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Use of inhibitors of the glycosylation process for the prevention and treatment of genetic diseases

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Publication number
WO2008068548A1
WO2008068548A1 PCT/IB2006/004074 IB2006004074W WO2008068548A1 WO 2008068548 A1 WO2008068548 A1 WO 2008068548A1 IB 2006004074 W IB2006004074 W IB 2006004074W WO 2008068548 A1 WO2008068548 A1 WO 2008068548A1
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cells
chemical
fig
glycosylation
mm
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PCT/IB2006/004074
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French (fr)
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Jean-Laurent Casanova
Guillaume Vogt
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Institut Necker
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445Non condensed piperidines, e.g. piperocaine

Abstract

The invention relates to use of a use of a therapeutically effective amount of at least one chemical compound for the preparation of a pharmaceutical composition for the prevention and/or treatment of diseases associated with genomic or mitochondrial, germ-line or somatic mutations in animals, including human beings, wherein said at least one chemical compound is a molecule inhibiting any step of the glycosylation process, preferably an inhibitor selected from the group consisting of inhibitors of glucosidase and inhibitors of mannosidase.

Description

USE OF INHIBITORS OF THE GLYCOSYLATION PROCESS FOR THE PREVENTION AND TREATMENT OF GENETIC DISEASES

FIELD OF THE INVENTION

This invention relates to the detection, prevention and/ or treatment of genetic defects or inherited diseases in a living matter. More generally, the invention relates to the prevention and /or treatment of diseases associated with genomic or mitochondrial, germ-line or somatic mutations in animals, including human beings, in plants and in fungi, with molecules inhibiting any step of the glycosylation process, whether by adding or removing carbohydrate moieties from the polypeptide backbone, especially with at least one inhibitor selected from the group consisting of inhibitors of glucosidase and inhibitors of mannosidase (e.g. inhibitors of glucosidase I, glucosidase II, endoplasmic reticulum mannosidase I and II and Golgi mannosidase I, and the like).

TECHNOLOGICAL BACKGROUND

CB. Guerra et al, Journal of Immunology, 160: 4289-4297 (1998) disclose that the DRA mutation (namely Pro^ — > Ser) is directly responsible for the class π-associated invariant chain peptides (CLIP) release and peptide- loading defect in 10.24.6 cells (i.e. cells of the HLA-DR hemizygous B lymphoblastoid cell line) Two different approaches were taken to evaluate the importance of aberrant DR glycosylation to the 10.24.6 phenotype. However only selected aspects of the said phenotype could be evaluated in tunicamycin-treated cells.

P.B. Fischer et al., Journal of Virology, 69 (No. 9): 5791-5799, (1995) disclose that the α-glucosidase inhibitor N-butyldeoxynojirrmycin (NB-DNJ) is a potent inhibitor of the VIH replication and syncytium formation in vitro. In the application of NBDNJ to the treatment of Gaucher disease envisaged by some previous authors, these molecules were considered to be inhibitors of the ceramide-specific glucosyltransferase. Other authors have considered them to be inhibitors of glucosylceramide synthase (see Platt et al, supra, concerning experiments with miglustat and Tierney et al., Journal of AIDS and Human Retrovirology, 10: 549-553 (1995) concerning experiments evaluating the safety of treatment in 130 HIV-positive patients), which initiates the glycosphingolipid biosynthesis pathway and catalyses the formation of glucocerebroside.

More recently, it has been suggested that miglustat or castanospermine (however to a lesser extent for the latter one) can also function as inhibitors of the complementation of the delF508 mutation in the endoplasmic reticulum and prevent the interaction of delF508-CFTR with calnexin and hence its entry in the degradation pathway (C. Norez et al, FEBS Letters 580: 2081-2086 (2006)). In other words, C. Norez et al. disclose that the orally active orphan drug named NBDNJ or miglustat, or to a lesser extent the drug castanospermine, prevents the delF508-CFTR/ calnexin interaction and restores cAMP-activated chloride current in epithelial cystic fibrosis (CF) cells, thus allegdly providing a basis for future clinical evaluation of miglustat in CF patients. However, even in this context, it was not conceivably and feasibly taught or even envisaged to use such drugs for diagnosing, preventing and/or treating genetic diseases, since it was not clearly established that (i) these compounds worked on polyclonal, fresh cells (ϋ) studied ex vivo from (iii) multiple patients, as the effect was only assessed in vitro on a tumoral cell line derived from a single patient. It was not either documented that (iv) the compounds were active in vivo (In one experiment the Whole Cell Patch Clamp did not even provide a proper comparison with the wild type). Moreover, (v) the functional complementation was not complete; (vi) the only cell type tested was extra-hemopoietic; (vii) the findings were not generalized to other mutants, whether in the same or another protein, (viii) the complementation was not documented biochemically, with a lack of demonstration that the proteins would reach the cell surface, and finally (ix) the two compounds tested inhibit the same enzymes, thus preventing a generalization to the glycosylation pathway. It was also to be feared that the results presented in the aforesaid publication of C. Norez et al. reflected an enhanced cell mortality, which would lead to the false interpretation that the compounds were able to complement the defect in living cells. Indeed in experiments using these teachings conducted by the present inventors, miglustat and castanospermine led to a false complementation of the CFTR mutation on dead cells, and were not effective on living cells.

