WO1995005473A1

WO1995005473A1 - Non-human chimeric mammalian model for human pulmonary diseases

Info

Publication number: WO1995005473A1
Application number: PCT/FR1994/001002
Authority: WO
Inventors: Bruno Peault; Michel Perricaudet
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1993-08-13
Filing date: 1994-08-12
Publication date: 1995-02-23
Also published as: AU7501894A; FR2708831A1

Abstract

The present invention relates to an experimental animal model represented by a non human chimeric mammalian as well as the utilisation of such animal model for the screening of compositions which could be active in the prevention or treatment of human pulmonary diseases.

Description

NON-HUMAN CHEMICAL MAMMAL MODEL FOR HUMAN PULMONARY CONDITIONS

The subject of the present invention is an experimental animal model represented by a chimeric non-human mammal, as well as the use of this animal model for the screening of compositions capable of being active in the context of the prevention or treatment of human pulmonary pathologies. .

Among the various pulmonary pathologies, cystic fibrosis is a genetic disease that is frequently encountered in humans. The clinical signs of this pathology are numerous. In particular, intestinal, pancreatic, and especially pulmonary disorders are observed. The lung is invaded by very thick mucus, which leads to respiratory failure, and facilitates the development of infectious foci in the lungs.

Gene transfer is increasingly appearing as a preferred therapeutic approach to cystic fibrosis. We know indeed the protein deficient in this pathology, the C.F.T.R. (Cystic Fibrosis Transmembrane Conductance Regulator), whose human gene was cloned in 1989 (Rommens,

J.M. et al., Science (1989) 245: 1099-1065).

Around 400 mutations responsible for his dysfunction have been located to date. The most frequent mutation is ΔF508 (deletion of a phenylalanine at position 508 of the CFTR); this unique deletion makes the CFTR protein non-functional.

The problem which arises in an acute way is that of the experimental testing of the compositions likely to be active against pulmonary pathologies in general, and more particularly against cystic fibrosis. In the particular case of cystic fibrosis, the problem that arises is that of experimenting with gene constructs including the CFTR protein, before their therapeutic use, in particular in order to know their tropism, their expression, and their possible toxicity.

The experimental models currently available to try to answer these questions are the following: - In vitro culture of human respiratory epithelial cells.

However, the results are difficult to extrapolate to the functioning of lung tissue in vivo. In addition, it has recently been shown that a site major expression of the CFTR protein in humans consists of the subepithelial serous glands, which are not known to be cultivated selectively.

- Animal models.

. There is no spontaneous animal model of cystic fibrosis. Two strains of "cystic fibrosis" mice were created by inactivating the gene

CFTR by homologous recombination (Snouwaert, J.N. et al., Science (1992) 257: 1083-1088), Ratcliff, R. et al., Nat. Gen. (1993), 4: 35-41. One of the limitations of these models is that the submucosal glands, major sites of expression of CFTR in humans, are absent or extremely rare in rodents.

. Xenogenic chimeras.

- The SCID-hu mouse (Science, 241, pp. 1632-1639, 1988), allows direct transposition in vivo of human fetal hematopoietic tissues into their natural three-dimensional structure. PCT / US 92 06309 essentially concerns the development of a human, embryonic or fetal bone blank, after implantation in the SCID mouse.

- In the "humanized" rat trachea model, cells of the human respiratory epithelium are cultured on the internal face of the rat trachea, which is then transplanted into the partially immunodeficient nude mouse (Engelhardt et al., J Invest Clin (1992) 90: 2598-2607). Only the superficial epithelium is therefore, in this model, of human origin. The submucosal glands are absent; all other respiratory cellular compartments are from the rat.

However, there is currently no experimental model containing human respiratory tissue, pathological or not, in particular

"cystic fibrosis", in its natural three-dimensional structure, and therefore making it possible to screen the compositions capable of being active against a determined pulmonary pathology, in particular against cystic fibrosis.

The object of the present invention is precisely to provide a reliable experimental model for the implementation of a method of screening for compositions capable of being active against the various pulmonary pathologies encountered in humans.

The subject of the present invention is any chimeric non-human mammal as obtained by transplanting a graft of human lung tissue, said chimeric mammal being characterized in that the graft comprises, where appropriate after differentiation, submucosal glands.

