WO1996021733A2 - Transfert of dna in cells with the view to correcting the adrenoleucodystrophy - Google Patents

Abstract

The invention relates to a recombinant vector usuable to introduce a nucleic acid sequence into a target cell, characterized in that it comprises: a nucleotide sequence derived from a cDNA on the gene whose mutuation is responsible for the adrenoleucodystrophy or the adrenomyelopathy and which codes for a membrane protein; a retroviral vector M48 wherein said nucleotide sequence is inserted. According to an another embodiment of the invention, the vector M48 is replaced by another retroviral, viral or chemical vector.

Description

TRANSFER OF DNA TO CELLS FOR CORRECTION OF ADRENOLEUCODYSTROPHY.

The invention relates to means which can be used for the transfer into target cells of a cDNA fragment coding for a protein whose absence or mutation is responsible for a genetic disease, adrenoleukodystrophy (ALD), but also adreneuromyomyopathy. The gene whose abnormality is responsible for these pathologies is located at the Xq28 end of the X chromosome. The present invention also relates to recombinant cells (target cells) modified by a cDNA fragment coding for a protein whose absence or the mutation is responsible for ALD or related pathologies, these cells being capable of being used for implantation in vivo for therapeutic purposes. Adrenoleukodystrophy is a lethal disease causing progressive demyelination of the central nervous system, and caused by mutations in a gene coding for a protein called ALDp, located at the peroxisomal membrane. This disease is associated with a defect in the oxidation of very long chain fatty acids, and consequently their accumulation in various types of tissue.

The gene whose mutation can lead in a patient to an adrenoceukodystrophy type pathology was isolated and characterized by Mosser et al. (Nature No. 361, 726-730, 1993).

Work done by Singh et al. in 1984 (Proc. natl. Acad. Sci. U.S.A. 81, 4203-4207) then Moser & Moser in 1991 [The Metabolic

Basis of Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS & Valle, D.; McGraw-Hill, New York) pp.1511-1532] have shown that the main biochemical disorder caused by ALD corresponds to a faulty oxidation of very long chain fatty acids, as well as their accumulation in cerebral white matter, adrenal glands, fibroblasts and plasma. A 1992 study by Singh et al. (J. biol. Chem. 267, 13306-13313) has shown that in the case of fibroblasts, very long chain fatty acids are normally transported through the peroxisomal membrane. At the same time, it has also been shown that a dysfunction of the synthetase responsible for the association of coenzyme A and very long chain fatty acids prevents β-oxidation of these. It seems that this synthetase is also located at the peroxisomal membrane (Hashmi et al., FEBS Lett. 196,247-250; Wanders et al., 1988 J; Inher. Etab. Dis. 1 1 Suppl 2, 173-177) . More recently, patent application FR 2.108.606 has described the identification as well as the isolation of the gene responsible for adrenoleukodystrophy. The applicants have demonstrated there that the metabolic disorders were due to the defective nature of a 75 kDa protein intended to integrate into the membrane of the peroxisome. Several therapeutic approaches, including even a lipid diet combining the administration of trioleate as well as glycerol trierucate, have not, however, made it possible to modify the development of brain lesions due to ALD.

The inventors had also shown that a bone marrow transplant could reverse the demyelination process, an indirect consequence of the absence of normal protein, if this transplant was performed at a relatively early stage of disease development (Aubourg et al, 1990, New Engl. J. Med. 322, 1860-1866). However, in order to circumvent the problems relating to histocompatibility, only an autologous transplantation of bone marrow cells remained possible.

The "normal" protein corresponding to the "normal" gene which, when mutated, can lead to an ALD type pathological condition, having no specific biological name, will be defined using the term corresponding to the disease: - the gene called "normal ALD gene" or "ALD gene" is the normal gene which, when mutated, can lead to a pathology of the adrenoleukodystrophy type in a patient. It was characterized in the publication of Mosser et al. (Nature n ° 361,726-730, 1993), - normal ALDp is the protein encoded by the ALD gene whose abnormalities are genetically responsible for adrenoleukodystrophy,

- ALD fibroblasts are fibroblasts of patients whose ALD gene is mutated and which express the pathological biochemical phenotype of ALD.

A correction of the defective gene has been envisaged by the inventors in particular to remedy certain difficulties linked to the technique of bone marrow transplantation. This correction involves the introduction into target cells intended to be administered to a patient, of a nucleic acid sequence capable of expressing in these target cells, the protein normally encoded by the gene whose mutation can lead to a ALD-like pathology, or a polypeptide derived from this protein, used to correct the abnormality correlated with ALD. The introduction of this nucleic acid, in particular of the gene coding for the ALDp protein or of the cDNA or of a fragment of this cDNA coding for the ALDp protein, must be carried out taking care to choose a vector compatible with l nucleic acid, in particular the cDNA fragment inserted on the one hand, and allowing the expression of a protein intended to integrate into the peroxisomal membrane on the other hand.