There was thus still a need for new means, especially chemical means, for the detection, prevention and /or treatment of genetic defects or inherited diseases in vivo as well as ex vivo.

SUMMARY OF THE INVENTION

It has now been found that compounds which are active as inhibitors of the glycosylation process, especially as inhibitors of glucosidase and/or mannosidase, can be used on living cells and are particularly useful in the prevention and treatment of genetic defects and inherited diseases.

The present invention provides chemical means for the preparation of compositions for treatment of genetic diseases and/ or inherited defects in animals, including human beings, and in plants and fungi, as well as the use of said compositions for in vitro, in vivo or ex vivo treatment of genetic defects in such humans, animals, plants, or fungi whereas the said defects are pathogenic mutations which are amorphic, hypomorphic, hypermorphic, or neomorphic, including mutations that exert their pathogenic potential through at least one post-translational modification. The invention more particularly relates to means for the detection and/or treatment of genetic defects and/or inherited defects, whereas said means comprise an inhibitory tool essentially comprising molecular inhibitors of at least one step of the N-glycosylation.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, as well as from the drawings and the appended claims.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows a clinical phenotype, IFNGR2 genotype and cellular phenotype of patients with MSMD.

Fig. Ia. Clinical phenotypes of consanguineous patients. Healthy individuals are shown in white. Patient 1 (Pl), with M. avium is shown in black.

Fig. Ib. Electrophoregram showing the mutations in exon 3 of the IFNGR2 gene in patient 1 (Pl) (382-387dup) and the wild-type sequence of a control (WT). The position of the insertion is the indicated by the asterisk on the WT sequence and the additional amino acids present in the mutant are shown in thickened line.

Fig. Ic. Novel mutation in the IFNGR2 gene (382-387dup). The IFNGR2 coding region is represented by vertical bars between exons designated by Roman numerals. The leader sequence (L: 1-22), extracellular domain (EG23-248), transmembrane domain (TM: 249-272) and intracellular domain (IC: 273-337) are indicated. Consensus sites for N-glycosylation are indicated by asterisks. Mutations which are underlined once cause complete IFNγR2 deficiency with no detectable expression of IFNγR2 at the cell surface. The mutation underlined twice causes complete IFNγR2 deficiency, with detectable surface expression of non functional IFNγR2 6. The mutation underlined thrice causes partial, as opposed to complete, IFNγR2 deficiency.

Fig. Id. Response of EBV transformed B cells to IFNγ and DFNα, as determined by EMSA analysis of nuclear proteins binding a GAS probe, from a positive control (C+), two patients (Pl, bearing mutation 382-387dup; P2, bearing mutation 663del27) and a negative control (C-; homozygous for the 278delAG IFNGR2 allele), in response to IFNγ or IFNα (105 IU/ml) treatment for 30 minutes and in the absence of such treatment. Fig. Ie. GAS probe-binding nuclear protein from EBV-B IFNγR2- deficient cells (278delAG) transiently transfected with vectors encoding wild-type (WT), 278delAG, 663del27 and 382-387dup IFNγR2, without (NS) or with 105 IU/ml of IFNγ, as determined by EMSA.

Fig. 2 shows biochemical properties of IFNγR2 under various chemical treatments.

Fig.2a. Hek-293 cells were transfected with 278delAG, WT, 382-

387duρ and 663del27 IFNγR2-tagged V5 contracts. They were then incubated alone or with kifunensine (lμM), NB-DNJ (1.5 mM) or castanospermine (2 mM) for 48 hours. Whole-cell extracts were generated and left untreated or digested with Endo-H over night. They were then subjected to SDS-PAGE and to immunoblotting with an anti-V5 antibody.

Fig. 2b. Maturation of N-linked oligosaccharides: the triglucosylated polymannose oligosaccharides (GlcNac: square, Mannose: circle, Glucose: circle enclosed in a square) transferred to the asparagine residues of proteins from the dolichol phosphate during the translation of mRNA on ribosomes. This oligosaccharide was then maturated by- incubation with a series of enzymes, including glucosidase I or II (G-I or G-II), glucosyl transferase (UGTI), erρ57, calnexin (CNX), calreticulin (Crt), ER-mannosidase I or II (ERM-I or ERM-II), M9-mannase (M9M), Golgi mannosidase I or II (GM-I or GM-II) and glucosyl transferase I (GnT-I). Some pathways for unfolded proteins lead to proteasomal or non proteasomal proteolysis.

Fig. 2c. IFNγR2-deficient SV40-transformed fibroblasts were transformed with the 278delAG, WT, 382-387dup and 663del27 IFNγR2- tagged V5 constructs, either without prior treatment, or 48 hours after treatment with NB-DNJ (1,5 mM), castanospermine (2 mM) or kifunensine (lμM). Cell surfaces were biotinylated and precipitates (IP-Strept) were analysed by western blotting with horseradish peroxidase-conjugated anti- V5 antibody.