Advantageously, the subject of the present invention is any chimeric non-human mammal as obtained by transplanting a tissue graft human pulmonary, said chimeric mammal being characterized in that the graft comprises, where appropriate after differentiation, all the cellular compartments constituting human respiratory tissue, and in particular the submucosal glands. According to an advantageous embodiment, the invention relates to a chimeric non-human mammal as defined above, in which the graft of human lung tissue is pathological.

According to an advantageous embodiment, the invention relates to a chimeric non-human mammal as defined above, in which the graft of human lung tissue is non-pathological.

The pathologies characteristic of the aforementioned human lung tissue graft, are of genetic origin or not.

Genetic pathology means any pathology caused by the dysfunction of one or more genes in the genome of an individual, and capable of being transmitted through the generations. As an example of a genetic lung disease characteristic of a graft according to the invention, there may be mentioned cystic fibrosis.

Pathologies not of genetic origin are those likely to be contracted through external factors, such as bacteria and viruses, or certain chemicals or other pollution components.

As an example of a non-genetic lung disease characteristic of a graft according to the invention, mention may be made of the various forms of lung tumors.

According to an advantageous embodiment, the invention relates to a chimeric non-human mammal as defined above, in which the graft of human lung tissue is of embryonic, fetal, or postnatal origin.

According to one embodiment of the invention, the human lung tissue grafted on the chimeric non-human mammal, consists of all or part of an embryonic or pulmonary fetal draft of human origin.

This pulmonary outline is made up of undifferentiated lung tissue, and is advantageously taken from embryos (corresponding to a development less than eight weeks post conception) or fetus from approximately 8 to approximately 15 weeks, originating from voluntary or therapeutic abortions of pregnancy.

All or part of the pulmonary outline can be grafted onto the animal. Preferably, the size of the fragments of pulmonary outline used to perform the graft, varies from approximately 1 to approximately 10 mm. Advantageously, the part of the grafted pulmonary blank corresponds to the proximal fraction of this blank, that is to say the fraction situated on the side of the opening of this pulmonary blank (which is in the form of a bag), so as to obtain a graft consisting of bronchi and / or bronchioles of tubular shape.

According to another embodiment of the invention, the pathological human lung tissue grafted on the non-human mammal, is an anatomically differentiated tissue, or cellulose differentiated; it can be taken from late fetuses, ranging in age from around 16 to around 35 weeks; it can be taken from newborns and infants of age ranging from birth to approximately 1 year (postnatal lung tissue), and for example of age ranging from birth to approximately 3 weeks. Lung tissue can come from surgeries, corpses of newborns or infants, or fetal corpses expelled spontaneously or medically. Advantageously, the tissue fragments used to perform the graft are chosen from the tissue constituting the bronchioles, so as to obtain tubular grafts, or the tissue constituting the alveoli. Preferably, the size of the tissue fragments used to perform the graft varies from about 5 to about 15 mm. Advantageously, a pulmonary blank taken from embryos, fetuses or lung tissue taken from newborn babies is used, because on the one hand, the younger the cells of the lung tissue, the better the cell proliferation, and the lower the risk of rejection of the graft, and on the other hand, the young cellular tissue contains fewer lymphocytes than of the adult pulmonary tissue, which has the advantage of limiting the risks of reaction of the graft against the host (GNH). In the case of the use of adult lung tissue, the host is preferably treated to limit the rejection reactions of the graft.

Furthermore, it should be noted that it has been unexpectedly found that undifferentiated or partially differentiated lung tissue develops after being grafted onto the host, until it reaches the same degree of differentiation as a human lung tissue. developed in utero (see detailed description below).

As such, it was unexpectedly found that the development of undifferentiated or previously differentiated lung tissue, grafted onto the host, takes place with a degree of differentiation such that it allows the formation of submucosal glands in the transplanted lung tissue. The pathological pulmonary embryonic draft or the differentiated pathological pulmonary tissue mentioned above respectively originate from an embryo, a fetus, a newborn, an infant or an individual suffering from the pulmonary pathology in question. This draft or this differentiated lung tissue can also come from an embryo or from an individual who is not suffering from the determined pathology which one wishes to find in the graft. In the latter case, the blank or the differentiated tissue are treated so as to present the characteristic signs of this pathology. This treatment of the draft or of the differentiated pulmonary tissue may consist of bringing the latter into contact with a vector capable of inducing the appearance of the determined pathology.