The inventors then found that the retroviral vector M48 derived from the oloney Mo-MLV retrovirus, and generally preferred for allowing the expression of genes coding for soluble proteins, could also allow the expression of a gene coding for a structural protein. , in this case a transmembrane protein. This solution, using the techniques of gene therapy, included a first step in studying the possibility of correcting _ ^'vitro abnormal phenotype skin fibroblasts which the cDNA of the gene responsible for ALD had been transfected. The inventors have observed that the correction of the effects on metabolism of adrenoleukodystrophy, requiring the integration of the normal protein into the membrane of the peroxisome, was achieved by the combination of the vector M48 and a fragment of the cDNA. encoding said protein. The invention therefore relates to a recombinant vector which can be used to introduce a nucleic acid sequence into a target cell, characterized in that it comprises:

- a nucleotide sequence derived from a cDNA of the gene whose mutation is responsible for adrenoleukodystrophy or adrenomyyelopathy and which codes for a membrane protein,

- a M48 retroviral vector into which said nucleotide sequence is inserted.

The nucleotide sequence derived from the ALD gene or its cDNA introduced into the vector is capable of coding for a functional ALDp protein in the target cells transformed with the vector.

By the term "functional" is meant in the context of the present invention, the ability of the protein thus produced to contribute to the correction at least in part of the effects on the metabolism resulting from the ALD pathology. Furthermore, the use for therapeutic purposes of the vectors of the invention, in particular retroviral vectors, as well as recombinant cells containing them, can be envisaged on animal subjects including man.

The present invention provides means for transfection in vitro or in vivo of cells expressing the gene whose abnormalities are responsible for adrenoleukodystrophy. In particular, the invention defines a recombinant retroviral vector which allows the expression of the normal ALD gene after integration into the genome of said cells called target cells.

According to a first aspect, the invention relates to recombinant retroviral vectors comprising a cDNA fragment coding for a protein capable of integrating into the peroxisomal membrane of the cells in which it has been expressed. These recombinant retroviral vectors have the capacity to infect cells and in particular eukaryotic cells. These vectors are defective and are obtained for example by co-transfection with a transcomplementation vector carrying a sequence coding for a retroviral envelope so as to form viral particles in packaging lines.

The vector chosen will also have a high capacity for infecting cells, and will preferably confer long-term stability of expression of the normal ALD gene present, where appropriate, in a form integrated into the genome of the cells.

According to a preferred embodiment of the invention, a recombinant vector using the M48 construction is characterized in that it comprises: - a fragment of a cDNA of the gene whose mutation is responsible for adrenoleukodystrophy or adrenoneuromyelopathy , and which codes for a membrane protein,

- A M48 retroviral vector into which said cDNA fragment is inserted at a BamHI restriction site. The vector called M48-ALD is a recombinant vector comprising the cDNA, coding for a so-called normal protein, constructed from the vector M48 described in particular in the patent application.

WO94 / 24298.

According to a first particular embodiment of the invention, the recombinant retroviral vector is such that the expression of the cDNA is carried out under the control of the mouse phosphoglycerate kinase promoter. Also according to the invention, the cDNA is a Spel-EcoRI fragment with a length of 2.43 kb.

This fragment corresponds to nucleotides 333 to 2750 of the DNA complementary to the ALD gene whose sequence has been published by Mosser et al. in 1993 (Nature 361, 726-730).

The expression of this cDNA fragment can be carried out according to another embodiment of the invention in another retroviral vector where the expression of the sequence is under the control of a viral LTR or of promoters active only in hematopoietic stem cells, glial cells of the central nervous system, in particular oligodendrocytes, microglial cells and Schwann cells.

A diagram of the recombinant retroviral vector according to the invention established from the vector M48 is shown in FIG. 1. Also according to the invention, a recombinant retroviral vector defined above can be further characterized in that the cDNA fragment code for a protein capable of integrating into the membrane of a cellular organelle, namely the peroxisome when this vector is introduced into a recombinant host cell. According to other embodiments of the invention, the retroviral vector M48 is replaced by another retroviral vector, in particular MFG.

In addition, as other examples of vectors which can be used in the context of the present invention, the retroviral vectors derived from the Foch29 virus (WO95 / 01447) as well as the retroviruses described in patent application EP 336 822 can be cited. other recombinant retroviral vectors has been widely described in the literature, in particular by Breakfield et al., New Biologis 3 (1991) 203, Bernstein et al. Broom. Eng. 7 (1985) 235, McCormick, BioTechnology 3 (1985) 689, as well as patent applications EP 453242 and EP178220. To construct recombinant retroviruses comprising a nucleic acid coding for ADLp, a plasmid comprising in particular the LTRs, the packaging sequence and said nucleic acid is constructed, then used to transfect a cell line known as packaging, capable of providing in trans the deficient retroviral functions in the plasmid which allow the preparation of defective infectious retroviral particles.

Generally, the packaging lines are capable of expressing the gag, pol and env genes. Such packaging lines have been described in the prior art; we will refer in particular to the lineage

PA317 (USA, 861, 719), to the yCRIP line (WO90 / 02806) and to the GP + envAm-12 line (WO89 / 07150). The recombinant retroviruses produced are then purified by conventional techniques.

In addition to the retroviral vectors defined above, the invention can be implemented using other vectors, in particular recombinant vectors. These vectors generally comprise the nucleotide sequence derived from the gene coding for the ALDp protein or derived from the cDNA derived from this gene, under the control of signals necessary for the expression of a functional protein in a target cell. According to a particular aspect of the invention, the DNA which one seeks to express in the target cells is for example carried by a viral vector. As such, mention will be made of vectors of the "Adeno-associated-virus" or adeno virus type.