Fig. 2d. IFNγR2-deficient SV40-transformed fibroblasts were transformed with the 278delAG, WT, 382-387dup or 663del27 IFN(R2-tagged V5 constructs, either without prior treatment or 48 hours after treatment with NB-DNJ (1.5 mM), castanospermine (2 mM) or kifunensine (lμM). Cell surfaces were biotinylated and total extract (-) or flow-through (+) from the biotynylated cells was analyzed by western blotting with horseradish peroxidase-conjugated anti-V5 antibody.

Fig. 3 shows chemical complementation of the cellular phenotype with multiple drugs

Fig. 3a. Response of EBV-B cells to IFNγ, as determined by electrophoretic mobility shift assay (EMSA) analysis of GAS probe-binding nuclear proteins from a positive control (C+), a negative control (C-; homozygous for the 278delAG IFNGR2 allele) and two patients (Pl, bearing mutation 382-387dup; P2, bearing mutation 663del27), in response to IFNγ

(2.4 x 104 IU/ml) for 30 minutes, 16 hours after incubation with NB-DNJ (1.5 mM), castanospermine (2 mM) or kifunensine (lμM).

Fig. 3b. SV-40-transformed fibroblasts from a positive control

(C+), two patients (Pl, bearing mutation 382-387dup; P2, bearing mutation 663del27), and a negative control (C-; bearing the 278delAG mutation) were incubated for 72 hours in complete culture medium with (black histogram) or without (white histogram) IFNγ (2.4 x 10^ IU/ml), with or without NB- DNJ (1.5 mM), castanospermine (2 mM) or kifunensine (IuM or 0.375μM). They were analyzed 48 hours later. The surface expression of HLA-DR molecules was determined by flow cytometry using a specific antibody.

Fig. 4 shows chemical complementation of other misfolded proteins not expressed on the cell surface

Fig. 4a. SV-40-transformed fibroblasts from P3, bearing mutation T168N were incubated for 72 hours in complete culture medium with (black histogram) or without (white histogram) IFNγ (2.4 x 10^ IU/ml), in the presence of DNJ (4.72 mM), NJ-I-S (0.74 mM), tunicamycin (0.121 μM) or DM (82 μM), and analyzed. The surface expression of HLA-DR molecules was determined by flow cytometry using a specific antibody. Fig. 4b. EBV transformed B cells from a positive control (C+), a negative control for IL2RG (C- with a premature stop codon in the region encoding the extracellular domain), and proteins encoded by IL2RG genes with missense mutations not expressed on the cell surface, such as L172Q, E68K, D39N, C62G,Y105C, E68G, R222C, Q144P and R285Q were incubated for 72 hours in complete culture medium with (right panel) or without (left panel) 4 uM of kifunensine. The surface expression of IL2-Rγ molecules was assessed by flow cytometry using an isotypic antibody (dashed line) or a specific antibody (bold line).

Figs. 4c, 4d. Hek-293 cells were transiently (c) or stably (d) mock transfected or transfected with WT, D39N, C62G, E68G, L172Q or R222C IL2Rγ-tagged V5 constructs. They were then incubated in the presence (+) or absence (-) of kifunensine (4 μM), for 48 hours. Cell surfaces were biotinylated and precipitates (JP-Strept) were analyzed by western blotting with horseradish peroxidase-conjugated anti-V5 antibody. The arrow indicates the MW of tWT molecules in the presence of kifunensine.

Fig. 4d. EBV-transformed B cells from a positive control (C+), a negative control for CD18 (C-), and G284S-CD18 cells were incubated for 48 hours in complete culture medium with castanospermine (2 mM), kifunensine (IuM (Kif) or 153 μM (Kif-o)), NM-DNJ (6.51 mM) or DM (82 μM). The surface expression of CDl 8 molecules was determined by flow cytometry using an isotypic antibody (dashed line) or a specific antibody (bold line)

Fig. 5 shows the chemical complementation of the cellular phenotype with multiple drugs.

Fig. 5a. IL-12p40 production by PBMCs from a positive control

(C+), the patient (Pl) bearing the 382-387dup mutation and the patient (P2) bearing the 663del27 mutation, after 48 hours of stimulation with complete medium (white), live BCG alone (grey), live BCG plus IFNγ (5,000 IU/ml) (black), or IFNγ (5,000 IU/ml) (dark grey), with or without NB-DNJ (1.5 mM) or castanospermine (2 mM), as detected by ELISA.

Fig. 5b. Production of IL-12ρ70 (the active form of IL12) in

PBMCs from a positive control (C+), the Pl and P2, after 48 hours of stimulation with complete medium (white), live BCG alone (grey), or live BCG plus IFNγ (5,000 IU/ml) (black), with or without NB-DNJ (1.5 mM) or castanospermine (2 mM), as detected by ELISA.