This vector can be the natural vector of this pathology, for example the bacteria or virus directly responsible for the disease in question. In the case of genetic pathologies, this vector can be chosen from those capable of inducing the appearance of said pathology, in particular either by the introduction of an antisense nucleotide sequence capable of blocking the expression of a gene determined by hybridization with this the latter, or by introduction into the genome of cells of nucleotide sequences coding for mutated proteins whose presence and / or inactive character are responsible for the pathology in question.

By way of illustration, the cystic fibrosis human lung tissue grafted onto the non-human mammal can also be obtained from embryonic, fetal or differentiated human non-cystic fibrosis lung tissue whose cells have been transformed using a vector suitable for obtaining lung cells in which the normal CFTR protein is no longer produced, or in which an inactive CFTR protein is produced. This transformation can in particular be carried out using a vector containing an antisense nucleotide sequence capable of hybridizing with the sequence coding for normal CFTR, thus blocking the expression of this protein, and / or using an adenovirus vector, the genome of which contains a DNA sequence coding for an inactive CFTR protein such as the CFTR protein comprising the ΔF508 mutation. Obtaining this inactive adenovirus - CFTR vector can be carried out according to the method illustrated in the article by Rosenfeld et al., Cell., 68, 143-155 (1992), using instead of the gene coding for normal CFTR, a gene coding for an inactive CFTR, in particular the gene coding for CFTR ΔF508. The chimeric non-human mammals according to the invention are preferably chosen from rodents, and advantageously from xenotolerant mice (that is to say capable of being transplanted stably by a graft from another species, in particular by a graft of human origin).

The aforementioned mice are preferably chosen from those with a deficient immune system, in particular from SCID mice (whose characteristic is to have neither B nor T lymphocytes) (Bosma, GC et al., Nature (1983) 301: 527-532) or NUDE (not containing T lymphocytes) available in particular from IFFA CREDO. Advantageously, SCID mice are used.

The lung tissue can be grafted under the renal capsule, or preferably under the skin of mammals according to the invention.

The present invention relates more particularly to any chimeric non-human mammal characterized in that it is transplanted by a graft of human lung tissue comprising tumor or cancer cells.

A more particular subject of the present invention is also any chimeric non-human mammal, characterized in that it is transplanted by a graft of human pulmonary tissue comprising cells, in particular epithelial and serous cells, in particular cells of submucosal glands, or the precursors of these cells, the genome of these cells being such:

- that it does not contain genes coding for a normal (active) CFTR protein, - or that it contains a gene coding for an abnormal CFTR protein

(inactive),

- Or that it contains a gene coding for an active CFTR protein, this gene cannot be expressed, in particular by blocking by an antisense sequence, the absence or inactivity of this CFTR protein being responsible for cystic fibrosis in the man (we will also speak, in what precedes and what follows, of "pulmonary cystic fibrosis" tissue).

The invention relates more particularly to a chimeric non-human mammal as described above, in which the grafted lung tissue is an embryonic, fetal or postnatal, pulmonary origin of human origin, which comprises cells whose genome contains a responsible gene cystic fibrosis. The absence or inactivity of the CFTR protein in the aforementioned cells is reflected in particular by a significant reduction, or even the cessation of the transport of chloride ions (Cl ^" ) across the membrane of these cells.

The subject of the invention is also the process for obtaining a chimeric non-human mammal as described above, this process comprising:

the transplantation, into an appropriate anatomical site of said mammal, of a human pathological pulmonary tissue as described above, in particular under the renal capsule or under the skin of the mammal, under anesthesia and under sterile conditions,

- where appropriate, in particular when the lung tissue is an embryonic or fetal blank, or a differentiated tissue of insufficient size, keeping the chimeric mammal alive in appropriate conditions (in particular sterile) in order to allow the lung tissue to reach a differentiated stage or sufficient size.