The "adeno-associated virus" (AAV), to which an adenovirus can serve as a replication aid, has DNA which seems to be capable of integrating into a particular site of chromosomal DNA.

The construction and expression of an AAV type vector have moreover been described in the specific case of a beta-globulin cDNA (Dixit M, Gene 1991; 104; 253-7). Adeno-associated viruses (AAVs) are relatively small DNA viruses that integrate into the genome of the cells they infect, stably and site-specifically. They are capable of infecting a wide spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. Furthermore, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as an origin of replication for the virus. The rest of the genome is divided into two essential regions carrying the packaging functions: the 3 ′ part of the genome, which contains the rep gene involved in viral replication and in the expression of viral genes; the 5 ′ part of the genome, which contains the cap gene coding for the capsid proteins of the virus.

The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo, in particular in hematopoietic cells, has been described in the literature (see for example WO 91/18088; WO 93/09239;

US 4,797,368, US, 139,941, EP 488,528). These applications describe various constructs derived from AAVs, in which the rep and / or cap genes are deleted and replaced by a nucleotide sequence of interest, and the use of these vectors to transfer in vitro (to cells in culture) or in vivo. (directly in an organism) said nucleotide sequence of interest.

The defective recombinant AAVs according to the present invention can be prepared by co-transfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing a nucleic sequence coding for the ALDp bordered by two regions inverted repeat (ITR) from AAV, and a plasmid carrying the packaging genes (rerj and cap genes) from AAV. A usable cell line is, for example, line 293. The recombinant AAVs produced are then purified by conventional techniques. They can then be used to infect specific target cells, chosen in particular from cells which have been described in the preceding pages as being susceptible to infection by retroviral vectors.

The constructions of recombinant vectors will be carried out so as to obtain a vector having a strong capacity of infection and allowing a long term stability of the expression of the protein ALDp.

This type of AAV vector, as well as the retroviral vector MFG notably described by OHASHI et al. (PNAS vol.85 n ^β 23 M 11332-11336- 1992), are particularly used in the modification of this hematopoietic stem cells. The invention also relates to recombinant cells transformed with a recombinant vector according to the invention which can be administered, or implanted in humans or animals, alone or in combination with an appropriate vehicle and producing in vivo the protein to which it has been referred to above. The invention further relates to recombinant cells characterized in that they are cells having the capacity to be tolerated immunologically by the organism to which they would be administered, these cells having been previously modified by a vector of the invention , carrying the sequence coding for the ALDp protein or, where appropriate, for a functionally equivalent derived polypeptide.

These cells can be cells originating from the organism in which they are to be implanted after transduction in vitro with the vector of the invention. They may also or alternatively be cells lacking on their surface, antigens recognized by the immune system of the organism in which they are implanted, also transduced in vitro.

Preferably, these recombinant cells are hematopoietic stem cells, in particular early cells, in particular the CD 34 ⁺ cell population. Preferably, these are hematopoietic stem cells autologous with respect to the patient in whom it is desired to implant them after their modification by a vector according to the invention. Hematopoietic stem cells can be removed and isolated by any technique known to those skilled in the art. These different techniques may involve physical separation steps, in particular by centrifugation or cell sorting (FACS), of selection by immunological compounds such as antibodies specific for cell markers (US 4,965,204; EP 17,381 or EP 30,450) or biochemicals, in particular membrane receptor ligands (EP 405,972). Among the immunological compounds that may be mentioned in particular are antibodies recognizing specific markers such as CD34 +. Among the techniques that can be used in the context of the present invention, some have been described, for example in patent applications EP 451 611, or WO93 / 02182.

The cells thus obtained can then be cultured under all sterile conditions allowing their conservation, their proliferation and / or their differentiation. The culture is advantageously carried out to maintain the cells during transformation, said transformed cells then being differentiated and / or activated in vivo, after their re-administration to the patient. The culture of the isolated cells can be carried out in various media known to those skilled in the art such as RPMI or IMDM, supplemented inter alia with serum, amino acids, and growth factors, in particular GM-CSF, M-CSF, the interleukins IL3, IL4, IL6 and IL11, or the SCF among others. The culture is carried out under sterile conditions, preferably at 37 ° C., as illustrated in the examples. It can be carried out in culture plates or in Teflon® bags.

The culture conditions can be adapted by a person skilled in the art, in particular as a function of the isolated cell population or of the desired use.

In addition to the aforementioned hematopoietic cells, the means described in the preceding pages allow the preparation of recombinant cutaneous fibroblasts. Can also be transformed, constituent cells of the nen system, in particular oligodendrocytes or microglial cells or Schwann cells.

As previously described, these cells can be modified by a recombinant vector coding for a determined polypeptide, in this case the ALDp protein, this vector allowing the expression of the foreign DNA fragment - here a cDNA - in the cells.

The modification of these cells by a viral vector, such as a retrovirus, carrying the cDNA coding for the ADLp can be carried out according to different protocols known to those skilled in the art, to a multiplicity of infections adaptable according to the vector used, as described in the examples below. Preferably, the cells are incubated in the presence of a supernatant of retrovirus producing cells. Depending on the processing conditions, the percentage of stem cells modified by insertion of the recombinant nucleic acid can reach

10%.