Figure 6. shows chemical complementation of the cellular phenotype with multiple drugs. SV-40-transformed fibroblasts from a positive control (C+), two patients (Pl, bearing mutation 382-387dup; P2, bearing mutation 663del27), and a negative control (C-; bearing the 278delAG mutation) were incubated for 16 hours in complete culture medium with (black histogram) or without (white histogram) IFNγ ( (2.4 x 10^ IU/ml), in the presence or absence of NB- DNJ (1.5 mM), castanospermine (2 mM), NN-DNJ (33.25 μM), NM-DNJ (6.5I mM), NB-DMJ (1.5 mM), kifunensine (lμM or 0.375 μM), DNJ-H (3.86 mM), DNJ (4.72 mM), DIM (7.72 mM), NF-DNJ (1 mM), DM (82 μM), 5-fgm (10.73 μM), 2-5-DM (2.36 mM) or NJl-S (0.74 mM). They were analyzed 48 hours later. The surface expression of HLA-DR molecules was investigated by flow cytometry using a specific antibody, "nd" = not done.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides a use of a therapeutically effective amount of at least one chemical compound for the preparation of a pharmaceutical composition for the diagnosis, prevention and/or treatment of human, animal, plant and/ or fungal diseases associated with genomic or mitochondrial, germline or somatic mutations, wherein said at least one chemical compound is a molecule inhibiting any step of the glycosylation process, especially as an inhibitor selected from the group consisting of inhibitors of glucosidase and inhibitors of mannosidase (e.g. inhibitors of glucosidase I, glucosidase II, endoplasmic reticulum mannosidase and/or Golgi mannosidase, and the like).

A special embodiment of the first aspect of the invention provides a use of a therapeutically effective amount of at least one chemical compound capable of complementing a IFNGR2 defect in animals, including human beings, as well as in plants and in fungi, for the preparation of a pharmaceutical composition for the prevention and /or treatment of genetic defects or inherited diseases in such a living matter, in vitro or ex vivo, whereas said at least one drug is at least one chemical compound capable of effecting a substantially complete chemical complementation of misfolding mutations in polyclonal cells.

According to a preferred embodiment of this aspect of the invention, the said at least one drug is an inhibitor of the synthetic pathway of the N-linked carbohydrates and/or of any other pathway encoding enzymes selected from the group consisting of glucosidases and mannosidases, and makes possible the glycosylation of the glycolipides.

According to a another preferred embodiment of this aspect of the invention, the said at least one drug is a chemical compound inhibiting enzymatic targets selected from the group consisting of glucosidase I, glucosidase II, endoplasmic reticulum mannosidase and Golgi mannosidase.

According to a most preferred embodiment of this aspect of the invention, the said at least one drug is selected from the group consisting of:

N-butyldeoxynojirimycin, castanospermine, kifunensine, N-butyldeoxy- mannojirimycin, deoxynojirimycin, deoxynojirimycin, l,4-dideoxy-l,4- irnino-D-mannitol, N-methyl-deoxynojirimycin, nojirimycin-1-sulfonic acid; N-(n-nonyl)deoxynojirimycin; and australine, or mixtures thereof, and/or any chemical compounds having a similar general structure, especially an indolizidine or a piperidinetriol structure.

In one aspect, the invention relates to the use of chemical substances selected from the group consisting of those listed in Table 1 (see below), the toxicity of which has been shown to be low or non-significant, either individually or in combination with each other or with other inhibitors of any step of the glycosylation process, as inhibitors of the quality control machinery of the endoplasmic reticulum (ER) and/or of the Golgi apparatus, permitting the normal expression of misfolded mutant proteins and ensuring the functioning of these proteins in cells, as a means for the chemical complementation of pathogenic mutations in animals, including human beings, as well as in plants and fungi, more particularly for the prevention and/ or treatment of genetic diseases and of inherited defects. In certain embodiments of this aspect of the invention, the composition for the prevention and/or treatment of genetic diseases comprises at least two such chemical compounds which are effective as inhibitors of glucosidases and for as inhibitors of mannosidase.

According to a preferred embodiment of this aspect of the invention, the said use is for the preparation of a pharmaceutical composition for the detection and /or treatment of genetic defects or inherited disorders in a living matter, whereas the said pharmaceutical composition is for preventing or treating at least one mutation selected from the group consisting of 663del27, 382-387dup, IL2RG, TGB2 (CD18), T168N, and analog mutations in the IFNGR2 gene.

In another aspect, the invention provides the said at least one inhibitor of the glycosylation process in a living matter, for use in a medicament for the prevention or the treatment of genetic defects or inherited diseases in animals, including human beings, in plants and/or in fungi. In a further aspect, the invention provides a pharmaceutical composition, which comprises such an at least one inhibitor, and optionally a pharmaceutically acceptable carrier.

According to a preferred embodiment of this aspect of the invention, the pharmaceutical composition comprises as a dose unit liable to provide a proper form of dosage for use or administration of about 8 μg to about 80 mg of at least one or a combination of said chemical compounds per liter of serum.