Preferably, the size of the graft must reach between approximately 5 mm and approximately 20 mm, in the case of an embryonic draft graft, and between approximately 5 mm and approximately 30 mm, in the case of an '' a differentiated tissue graft. The invention also relates to the use of non-human mammals as described above, for the implementation of a method for screening for compositions capable of being active in the context of the prevention or treatment of pulmonary pathologies in humans, and more particularly in the context of the pulmonary pathologies described above, in particular pulmonary pathologies in which the cells of the submucosal glands are involved, such as cystic fibrosis.

A subject of the invention is also a method for screening compositions capable of being active in the context of the prevention or treatment of the above-mentioned human pulmonary pathologies, and in particular capable of transfecting lung cells and more particularly epithelial cells and cells. serosa of the submucosal glands, characterized in that it comprises:

- bringing at least one of these compositions into contact with the graft, present in a chimeric non-human mammal as described above, for a determined time,

- detecting the possible restoration of the functioning mechanism of the lung tissue in the graft, corresponding to the normal functioning mechanism of the lung tissue in an individual not suffering from the pathology in question, this restoration being the witness to the effectiveness of the compositions tested against said pathology.

Advantageously, the compositions referred to above are such that they make it possible to introduce - either the gene which codes for a protein restoring normal functioning of the lung, in particular which codes for CFTR, in the context of cystic fibrosis,

- either an antisense nucleotide sequence capable of inhibiting the transcription of a nucleotide sequence responsible for a pulmonary pathology,

- Or a gene coding for a "marker" protein or CFTR, conveyed by different vectors (recombinant virus or non-viral "inert" vector, such as a liposome) in the aforementioned lung cells. The compositions can be brought into contact with the graft by micro-injection of these compositions into the graft, or into the tissue before transplantation.

Advantageously, in particular in the case of grafts corresponding to bronchioles or alveoli, contact between the graft and said compositions can be obtained by spraying the latter in the lumen of these bronchioles or alveoli, in particular by using aerosols.

The detection of the possible restoration of the functioning mechanism of the pulmonary tissue, within the framework of the aforementioned screening method, is advantageously carried out by removal of a part of the graft which is placed in culture in vitro in an appropriate medium, then the possible restoration of the functioning mechanism of the lung tissue in the graft is detected, corresponding to the normal functioning mechanism of the lung tissue in an individual not suffering from the pathology in question.

In the case of cystic fibrosis, we will detect the possible restoration of the mechanism corresponding to that controlled by the CFTR protein in the lung tissue of a healthy man (ie not suffering from cystic fibrosis).

Detection of the possible restoration of the aforementioned pulmonary functioning mechanism in the case of cystic fibrosis can be carried out:

- by detecting the normal functioning of the Cl "ion transport mechanism (by measuring the transmembrane currents of an isolated cell or a fragment of cell membrane, in particular according to the technique described by Hamill et al., (1981), Pflϋgers Arch 391: 85-100; using radioactive labeled Cl "ions, in particular according to the technique described by Nenglarik et al. (1990), Am. J. Physiol. 90: C358-C364), or by measuring the difference in total transepithelial potential, using micro-electrodes placed, for one under the skin of the non-human host mammal, and for the other, in situ, in the lumen of the grafted bronchopulmonary tissue, witnessing the presence of normal CFTR, or of a compound exhibiting an activity analogous to that of the protein

CFTR,

- Or by detection or assay of the amount of normal CFTR protein produced in the graft, in particular using antibodies directed against this protein, in particular according to the method described in the article by Rosenfeld et al., cited above. above.

The invention also relates to the use of a recombinant adenovirus vector comprising, at one of the sites of its genome which are not essential for its replication, a gene coding for the normal CFTR protein in humans, as a control control. of the restoration of the functioning of the pulmonary tissue within the framework of the implementation of a screening method as described above.

A subject of the invention is also the pharmaceutical compositions as obtained by implementing a screening method mentioned above. The invention will be further illustrated with the aid of the following detailed description of the preparation of chimeric SCID mice according to the invention, transplanted with a human pulmonary cystic fibrosis blank, and the demonstration of their aptitude as an animal experimental model using an adenovirus whose genome contains the gene coding for β-galactosidase. In this description, one can also note the interest presented by the sample and the various modes of analysis of the substances secreted in the grafted tissues (mucus).