The modified cells thus obtained can then be packaged for immediate use, and / or stored for later use. For immediate re-administration, the cells are generally suspended in a phosphate buffer or in physiological saline, at a concentration varying from 10 ⁵ to 10 ⁹ cells per dose. For their conservation, the cells can be frozen, preferably in the presence of preservatives such as glycerol or DMSO. According to another embodiment of the invention, the vector which can be used can also be a chemical vector, and in particular a vector promoting the entry of a nucleic acid into a cell and the integration of said nucleic acid into the cell genome. Among the synthetic vectors described in the literature, it is preferred to use, within the framework of the invention, cationic polymers of polylysine type, (LKLK) n,

(LKKL) n, immine polyethylene and DEAE dextran or lipids cationic or lipofectants. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, mention may be made of lipopolyamines such as lipofectamine or transfectam, various cationic or neutral lipids such as DOTMA, DOGS or DOPE, as well as peptides of nuclear origin.

In addition, various synthetic vectors derived from AAVs have been described, based in particular on the use of the integrative properties of ITRs of AAVs. Mention may be made, for example, of AAV-liposome vectors as described in application WO95 / 07995 or any other plasmid vector containing an ITR sequence of AAV, capable of integrating into the genome of a cell.

The cells to be transformed can for example be modified by the methods known as homologous recombination. The penetration of the vector into the cells is carried out using electroporation or, as described below, DNA-calcium phosphate co-precipitation.

The subject of the invention is also generally, the use of a sequence coding for the ALDp protein, for the preparation of compositions for therapeutic use, for the correction in a patient, of metabolic abnormalities due to a pathology of adrenoleukodystrophy type. This therapeutic use involves the introduction into a cell of a nucleic acid sequence and the treatment of the patient with these cells.

In addition to the techniques described above, any method involving the entry of a nucleic acid, either alone or via a recombinant virus, therefore also falls within the scope of the invention.

The invention also relates to the use of vectors or recombinant cells according to the invention, for the preparation of a medicament for the treatment of diseases capable of being corrected by the expression of the cDNA fragment as defined above. Furthermore, the present invention relates to a method for the introduction into a body, of a cDNA or of a cDNA fragment coding for the ADLp, said method comprising:

the insertion in vitro of said cDNA, preferably by means of a retroviral vector or AAV, into a population of cells having the capacity to be tolerated immunologically by said organism, and

- the introduction into the organism of said cells.

Preferably, said cells are cells autologous with respect to the organism, preferably hematopoietic cells, in particular of the CD34 + population.

This introduction method is particularly applicable to an organism of the human type.

DESCRIPTION OF FIGURES Figure 1: schematic representation of the retroviral construction M48-

ALD. The gene complementary to the ALD gene, from position 333 to 2750 of the sequence published by Mosser et al, Nature, 361, 726-730, 1993, was inserted into the BamHI cloning site of the vector M48 at position 2727 of this vector. Figure 2: Integration of the intact M48-ALD provirus into fibroblasts

ALD.

The genomic DNA of normal cutaneous fibroblasts as well as cutaneous fibroblasts ALD, digested with Kpn1 as well as the plasmid vector were probed at the level of exon 13 of the cDNA. Lane 1: genome at 0.2 copies per cell of the plasmid M48-ALD; lane 2: normal fibroblasts; lane 3: ALD fibroblasts which have not undergone transduction; lane 4: ALD fibroblasts which have been transduced by the vector Glu-3; lane 5: ALD fibroblasts which have been transduced by the vector M48-ALD. Figure 3: Immunological transfer of ALDp into ALD fibroblasts before and after correction with the vector M48-ALD. Line 1: normal fibroblasts; lane 2: fibroblasts of patient 1 before transduction; lane 3: fibroblasts of patient 1 two weeks after transduction; lanes 4 and 6: fibroblasts of patient 2 before transduction; lane 5: fibroblasts of patient 2 two weeks after transduction; lane 7: fibroblasts of patient 2 two months after transduction.

Figure 4: expression and localization of ALDp in ALD fibroblasts before and after correction with the vector M48-ALD.

A and C: fibroblasts from 2 patients with ALD before correction; B and D: fibroblasts from patient 1 with ALD. Figure 5: lignoceric acid oxidation (A) and hexacosanoic acid content (B) in fibroblasts from patients with ALD before and after correction with the vector M48-ALD.

-: fibroblasts which have not undergone transduction; (1): fibroblasts having undergone transduction and after ten days of culture; (2): fibroblasts having undergone transduction and after two months of culture.

Figure 6: M48 vector in pBR 322 according to Danos and Mulligan.

Figure 7: cDNA sequence of the protein involved in ALD.