A preferred dose for administration to human beings is about 1 mg to about 10 g for a body weight of about 60 kg. The inventors have actually found that chemical substances acting efficiently either individually or in combination (with each other or with other glycosylation inhibitors) as inhibitors of the glycosylation process, especially as agents inhibiting glucosidase I, glucosidase II, endoplasmic reticulum mannosidase I and II, and Golgi mannosidase I, or more generally those selected from the group consisting of the substances listed in Table 1 or mixtures thereof, permit the normal expression and traffic of misfolded mutant proteins, ensuring the appropriate functioning of these proteins in living cells. Without wishing to be bound to any particular theory, we believe that in the applications cited above, chemical compounds of the recommended classes are suitable, alone or in combination with other chemical compounds of the same classes and/or with other theoretically active agents, for abolishing the retention or intracellular degradation of "misfolded" (i.e. incorrectly coiled or folded) proteins, which do not have the correct three- dimensional structure. Morbid mutations of genes often exert their pathogenic properties by means of modifications to the primary, secondary, tertiary and quaternary structures and any gain of post-translational modification (such as glycosylation, phosphorylation, methylation, etc.) of the corresponding protein. In most cases, these mutations respect the reading frame of the coding region (missense mutations, in-frame insertions, duplications and deletions, and certain splicing mutations). However, some of these mutations may create a premature stop codon in the reading frame (nonsense mutations, out-of-frame insertions, duplications and deletions and certain splicing mutations). When the resulting mutant proteins enter the secretory pathway of the cell, their incorrect conformation generally leads to them being targeted to a so-called degradation pathway. They are likely to be retained or misdirected, and they escape the normal control system. The molecules of the classes indicated above can block this degradation process and allow the intra- and extracellular trafficking of mutant proteins. They constitute ligands with pharmacological selectivity, and are able to restore the correct targeting and functioning of misfolded proteins, including receptors. There is in no way a direct interaction with the mutation. We infer that they can be included in the development of new treatments for genetic diseases ("conformational diseases") by means of chemical complementation of the effects of the morbid alleles on intracellular protein trafficking.

To date, only a few of such substances inhibiting a glycosylation step have been used by a few authors, and notably either as tools in experimental protocols, or for the partial complementation of cells overexpressing morbid alleles, in an experimental system of stable or transient transfeetion or using a tumoral cell line. None of these drugs has been successfully used in polyclonal cells naturally carrying disease-causing alleles. Figures 1, 2a and 3 prove that the components of the aforementioned classes provide the first demonstration of chemical complementation of any kind in polyclonal cells, whether ex vivo or in vitro, naturally expressing a disease-causing allele.

According to this invention, the use of such substances can be accomplished in vitro, ex vivo and in vivo (e.g. with antibodies, with injection of EFNγ (IMUKUN)). The treatment is based on the chemical complementation of a cellular phenotype, which could be used in prevention or curative treatment, in a temporary or prolonged manner.

It is likely that glycosylation inhibitors or glucosidase inhibitors act on one or several sites of N- glycosylation, within proteins having an unsuitable three-dimensional structure, that are recognized by the protein recognition system or act directly on the system for recognizing misfolded proteins in themselves. In both cases, these glycosylation inhibitors operate in a general manner, which is not restricted to the IFNγR2 protein, as their mode of action is of a general nature and may therefore have to do with a large number of disease-causing alleles encoding many different mutant proteins. In such a use in vivo or ex vivo, the dose unit of miglustat and/or of kifunensine should preferably be such as to provide approximately 8 μg to 80 mg active compound or combination of active compounds per liter of serum, whereas the adrnirύstration thereof can be prescribed for an application on a shorter duration than usual in the field. Without wishing to limit the invention to a particular number of administrations per day, generally two to four administrations per day are envisioned.

For example, however only by way of non-limiting example, the oral administration of three doses of about 1 mg to about 2 g each per day of miglustat or of another similar glycosylation inhibitor could be recommended. For castanospermine or for similar glycosylation inhibitors of the same category, substantially the same doses can be recommended, but preferably the doses can be of the order of 200 mg to 2 g in capsules, with the optimal dose being approximately 1 g. Actual dosage levels of active ingredients in the pharmaceutical compositions prepared according to this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient or other living matter, as well as particular compositions, and a particular mode of administration. The selected dosage level will depend upon the activity of the particular compound (which can be checked and assessed by those with an ordinary skill in the art on the basis of their own general knowledge and of the present description), the route of administration, the severity of the condition to be treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved, to be determined within the sound judgment of the clinician.

Pharmaceutically active formulations or compositions may also contain one or several other therapeutically effective substances, together with adjuvants (such as preservatives, wetting agents, emulsifying agents, and dispersing agents), or conventional pharmaceutically acceptable vehicles and/or supports/carriers.

The composition of this invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions.

The pharmaceutical compositions of this invention can be administered to humans and other mammals orally, rectally, parenterally (i.e. intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray. In specific embodiments, the composition is to be administered subcutaneously, orally, or intravenously.

If desired for more effective distribution, the compound(s) can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres. This invention can give rise to very diverse applications, and it may for example be used for the prevention and the treatment of germ-line and somatic, genomic and mitochondrial diseases in animals, including human beings, in plants and in fungi. The treatment using means according to this invention is primarily aimed at and effective for the chemical complementation, and/or for the diagnosis of the efficiency of a chemical complementation, of a cellular phenotype, which could be used in prevention or curative treatment, in a temporary or prolonged manner. In practice, it should be emphasized that the complementation carried out by the means according to this invention is a total complementation, and not only a partial one, as according to the teaching of C. Norez et al. (supra). Moreover, it was obtained on both fresh polyclonal and cultured clonal cells from diverse origins, whether hemopoietic (blood, B cell line) and extra-hemopoietic (fibroblasts).