- Animals

The C.B.17 scid / scid (SCID) mice are produced, bred and handled in a special animal facility, at the Institute of Cell Embryology and

Molecular of the CNRS and the Collège de France. These animals are raised in isolators supplied with sterilized and conditioned air. The farming equipment is sterilized by autoclaving, with the exception of food which is by γ irradiation. All mouse manipulations are carried out under laminar flow hoods, by specially trained and equipped personnel. - Experimental equipment

The lung sketches were taken from embryos and fetuses from voluntary or therapeutic termination of pregnancy. Postnatal tissue obtained after autopsy was also used. The gene transferred into these experiments is the adenovirus-βgalactosidase construction, described below.

- Transplantation of the pulmonary outline in the SCID mouse.

The lung tissue is transplanted, under a binocular magnifier and using microsurgical instruments, under the renal capsule or under the skin of anesthetized SCID mice (4 to 8 weeks). All handling is carried out under sterile conditions.

- Construction of V recombinant adenovirus, Ad-RSV-gal, by in vivo recombination.

This construction is described in detail in the article by Leslie D. Stratford - Perricaudet et al., Published in J. Clin. Invest., 90, 626-630, 1992.

In summary, this recombinant adenovirus was constructed by homologous recombination between an appropriate plasmid and the genome of the adenovirus type 5 (Ad5). In this construction, the β-galactosidase gene is placed under the control of the promoter RSV (Rous Sarcoma Virus). The plasmid pAdRSNβgal used contains the PvuII segment of the left end of Ad5 (segment located between positions 0 and 1.3 of the plasmid in FIG. 1) comprising the inverted terminal repetition, the origin of replication, signals d packaging, and the Ela amplifier. This fragment is followed by an nlslacZ gene (described in

Bonnerot, c. et al., Proc. Νatn. Acad. Sci. USA, 1987, 84, 6795-6799) coding for β-galactosidase, and with a fragment of the adenovirus Ad5 located between positions 9,4 and 17 of the plasmid of FIG. 1.

The values of positions 1,3, 9,4 and 17 indicated above are units indicating the number of base pairs included inside these fragments, one unit representing 360 base pairs.

The Ad5 sequence located between positions 9.4 and 17 above allows recombination with the adenovirus dl324 treated with the restriction enzyme Clal (corresponding to an E3 deletion mutant; the deletion being carried out between positions 78.4 and 84.3 of the adenovirus genome represented in FIG. 1), after transfection of 293 cells (human embryonic kidney cells transformed by adenovirus and mentioned above) in order to generate the recombinant vector Ad-RSV-βgal . The nlslacZ gene is controlled by the promoter RSN LTR and possesses the polyadenylation signal of the virus SN40. The recombinant virus thus obtained is incapable of replicating due to the deletion of the E1 genes.

- Transfer of genes to the lung tissue grafted in the SCID mouse.

The gene construct in solution in PBS is injected directly into the grafted tissue, exposed after anesthesia of the animal. The injection was carried out either with a 20 μl Hamilton syringe, or with a standard lml syringe fitted with a fine needle (27G). Depending on the size of the graft, volumes of 20 to 100 μl were injected.

The skin of the rootstock animals is then closed using surgical staples, and they are replaced in the breeding.

- Histoenzymatic analysis of human lung tissue transduced in vivo in the SCID mouse.

One or two weeks after the injection of the gene construct, the rootstock animal is sacrificed by cervical dislocation. The graft is removed, stripped of its adhesions and fixed for 2/3 hours in a 10% formaldehyde solution in PBS, then after rinsing, placed in PBS containing 30% sucrose, after which the tissue is frozen in the nitrogen in Tissue-Tek inclusion medium. Slices with a 10 μm thick cryostat are mounted on glass slides and incubated overnight, at 30 ° C., with the substrate solution of the β-galactosidase enzyme:

20μl K 200 mM ferrocyanide 20μl K 200 mM ferricyanide lμl MgCl ₂

950μl PBS lOμl X-gal

The sections are then rinsed with PBS, counterstained with hematoxylin or eosin, then mounted for observation.

- Differentiation of immature pulmonary structures and maintenance of differentiated structures of the human lung in the SCID mouse.