Example Cell Culture

These cultures were carried out from immortalized fibroblasts and "normal" fibroblasts. The experimental results concerning the expression of normal ALP and discussed later relate therefore to non-immortalized fibroblasts. The inventors first carried out cultures of cutaneous fibroblasts from patients hereinafter called JL and SM, respectively suffering from an infantile cerebral form of ALD and adrenomyeloneuropathy. These cultures presented fibroblasts having a high content of very long chain fatty acids, fatty acids having in this case not undergone the expected oxidation due to abnormalities of the ALD gene. The cells were kept in a DMEM medium supplemented with a solution at 10% by volume of inactivated fetal calf serum. These cell lines are characterized either by a virtual absence of ALDp during immunofluorescence or "immunoblotting" experiments, or by a presence of non-functional and therefore defective ALDp.

Fibroblasts from patients JL and SM were immortalized by the use of a viral construct containing a gene coding for the large T antigen of SV40 and recombined by the selection marker gene coding for neomycin phosphotransferase. The cells producing the retrovirus (SV40 pop) were derived from the pZipNeoSV (X) vector system described by Jat et al. in 1986 (Mol. Cell Biol. 6, 1204-1217).

The ALD fibroblasts, immortalized or not by SV40 as well as the packaging cell line ψ CRIP were cultured in a DMEM medium with 10% of newborn calf serum. All media were supplemented with 2mM glutamine, penicillin at 50 units / ml and streptomycin sulfate at 50 mg / ml.

Construction and packaging of the retroviral vector M48-ALD

For the construction of the retroviral vector M48-ALD, the inventors isolated the 2.43 Kb Spel-EcoRI fragment from the full-length cDNA coding for normal human ALDp (FIG. 7). After having been completed and then linked to a Bell "linker", this fragment was then inserted in the sense orientation at a BamHI restriction site of the M48 retroviral vector derived from the MoMuLV Moloney murine leukemia retrovirus. The insertion required the use of linkers resulting in a modification of the sequences upstream (5 ') and downstream (3') of the cDNA of the ALD gene without the latter itself being modified. These modifications are found in FIG. 1. In this construction M48-

ALD, cDNA expression is under the control of the mouse phosphoglycerate kinase (PGK) gene promoter. In parallel, a GLU-3 control vector was obtained by inserting the β-glucuronidase gene into the same M48 vector. The vector PGK-neomycin, containing a neomycin resistance gene derived from TN5 (Moullier et al., 1993, Transplantation 56, 427-432) under the control of the PGK promoter, was used for selection with G418 (Adra,

C.N., 1987, Gene, 60, 65-72).

The vectors M48-ALD and "PGK-neomycin" were co-transfected into an amphotropic packaging cell line γCRIP by means of DNA-calcium phosphate co-precipitation. The transfected cells were selected in a medium containing

G418 (1 mg of active base per milliliter, GIBCO / BRL). Clones were isolated by "ringcloning" and amplified to generate cell lines. The recombinant M48-ALD clones were selected on the basis of positive immunofluorescence tests with anti-ALDp antibodies. The absence of the "Helper" virus was tested by virus mobilization tests.

Transfer of the cDNA fragment

In order to transfer the cDNA of the ALD gene, ALD skin fibroblasts were first seeded at 5% confluence during the sixteen hours before infection. The cells were then incubated for eight hours with ten milliliters of M48-ALD or Glu-3 vector supernatant freshly filtered in the presence of polybrene

(8 mg / ml, Sigma). The infection is carried out twice, two consecutive days. After infection of the ALD fibroblasts, the clones producing the highest amount of recombinant M48-ALD virus were selected based on the number of integrated copies of the intact M48-ALD provirus.

Southern blot analysis

A Southern blot then revealed that this infection resulted in the integration of approximately one copy of provirus per genome. Genomic DNA had been isolated and digested with the restriction enzyme Kpnl. This analysis is more fully detailed in the publication by Sarde et al. Genomics 22, 13-20, 1994. The probe used (Ex13) corresponds to nucleotides 1221 to 1829 of the cDNA of the ALD gene.

Mono and polyclonal antibodies

The production, as well as the characteristics of polyclonal antibodies against normal human ALDp (1D6 and 2B4), have been described in the publication by Mosser et al. (1994, Hum. Mol. Genêt. 3, 265-271).

These two antibodies did not cross-react with mouse ALDp.

Analysis by immunoblottino and immunocvtofluorescence An analysis by "immunoblotting" and immunocvtofluorescence was then carried out according to a protocol described in this same document, in which the entire cell extraction process is also found.

Thus, 50 × 10 ³ fibroblasts per milliliter were cultured for sixteen hours in culture plates (Nunc), to then be fixed in 2% formaldehyde for four minutes, and permeabilized in PBS-Triton at a rate of 0, 1%. The cells were then incubated at room temperature either with the antibodies (1D6 or 2B4) or with a keto-thiolase or peroxisomal catalase anti-serum in PBS-Triton at 0.1%, and with 500 μg / ml (of IgG of goose. Biotinylated antibodies of mice for 1D6 and 1D4, and of rabbit for ketothiolase or peroxisomal catalase were added at a rate of 10 μg / ml ((Vector), and a specific labeling was revealed by the use of streptavidin Cy3 (Jackson Immunoresearch Laboratories) or streptavidin fluorescein (Vector).

For the co-localization studies, cells were first treated with 1D6 or 2B4 antibodies, then with the peroxisomal catalase anti-serum.

