally similar to them. A DNA molecule as described. 17. The DNA molecule according to any one of claims 1 to 16, for application in gene therapy. 18. A method according to claim 18, wherein said expression is performed inside said host cell.
A host cell comprising the DNA molecule according to any one of claims 1 to 17. 19. A composition comprising a DNA molecule according to any one of claims 1 to 17 conjugated to a vehicle that allows entry into human cells and expression in human cells. 20. The composition according to claim 19, wherein the DNA molecule is associated with a cationic lipid vehicle that favors expression. 21. The composition according to claim 19, wherein the DNA molecule is associated with a pharmaceutically acceptable vehicle that allows for administration in vivo by a systemic route. object. 22. The method according to claim 19, wherein the DNA molecule is associated with a pharmaceutically acceptable vehicle that allows administration by a systemic route through the respiratory tract. Composition. 23. The method according to claim 19, further comprising a DNA molecule encoding glutathione-S-transferase in such a form as to enable its expression in vivo. The composition according to claim 1. 24. The DNA molecule according to any one of claims 1 to 17, or the DNA molecule according to any one of claims 19 to 23, for producing a pharmaceutical composition suitable for treating pancreatic fibrosis. Use of the composition. 25. A polypeptide having a sequence corresponding to the sequence of the expression product of the DNA sequence according to any one of claims 1 to 14. 26. The composition of claim 25 in combination with a pharmaceutically acceptable vehicle.
A composition comprising the described polypeptide. 27. The composition according to claim 26, wherein the pharmaceutically acceptable vehicle allows administration by a systemic route. 28. The composition according to claim 26, wherein the pharmaceutically acceptable vehicle is of a type that allows administration via the respiratory tract. 29. The composition according to claim 28, which is releasable in aerosol form or another formulation form which can be administered by inhalation. 30. The composition according to claim 26, which also contains glutathione-S-transferase. 31. Use of the polypeptide composition according to claim 25 or any one of claims 26 to 29 for the production of a medicament for treating pancreatic fibrosis.