From a human, embryonic or fetal endomesodermal rough draft (between approximately 6 and approximately 13 weeks of development), we observe the development in the SCID mouse of a human respiratory tissue in which all the cellular compartments put in place during normal development in utero are present: an epithelium ciliated secretory pseudostratified respiratory system, smooth peribronchial musculature, peribronchial cartilaginous rings, submucosal glands with secretory activity and, in the longer term, an alveolar respiratory structure. These two types of structure present a peripheral vascularity of murine origin and an internal vascularization comparable to that encountered in human tissues developed in utero.

The kinetics of time is comparable to that of development in utero. More differentiated fetal lungs (around 20 weeks) are also transplanted into the SCID mouse where they complete their differentiation.

All of these aspects were verified by histological, electron microscopy and immunohistochemistry analyzes familiar to those skilled in the art. From a functional point of view, it was possible to study in these grafts, the activity of mucociliary beat and the difference in transepithelial potential.

- Measurement of the potential difference (PDD) in situ on grafted tissue.

The mouse is anesthetized, the tissue placed in graft at least two weeks before, is exposed after incision of the host, using microsurgical instruments. A reference microelectrode is placed under the host's skin, while a second microelectrode is introduced into the lumen of the transplanted bronchopulmonary tissue: the potential difference between these two electrodes is due to ion transport across the epithelial barrier of the graft ; a voltohmmeter makes it possible to measure this difference in potential and to deduce therefrom the transepithelial ionic exchanges, and in particular those concerning the Cl "ion, whose secretion is defective in cystic fibrosis patients. This method therefore provides a functional test for the whole graft scale.

- Methodology for the collection of substances secreted from the grafted tissues.

The grafted bronchopulmonary tissue is exposed, as described in the measurement of PDD in situ. An incision is also made on one of its luminal surfaces, in order to allow the pumping of secretion collected in the lumen of the graft. This pumping is carried out using a glass microcapillary, in which slides a steel piston provided with an O-ring at its end: even a very viscous liquid can thus be removed. Analysis of the physicochemical properties of mucus is an indication of the proper functioning of the lung cells that make up the graft. - Methodology of functional tests to detect the restoration of the functioning mechanism of lung cells corresponding to that controlled by the CFTR protein in the lung cells of a healthy man.

These study methods are the membrane patch clamp and the measurement of 36Q- efflux on isolated cells. The functional restoration of the CFTR channel protein is tested by the application of Cl "secretion agonists which use the intracellular cAMP pathway.

. Cell isolation

The epithelial cells are isolated from samples of all or part of the grafts by enzymatic digestion using protease and mechanical agitation according to the protocol for isolating epithelial cells from polyps.

(Chevillard et al. (1991), Cell. Tissue. Res. 264: 49-55). They are kept in primary culture.

. 36α ^~ efflux measurement

The use of radioactive Cl (36Q ~) makes it possible to follow the efflux of Cl "over time in previously charged cells (Nenglarik et al., Already cited) Cells having reached a confluence rate of approximately 90 % are incubated in efflux buffer containing 36Q. After rinsing, aliquots of the supernatant are removed regularly and the radioactivity of each sample is measured. The addition of different active substances during the experiment makes it possible to measure the functionality of the mechanisms for regulating the efflux of Cl "in a reconstituted epithelium.

. Membrane patch clamp

The patch-clamp technique allows the measurement of the transmembrane currents of an isolated cell or a fragment of membrane. Several configurations of the patch-clamp technique are used (Hamill et al., Already cited).

* Permeabilized patch configuration

In the cell-attached configuration, all of the currents are recorded. The membrane located under the pipette is permeabilized allowing electrical continuity between the pipette and the interior of the cell (Horn and Marty (1988), J. Gen. Physiol. 92: 145-159). The addition of different active substances during the experiment makes it possible to measure the functionality of the channels responsible for the exit of Cl ^" in a cell.

* Cell-attached configuration

When the pipette is stuck on the cell, the activity of the CFTR channels present in the membrane fragment can be recorded directly. * "Inside-out" configuration

When the fragment of membrane situated under the pipette is isolated from the cell, its external face is in contact with the intrapipette medium and its internal face with the medium of the bath. Under these conditions, the experimenter has access to the intracellular side of the channels present in the membrane fragment.