Three series of experiments were carried out in order to exclude non-specific cross-reactions between primary antibodies and secondary. Cells were incubated with: 1) monoclonal antibodies 106 and rabbit antibodies labeled with fluorescin-avidin D; 2) a catalase antiserum and mouse antibodies labeled with Cy3-Streptavidin; 3) anti-isotype antibodies (IgGI) from biotynilized mice and Cy3-Streptavidin.

Determination of the levels of very long chain fatty acids and of the β-oxidant activity of the peroxisome

The concentrations of very long chain fatty acids were then measured in skin fibroblasts by the use of a technique combining mass spectrometry and gas / liquid chromatography, according to a protocol described by Aubourg P. et al. (1985, J. Lipid Res. 26, 263-267). The ability of fibroblasts to oxidize lignoceric acid to acetate was measured according to a protocol described by Aubourg P. et al. (1993, Ped Res. 34, 270-276). Retroviral transduction of cDNA coding for normal ALDp in fibroblasts of patients with ALD

This stage made it possible to assess whether this transfer was able to correct the disorders generated by this disease. As previously described, the construction of the invention was co-transfected with the plasmid PGK-neomycin, conferring resistance to G418 in yCRIP envelope cells, this allowing the isolation of several cell lines producing the amphotropic virus M48 -ALD.

ALD skin fibroblasts were then transduced with either a supernatant of yCRIP cells producing M48-ALD, or a supernatant of Glu-3-yCRIP cells. Immunocytofluorescence-based studies have shown that 75 + 6% of fibroblasts in patient JL and 66 + 4% of fibroblasts in patient SL express the normal recombinant protein ALDp after two cycles of infection. The proviral integration of intact M48-ALD was then demonstrated by Southern blot analysis on ALD skin fibroblasts (Figure 2).

In vitro expression of normal ALDp in fibroblasts before incorporating the vector In order to determine the effect of retroviral transduction on the expression of the recombinant protein ALDp, transduced ALD fibroblasts were used. in culture at confluence in a fresh medium, to then be studied by immunocytofluorescence and immunoblotting. Immunoblotting of normal fibroblasts with antibodies 1 D6 and 2B4 revealed a specific protein of 75 kDa: ALDp (FIG. 3, line 1) (Mosser et al., 1994, Hum. Mol. Genêt. 3, 265- 271). This 75 kDa band was absent in fibroblasts from patient JL (Figure 3, lines 4 and 6). After transduction with the vector M48-ALD, an ALDp band at 75 kDa was present in the fibroblasts of the two patients with ALD (FIG. 3, lines 3 and 5). Based on the measurement of the intensity of the band, it can be observed that the expression of the ALDp is increased by five times compared to its normal value in these fibroblasts. The 75 kDa band was not detectable in ALD fibroblasts having been transduced by the control vector Glu-3.

By using indirect immunocytofluorescence, the 1 D6 and 2B4 antibodies made it possible to detect, before transduction with the vector M48-ALD, a dotted line of cytoplasmic fluorescent spots in the fibroblasts of MS patients (FIG. 4), however less intense than those observed in normal fibroblasts, but could not reveal any dotted fluorescent signal in JL fibroblasts.

After transduction with the vector M48-ALD, a marked dotted coloration reappeared in the fibroblasts of patient JL (FIG. 4B) and the dotted pattern increased notably in the SM fibroblasts (FIG. 4D). These changes were not observed in cells transduced by the Glu-3 control vector. A double indirect immunocytofluorescence confirmed that the recombinant ALDp was indeed peroxisomal. The fibroblasts of the patient JL having undergone a transduction by the vector M48-ALD, presented a red dotted signal when an indirect immunocytofluorescence was carried out with anti-ALDp antibodies labeled with Cy3 (FIG. 4E), and a green dotted signal when a Indirect immunocytofluorescence was performed by a peroxisomal catalase antiserum labeled with fluorescein (Figure 4F). A superimposed image showed a yellowish brown spot between the green and red dotted signals (Figure 4G), indicating that the recombinant ALD as well as the endogenous peroxisomal thiolase were co-located at the peroxisome level, in the same way as had been observed. by Mosser et al. (1994, Hum. Mol. Genêt. 3, 265-271) in normal fibroblasts.

No cytosolic aggregates were observed in the two ALD cell lines, indicating that most, if not all of the recombinant ALDp, had been properly incorporated into the peroxisomal membranes. This attests to the fact that the ALD fibroblasts have a system capable of integrating at the level of the peroxisomes large quantities of ALDp. Long-term expression of normal ALDp in fibroblasts before incorporating the vector

The inventors then studied the long-term expression of normal ALDp in fibroblasts treated with the vector of the invention. The intensity of the band at 75 kDa detected by "immunoblotting" did not show any significant difference in skin fibroblasts

ALD having been transduced by the M48-ALD vector and having been cultured for a period of two months (that is to say corresponding to five passages) (FIG. 3, lines 5 to 7). By immunofluorescence techniques, no reduction in the percentage of expression of recombinant ALDp in JL and SM patients was observed during this two-month period. Correction of the metabolism of very long chain fatty acids in fibroblasts before incorporating the vector

The β-oxidation of lignoceric acid (C24: 0) had undergone a reduction of 27% and 35% respectively in the fibroblasts of patients JL and SM. After transduction with the vector M48-ALD of the invention, these fibroblasts exhibited normal lignoceric acid degradation rates (FIG. 5A).