DESCRIPTION OF PREFERRED EMBODIMENTS

The description which follows is intended to illustrate certain embodiments of the invention and are not limiting in nature. They refer to specific experiments conducted by the inventors, unless otherwise stated. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims. Germline mutations may cause human disease by various mechanisms. Missense and other in-frame mutations may be deleterious because the mutant proteins are not correctly targeted, do not function correctly, or both. We studied a child with mycobacterial disease due to homozygosity for a novel in-frame microinsertion in IFNGR2. Most of the IFN-γR2 protein was retained within the cell, and that expressed on the cell surface had an abnormally high molecular weight. Consequently, the patient's cells did not respond to IFN- . We tested 29 compounds affecting maturation by N-glycosylation in the secretory pathway. Up to 13 of these compounds reduced the molecular weight of surface-expressed IFN-γR2 mutant molecules and restored cellular responsiveness to IFN-γ. Some of these compounds also complemented missense mutations in other genes, such as IL2RG, associated with X-linked severe combined immunodeficiency. Modifiers of N-glycosylation can therefore complement human cells carrying in-frame mutations in genes encoding proteins subject to trafficking via the secretory pathway. Some of these compounds are available for clinical use, paving the way for clinical trials of chemical complementation for various human genetic traits.

Mendelian susceptibility to mycobacterial diseases (MSMD) is a primary immunodeficiency that selectively weakens host resistance to mycobacteria, such as BCG vaccines and environmental mycobacteria 1^ . There are six known MSMD-causing genes (IL12B, ILlZRBl, NEMO, IFNGRl, IFNGR2, STATl), all involved in IFN-γ-mediated immunity. These genes display high levels of allelic heterogeneity, resulting in the definition of 13 different disorders 3"5 . We studied a Lebanese child with MSMD (Pl) (Fig. Ia). Pl is homozygous for a previously unidentified in-frame microduplication of six nucleotides (382-387) in IFNGR2 (designated 382- 387dup). This mutation is predicted to result in the duplication of amino acids T128 and M129 in the protein (WVTMPW > WVTMTMPW) (Fig. Ib, c). This duplication neither creates nor deletes known consensus sites for posttranslational modifications; in particular, it is not a gain-of-glycosylation mutation 6 . It was not found in 50 and 77 unrelated healthy individuals of European and Arabian descent, respectively. The parents and one sibling are heterozygous for the mutation, which was not found in the other sibling. No IFN-γ-activated sequence (GAS)-binding proteins were detected in electrophoretic mobility shift assays (EMSA) with EBV-transformed B cells from Pl stimulated with IFN-γ (Fig. Id). Similarly, no such proteins were detected in cells from two previously described children homozygous for other mutations: an in-frame 27 bp microdeletion of nucleotides 663 to 689 (designated 663del27, P2) 6 , and the 278delAG null frameshift deletion (negative control, C-) 7 (Fig. Ic, d). IFN-γR2-deficient EBV-transformed B cells homozygous for the 278delAG allele 7 were complemented by the wild-type (WT) allele, but not by any of the three mutant IFNGR2 alleles (Fig. Ie). Thus, Pl, like P2, carried an in-frame IFNGR2 mutation causing complete functional IFN-γR2 deficiency 6. We explored the intracellular trafficking of mutant IFN-γR2 from Pl and P2, by transfecting Hek293 cells with IFNGR2 alleles encoding V5- tagged molecules and detecting IFN-γR2 in the lysates of these cells by western blotting (Fig. 2a). Both mutant proteins segregated into two major groups of low- (around 55 kDa) and high- (about 80 kDa) molecular weight (MW). In both cases, most proteins were of low MW and failed to mature normally, as shown by their sensitivity to Endo-H 8 (Fig. 2a). The high MW of mature, Endo-H-resistant proteins reflected excessive N-glycosylation, as shown by their digestion with PNGase-F (data not shown). This indicated that the two IFNGR2 in-frame alleles encoded misfolded proteins that were abnormally targeted, or N-glycosylated, or both. Glucosidases I and II contribute to the folding of glycoproteins in the endoplasmic reticulum (ER), their concomitant quality control by calnexin-cahreticulin, and their subsequent trafficking 9Λ5 . ER-mannosidase I is also involved in glycoprotein folding, quality control, and trafficking (Fig. 2b) 9"15 . We therefore hypothesized that NB-DNJ (Zavesca®) and castanospermine 16 — which inhibit glucosidases I and II — and kifunensine 16 — an inhibitor of ER-mannosidase I — may improve the glycosylation or trafficking of the non-native IFN-γR2 molecules (Fig. 2b, Table 1 below). NB-DNJ and castanospermine had no detectable impact on WT IFN-γR2 glycoptoteins (about 60 kDa), but most of the mutant molecules, including Endo-H- resistant proteins of high MW, displayed a shift in MW to about 60 kDa (Fig. 2a). In contrast, kifunensine impaired the maturation of both WT and mutant molecules, which became sensitive to Endo-H (Fig. 2a). We assessed the surface expression of IFN-γR2, by biotinylating surface-expressed proteins in IFN-γR2-deficient fibroblasts 7 transfected with various V5-tagged IFN-γR2 constructs (Fig. 2c, d). Most WT molecules were expressed at the surface of the cell, except in the presence of kifunensine, which reduced their surface expression. In contrast, only a small fraction of mutant molecules were surface-expressed, even after treatment with NB-DNJ, castanospermine, or kifunensine. However, following treatment with any of these three drugs, mutant IFN-γR2 molecules of about 60 kDa, corresponding to the MW of WT molecules, appeared on the cell surface (Fig. 2c). The maturation of the 382- 387dup and 663del27 IFN-γR2 molecules in the presence of NB-DNJ, castanospermine or kifunensine thus resulted in the expression of a detectable fraction of cell surface-expressed receptors with a MW similar to that of WT proteins under the same conditions - with 663del27 molecules being somewhat lighter owing to the deletion of nine residues.