These last two configurations make it possible to test the functionality of a reduced number of CFTR protein-channel.

- Results: gene modification of human lung tissue grafted in SCID mice.

After transfer of the β-galactosidase gene, carried by an adenoviral vector, to the human fetal respiratory tissue grafted in the SCID mouse, the transduction of numerous lung cells was observed, one or two weeks after gene transfer. The enzymatic reaction appears in the form of an intense dark blue nuclear coloration. The majority of transduced cells are epithelial cells lining the lumen of the bronchiolar ducts. More rarely, cells of the uniform type are observed in the graft, and the transduced cells appear rather in foci corresponding probably to the injection sites of the adenoviral construction.

Late fetal tissue (at least 16 weeks of age) and postnatal tissue have also shown transduction of constituent cells of the submucosal glands, the preferred site for expression of the CFTR protein in healthy individuals.

To address the quantitative aspect of gene transfer in this model, different multiplicities of infection (ratio between the number of target cells and the number of gene particles): 1/1, 1/10 and 1/50 were used and these experiments have shown that the test is quantitative, that is to say that the number of infected cells is proportional to the quantity of adenovirus injected.

Technically, the advantage of the present invention resides in the fact that the gene construct that we want to test is transferred into a tissue intact human lung, developed in its normal three-dimensional structure. In addition, such in vivo cultures can be maintained for a very long time (the longest transplant period is currently 16 months). Already, we have been able to show the transduction of the two types of cells preferentially expressing the CFTR protein in vivo, namely, the epithelial cells and the cells forming the submucosal glands.

- Results concerning the ciliary beats.

The grafted lung tissue was collected in the host, the respiratory epithelium was placed in in vitro culture, and the beating of the eyelashes of the respiratory epithelium recorded by cinematographic microvideo. This experiment showed the existence of ciliary beats of frequency comparable to that which can be recorded on a control respiratory epithelium.

Description of the figures.

Figure 1

FIG. 1 represents a construction of a recombinant vector corresponding to the adenovirus of the Ad5 type in the genome of which is inserted the gene for β-galactosidase under the control of the promoter RSN.

Figure 2

It concerns the development of the human fetal pulmonary outline in the SCID mouse. It is a rudiment of 8.5 weeks of development transplanted for 9.5 weeks, in a subcutaneous situation, in the SCID mouse.

Figure 3

It concerns the embryonic pulmonary outline of 8 weeks of development, implanted for 18 weeks in the SCID mouse. The bronchioles are bordered by a ciliated pseudostratified epithelium.

The lamina propia (green coloration) includes smooth peribronchial muscles (M), cartilage (C) and submucosal secretory glands (G).

Coloration with Trichrome de Masson.

Figures 4A and 4B

They relate to the expression of a transgene in human lung tissue developed in the SCID mouse. A- 11 week developmental bronchial sketch transplanted 4 weeks into the mouse, then infected intraluminal with 7.10 ⁸ pfu of Ad-RSNβGal. Six days after infection, the majority of the cells of the respiratory epithelial border express the transgene (dark blue nuclear coloration resulting from the reaction of the enzyme β-galactosidase with one of its substrates:

X-Gal).

B- Bronchial segment of a child who died 11 days after birth transplanted for 4 weeks into the SCID mouse, then infected intraluminally with 5.10 ⁷ pfu of Ad-RSNβGal. Eight days later, the basal nuclei of the submucosal secretory glands express the transgene.

A and B: hematoxylin counterstaining.

Claims

1. Chimeric non-human mammal as obtained by transplanting a graft of human lung tissue, said chimeric mammal being characterized in that the graft comprises, where appropriate after differentiation, submucosal glands.

2. Chimeric non-human mammal according to claim 1, characterized in that the graft of human pulmonary tissue is of embryonic, fetal, or postnatal origin.

3. Chimeric non-human mammal according to one of claims 1 or 2, characterized in that the graft of human lung tissue is pathological.

4. Chimeric non-human mammal according to one of claims 1 or

2, characterized in that the graft of human lung tissue is non-pathological.

5. Chimeric non-human mammal according to one of claims 1 to 3, characterized in that the pathologies characteristic of grafts of human lung tissue, are of genetic origin or not.