To estimate the phenotypic correction more precisely, the concentrations of very long chain fatty acids were measured in ALD fibroblasts before and after transduction by the vector M48-ALD.

FIG. 5B shows that the content of very long chain fatty acids, in fibroblasts which have not undergone transduction from patients SM and JL, was 3.5 to 4 times higher than that of normal fibroblasts. Transduction by the vector M48-ALD normalized the concentration of hexacosanoic acid in these fibroblasts (FIG. 5B), as well as the relationships between fatty acids C26: 0 / C22: 0 and C24.0 / C22.O. No change was however observed in ALD fibroblasts having undergone transduction with the control vector Glu-3. The very long chain acid levels of transduced transduced ALD fibroblasts cultured for three months remained normal

(Figure 5B).

From a histological point of view, the work done by Moser and Moser in 1991 [The Metabolic Basis of Inhe ted Disease (Scriver, CR, Beaudet, AL, Sly, WS & Valle, D .; McGraw-Hill, New York) pp.1511-1532] could have led to the following observations.

Demyelinating lesions caused by ALD in the brain are diffuse. They often concern the occipital lobe and / or the frontal lobe, as well as the internal capsules. It is likely that the demyelination observed in ALD is linked to the accumulation of very long chain fatty acids in the various lipid fractions of myelin, thus causing an alteration of their physical properties as well as a destabilization of the myelin membrane.

Non-replicating adenoviral or herpetic vectors have been proposed for stereotactic injections with the aim of introducing foreign genes into different areas of the brain. However, this injection approach is not suitable for ALD since it would require numerous injections at different sites in the brain.

Recent work by Altman (Altman, J. 1994, Trends Neurosci. 17, 47-49) has shown that up to 20% of glial cells are microglia cells. Although replacement of fixed microglia cells is still a controversial issue, several findings suggest that monocytes derived from peripheral hematopoietic cells may cross the blood barrier of the brain, penetrate the cervical parenchyma and differentiate into microglial cells (Altman,

J. 1994, Trends Neurosci. 17, 47-49; Hickey, W.F. & Kimura H. 1988, Science 239, 290-292).

Bone marrow transplantation allows the improvement of several neurodegenerative disorders, and this is explained by the replacement of abnormal microglial cells thanks to the contribution of new hematopoietic cells [Krivit, W. & Shapiro, E. 1991, in Treatment of genetic disorders (Desnick, RJ; Churchill Livingston, New York) pp.203-221].

The work of Aubourg et al. (1990 New Engl. J. Med. 322, 1860- 1866) have shown that concerning ALD, an allogenic bone marrow transplant can reverse, or stop, demyelination when it is performed on children with demyelinating lesions moderate and slow progression. However, this procedure requires a perfectly histocompatible donor, while carrying significant risks, including a violent reaction of the donor cells against the host cells. In order to circumvent these problems, the inventors have considered that an autologous bone marrow transplant, following the insertion of the normal ALD gene, could constitute an alternative approach. A first step allowed us to determine if the in vitro transfer of normal ALD cDNA could correct the phenotype of ALD fibroblasts.

We therefore constructed a recombinant retroviral vector M48-ALD allowing the insertion as well as the expression of the normal cDNA of human ALD. This construction was subsequently tested in cutaneous fibroblasts from unrelated patients. Before transduction by the vector M48-ALD, the ALDp was absent in one of the mutated cell lines and present but defective in the other.

After transduction by the recombinant vector M48-ALD, the abundantly expressed recombinant ALDp was normally incorporated into the peroxisomes of the fibroblasts of the two patients. The cellular distribution of recombinant ALDp was diffuse as in normal fibroblasts, and co-localized with peroxisomal ketothiolase. Although the ALDp was overexpressed, no aggregation of ALDp was observed in the cytosol, as is the case in a particular Zellweger cell line (GM6231). These results confirm the fact that most, if not all of the recombinant ALDp proteins, were correctly located at the peroxisome level.

It has been shown by Moser and Moser [The Metabolic Basis of Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS & Valle, D.; McGraw-Hill, New York) pp.1511-1532] and by Singh and al. (Proc. Natl.

Acad. Sci. U.S.A. 81, 4203-4207) that the oxidation of lignoceric acid, a substrate for peroxisomal β-oxidation, was reduced by 20 to 35% of normal in ALD fibroblasts.

Gene transfer using the means described in the present invention has made it possible to completely normalize this oxidation defect. As a result, the content of very long chain fatty acids is returned to normal in the fibroblast cell lines of patients in whom these fatty acids had been accumulated. These results indicate that the recombinant ALDp localized at the level of the peroxisomal membrane was functional and allowed a total restoration of the activity of the synthetase which makes it possible to associate the very long chain fatty acids deficient in ALDp. Stable expression of ADLp as well as prolonged metabolic correction was obtained for three months.

Since retroviral vectors can efficiently transfer, as well as express, physiological amounts of glucocerebrosidase in long-lived hematopoietic cells of the bone marrow in patients with Gaucher disease (Nolta JA et al., 1992, J. Clin Invest. 90, 342-348), similar results should be obtained with ALDp in patients with adrenoleukodystrophy.