We therefore investigated whether cells from Pl or P2 could be functionally complemented, by culturing EBV-transformed B cells with NB- DNJ, castanospermine or kifunensine for 16 hours, before IFN-γ stimulation for 30 minutes (Fig. 3a). WT cells responded to IFN-γ in the presence of each of these compounds, as shown by EMSA. Cells from a negative control (C-) 7 and cells from P2 (663del27) did not respond to IFN-γ, even in the presence of these drugs. However, cells from Pl (382-387dup) regained responsiveness to IFN-γ upon treatment with NB-DNJ or castanospermine. Surprisingly, they responded to an even greater extent, with responses reaching normal levels, when treated with kifunensine (Fig. 3a). We then assayed a more distal event, using flow cytometry to test the induction of HLA-DR on the surface of SV40-transformed fibroblasts (Fig. 3b). Cells from Pl, unlike those from all other patients, responded normally to IFN-γ following treatment with any of the three molecules tested, kifunensine was particularly efficient, as it was effective at concentrations below 1 μM and no toxicity was observed, even at millimolar concentrations. Finally, we stimulated freshly prepared peripheral blood mononuclear cells (PBMCs) with live BCG, BCG plus IFN-γ, or IFN-γ alone, and determined IL-12p40 and p70 levels by ELISA 17 , in the presence or absence of the only commercially available compound, NB-DNJ (Zavesca®). NB-DNJ restored the responsiveness to BCG, and, to a greater extent, to BCG plus IFN-γ, of PBMCs from Pl (Fig. 5). Thus, IFN-γR2-deficient cells from Pl, whether immortalized lymphoid and fibroblastic cell lines or freshly prepared blood cells, can be complemented by modifiers of glycosylation.

We assessed the specificity of enzyme inhibition, by transfecting human cells defective for the glucosidase I or glucosidase II gene with various tagged EPN-γR2 constructs 18-20 . The 382-387dup IFN-γR2 molecules were not of normal MW (60 kDa) in cells deficient for either glucosidase I or glucosidase π, suggesting that NB-DNJ and castanospermine complemented the patient's cells by inhibiting at least these two enzymes (data not shown). We then tested most of the commercially available compounds known to inhibit glycosylation, at various steps (Table 1 below, Fig. 2b). We incubated SV-40-transformed fibroblasts separately with each of the 29 drugs and IFN-γ for 72 hours. Cell-surface HLA-DR expression was assessed by flow cytometry (Fig. 6). None of the compounds impaired the response of control cells or restored IFN-γ responsiveness in fibroblasts from P2 and C-. However, 13 compounds restored IFN-γ responsiveness in Pl cells (Suppl. Fig 2). Six drugs fully complemented Pl cells when tested at the recommended inhibitory concentrations (NB-DNJ, castanospermine, NM- DNJ, NB-DMJ, kifunensine, and 2-5-DM) (Fig. 6). Eight other compounds had a detectable, but weaker effect. In contrast, 16 inhibitors did not complement the patient's cells (these compounds included tunicamycin, an inhibitor of N-glycosylation that blocks assembly of the lipid-linked oligosaccharide precursor). The inefficacy of australine, DMJ, and swainsonine suggested that the inhibition of glucosidase I, ER-mannosidase, or Golgi-mannosidase II alone was insufficient to complement cells from Pl (Table 1 below, Fig. 2b). We tested combinations of two compounds, each used at its minimal effective concentration (Tables 1 and 2 below). We observed no detectable inhibitory effect, but additive effects were observed for a number of combinations and synergic effects were observed in several cases. Interestingly, australine with bromoconduritol or miglitol (Table 1) did not complement the cells, suggesting that NB-DNJ and castanospermine also inhibited another enzyme. Further evidence to support this hypothesis is provided by the synergy conferred by NB-DGJ. Nonetheless, australine and castanospermine acted in synergy, suggesting that a potent and combined inhibition of glucosidase I and II is efficient. Paradoxically, kifunensine and swainsonine together complemented the cells whereas DMJ alone did not (Table 1). The complementation of cells from Pl may reflect the combined inhibition of several enzymes, such as glucosidases I and II, and ER-mannosidase I, in particular.