6. Chimeric non-human mammal according to one of claims 1 to

3, characterized in that the characteristic pathologies of grafts of human lung tissue are the different forms of lung tumors, or cystic fibrosis.

7. Chimeric non-human mammal according to one of claims 1 to 3, 5 or 6, characterized in that the graft of human pathological pulmonary tissue consists of all or part of a human embryonic pulmonary outline or a fragment of differentiated human lung tissue, this blank or fragment of differentiated tissue from an embryo or an individual suffering from the pathology in question, or from an embryo or an individual who is not suffering from the pathology in question, the draft or differentiated tissue being, in the latter case, treated so as to present the characteristic signs of this pathology.

8. Chimeric non-human mammal according to one of claims 1 to

7, characterized in that it is chosen from rodents.

9. Chimeric non-human mammal according to one of claims 1 to

8, characterized in that it is chosen from xenotolerant mice.

10. Chimeric non-human mammal according to one of claims 1 to

9, characterized in that it is chosen from mice having a deficient immune system, in particular from SCID mice.

11. Chimeric non-human mammal according to one of claims 1 to 9, characterized in that it is chosen from mice having a deficient immune system, in particular from NUDE mice.

12. Chimeric non-human mammal according to one of claims 1 to

11, characterized in that the lung tissue is grafted under the renal capsule or under the skin.

13. Chimeric non-human mammal according to one of claims 1 to 3, or 5 to 12, characterized in that it is transplanted by a graft of human lung tissue comprising cells, in particular epithelial and serous cells, in particular cells of subepithelial glands, or the precursors of these cells, the genome of these cells being such:

- that it does not contain genes coding for a normal (active) CFTR protein,

- or that it contains a gene coding for an abnormal (inactive) CFTR protein,

- or that it contains a gene coding for an active CFTR protein, this gene cannot be expressed, in particular by blocking by an antisense sequence, the absence or inactivity of this CFTR protein being responsible for cystic fibrosis in man.

14. Chimeric non-human mammal according to claim 13, characterized in that the grafted pulmonary tissue is an embryonic, fetal or postnatal, pulmonary origin of human origin, which comprises cells whose genome contains a gene responsible for cystic fibrosis.

15. Use of chimeric non-human mammals according to one of claims 1 to 14, for the implementation of a method of screening compositions capable of being active in the context of the prevention or treatment of pulmonary pathologies in l man, and more particularly pulmonary pathologies in which the cells of the submucosal glands are involved, in particular cystic fibrosis.

16. Method for screening compositions capable of being active in the context of the prevention or treatment of human pulmonary pathologies, in particular compositions capable of transfecting lung cells and more particularly epithelial cells and serous cells of the submucosal glands , characterized in that it comprises:

- bringing at least one of these compositions into contact with the graft, present in a non-human chimeric mammal according to one of claims 1 to 14, for a determined time,

the detection of the possible restoration of the mechanism of functioning of the pulmonary tissue in the graft, corresponding to the normal mechanism of functioning of the pulmonary tissue in an individual not carrying the pathology in question, this restoration being the witness of the effectiveness of the compositions tested against said pathology.

17. Screening method according to claim 16, characterized in that the bringing together of the compositions with the graft is done by micro-injection of these compositions into the graft, or by spraying of these compositions in the light of any bronchioles or alveoli present in the plugin.

18. A method of screening compositions capable of being active against cystic fibrosis according to one of claims 16 or 17, characterized in that the detection of the possible restoration of the functioning mechanism of the pulmonary tissue, corresponding to the mechanism controlled by the CFTR protein in the lung tissue of a healthy man, is produced by removing a part of the graft which is cultured in vitro in an appropriate medium, then:

- by detecting the possible restoration of the functioning of the transport of Cl "ions (by measuring the transmembrane currents, or using labeled Cl" ions) in a radioactive manner, indicating the presence of normal CFTR, or of a compound exhibiting an activity analogous to that of the CFTR protein, - Or by detection of the assay of the amount of normal CFTR protein produced in the graft, in particular using antibodies directed against this protein.

19. Pharmaceutical compositions as obtained by implementing a screening method according to one of claims 16 to 18.