Correction of CD 34+ cells of patients affected by ALD by transfer of normal complementary DNA to the ALD gene using the retroviral vector M48-ALDP

The vector M48-ALDP was used to correct in vitro CD34 + cells from a patient with ALD whose mutation in the ALD gene led to a complete absence of ALD protein (ALDp).

The CD34 + cells were purified on CelIPro columns from a sample of 100 ml of frozen marrow from an ALD patient. 5 × 10 ⁶ CD34 + cells were thus purified, and 3 × 10 ⁶ CD34 + cells were infected with supernatant of ψCRIP clones producing recombinant M48-ALDP retroviruses under the following conditions: 5 infection cycles over 36 hours, in the presence interleukins IL3 (10 ng / ml), IL6 (10 ng / ml), SCF (50 ng / ml), and 2 x 10 ⁴ cells / ml of supernatant. Analysis of cells in indirect immunoflorescence carried out at 24 hours with an anti-ALDp antibody showed that 21% of the cells expressed the protein

ALDp which was correctly targeted in peroxisomes. These cells transduced as well as CD34 + cells from the same uninfected ALD patient were then studied in a long-term in vitro culture system of bone marrow (Long term culture of human myeloid cells, Sutherland HJ, Evans. 1., p. 139- 163 (Culture of hematopoietic cells, Ed. Freshney RI, Pragnell IB and Freshney MG, Wiley-liss, 1994)). Cell differentiation tests in methylcellulose medium were carried out at 2, 5 and 7 weeks. These studies made it possible to demonstrate that after 7 weeks of culture followed by 2 weeks of culture in the middle of methylcellulose, 12% of granular and or macrophagic colonies expressed the recombinant ALDp protein.

All of these results, which have been duplicated on a bone marrow sample from a second patient with ALD, demonstrate that the vector M48-ALDP allows:

1) infect approximately 10% of the true stem cells in the CD34 + cell pool;

2) to stably express the ALDp protein for 7 weeks in a long-term bone marrow culture system, this protein being normally targeted in peroxisomes.

Claims

1. Recombinant vector which can be used to introduce a nucleic acid sequence into a target cell, characterized in that it comprises:

- a nucleotide sequence derived from a cDNA of the gene whose mutation is responsible for adrenoleukodystrophy or adrenomyyelopathy and which codes for a membrane protein, - a retroviral vector M48 into which said nucleotide sequence is inserted.

2. Vector according to claim 1, characterized in that it is a recombinant retroviral vector characterized in that it comprises:

- a fragment of a cDNA of the gene whose mutation is responsible for adrenoleukodystrophy or adrenoneuromyelopathy and which codes for a membrane protein,

- A M48 retroviral vector into which said complementary DNA fragment is inserted at a BamHI restriction site.

3. Recombinant retroviral vector according to claim 2 characterized in that the cDNA is a Spel-EcoRI fragment with a length of 2.43 kb.

4. Recombinant retroviral vector according to claim 2 or 3, characterized in that the expression of the cDNA is under the control of an LTR or of any other promoter active in hematopoietic stem cells, glial cells, in particular oligodendrocytes and microglial cells.

5. Recombinant retroviral vector according to claim 2, characterized in that it corresponds to the diagram in FIG. 1.

6. Recombinant retroviral vector according to claim 2, characterized in that the cDNA fragment codes for a protein capable of integrating into the membrane of a cellular organelle, the peroxisome.

7. retroviral vector according to any one of claims 2 to 6, characterized in that it is trans-complemented with a vector, in particular a plasmid carrying a sequence coding for the envelope of a retrovirus, under the control of elements of expression and regulation.

8. Vector according to claim 1 or claim 2, characterized in that the retroviral vector M48 is replaced by an adeno-associated virus (AAV).

9. Vector according to claim 8, characterized in that the nucleotide sequence derived from a cDNA of the gene whose mutation is responsible for adrenoleukodystrophy or adrenomyyelopathy and which codes for a membrane protein under the control of two sequences inverted repeaters placed respectively upstream and downstream of the nucleotide coding sequence.

10. Recombinant cells characterized in that they are cells having the capacity to be tolerated immunologically by an organism to which they would be administered, modified by a vector according to any one of claims 1 to 9.

11. Recombinant cells according to claim 10, characterized in that they are cutaneous fibroblasts.

12. Recombinant cells according to claim 10, characterized in that they are hematopoietic stem cells, in particular early cells, in particular the CD 34 ⁺ cell population.

13. Recombinant cells according to claim 10, characterized in that M48 is replaced by an AAV.

14. Recombinant cells according to any one of claims 10 to 13, characterized in that they are cells constituting the nervous system, in particular oligodendrocytes, microglial cells, or Schwann cells.

15. Use of a vector or a recombinant cell according to any one of claims 1 to 14, for the preparation of a medicament for the treatment of diseases likely to be corrected by expression of the protein (ALDp) normally encoded by the ALD gene.

16. Method for the introduction into an organism of a cDNA or of a cDNA fragment coding for ADLp, said method comprising:

the insertion in vitro of said cDNA, preferably by means of a retroviral vector or AAV, into a population of cells having the capacity to be tolerated immunologically by said organism, and

- the introduction into the organism of said cells.