In an attempt to generalize these findings, we tested fibroblasts from another patient (P3) previously reported to carry the T168N missense IFNGR2 mutation 6. Tunicamycin complements P3 cells, because T168N is a gain-of-glycosylation mutation 6 , but does not complement Pl and P2 cells (data not shown). Three other compounds also complemented P3 fibroblasts in terms of IFN-γ-induced HLA-DR expression: DNJ, NJ-I-S, and DM (Fig. 4a). A modest effect was observed with NB-DNJ and castanospermine (data not shown). These differences in efficacy of various modifiers of N- glycosylation in Pl and P3 cells likely reflect intrinsic differences in the glycosylation, folding, and trafficking of the two mutant IFN-γR2 glycoproteins, as T168N is a gain-of-glycosylation mutation, whereas 382- 387duρ is not. We then studied 10 missense IL2RG mutations associated with another primary immunodeficiency, X-linked severe combined immunodeficiency, which is characterized by a lack of autologous T-cell development. The IL2RG gene encodes the surface-expressed common cytokine γ chain, which is shared by at least six interleukin receptors. The mutations studied were not gain-of-glycosylation mutations. Five mutant EBV-transformed B-ceU lines bearing E68K, Y105C, E68G, R222C and Q144P alleles displayed an increase in IL-2R-γ chain expression at the cell surface following treatment with kifunensin, as shown by flow cytometry (Fig. 4b). We also transiently expressed V5-tagged WT, D39N, C62G, E68G, L172Q and R222C IL2RG alleles in Hek 293 cells. Cell surfaces were biotinylated and streptavidin precipitates analyzed by western blotting. Kifunensine increased IL-2R-γ expression and restored the expression of this molecule at the cell surface (Fig. 4c). We then established stable transfectants, which expressed both WT and mutant IL-2R-γ proteins on the cell surface (Fig. 4d). Remarkably, in the presence of kifunensine, the molecular weight of the mutant proteins was similar to that of WT proteins (Fig. 4d). We then treated EBV-transformed B cells from a child with a primary immunodeficiency known as leukocyte adhesion deficiency type I, or CD18 deficiency. The cells carrying the G284S missense mutation in ITGB2 were treated separately with castanospermine, kifunensine, NM-DNJ, and DM. Dose-dependent complementation was observed with kifunensine (Fig. 4e). Finally, no complementation was observed with seven missense IL12RB1 mutations 21 or with the delF508 CFTR mutation 22 (data not shown). Nevertheless, twelve in-frame mutations in three disease-causing genes were successfully complemented by up to 13 drugs affecting the N-glycosylation process.

Chemical chaperones, such as DMSO and glycerol, have been reported to complement some cell lines in vitro, albeit with low specificity and high toxicity, preventing their use in clinical trials 22-25 . None of these compounds complemented the cell lines tested in our study (data not shown). Curcumin was reported to complement delF508-CFTR in one study 24 , but not in another 25 , and it did not complement the cell lines studied here (data not shown). Pharmacological chaperones, designed to specifically complement target proteins, are less toxic and more promising, but their specificity prevents their broad application 22Α27 . Our report of modifiers of N-glycosylation acting as chemical chaperones is consistent with the well established role of glycans in protein folding and quality control in the endoplasmic reticulum 9"13 ' 15. Our successful complementation of misfolding mutations by this novel class of chemical chaperones opens up new avenues of research. Several modifiers of N-glycosylation were effective, and they complemented several misfolding mutations in several genes. Moreover, unlike most other previously reported chemical and pharmacological chaperones, which were active on transfected or tumor cells over-expressing the mutant protein, these compounds complemented germline cells in vitro and ex vivo. Our findings raise hopes that chemical modifiers of N-glycosylation could be curative in a fraction of patients bearing mutations in genes encoding proteins subject to trafficking via the secretory pathway, not only in patients with gain-of-glycosylation mutations 6 but also in patients bearing other types of misfolding mutations, kifunensine is a good candidate, as it has a wide window of efficacy without toxicity in vitro. Derivatives of castanospermine are also potentially useful 28. NB-DNJ is especially promising, as no overt toxicity has been reported for this molecule in human clinical trials 29 and this molecule is currently used in patients with Gaucher's disease 3031.

Table 1 : Chemical compounds used and their known targets

to

O

Notes for Table 1:

Chemicals compounds complementing the 382-387 mutation are indicated in black in column "C". The fall name of each chemical compound is given in the column "compound" and its abbreviation is indicated in column "Abbr.". GIu-I, GIu-II, ERM-I, ERM-2, M9M, GM-I, GnT-I and GM-II are enzymes known to be associated with the maturation of N-glycosylation. The effects of the chemical compounds tested on these enzymes are indicated in color: red = inhibition, blue = no effect and white = undescribed effects on N-glycosylation enzymes.

Ref:

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2. Elbein, A. D. Glycosidase inhibitors: inhibitors of N-rinked oligosaccharide processing. i^SEδ J 5, 3055-63 (1991).

3. Shailubhai, K., Pratta, M. A. & Vijay, I. K. Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland. Biochem J247, 555-62 (1987).

4. McDowell, W. & Schwarz, R. T. Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie 70, 1535-49 (1988).

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6. Helenius, A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. MoI Biol Cell 5, 253-65 (1994).

7. Liu, Y., Choudhury, P., Cabral, C. M. & Sifers, R. N. Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. J Biol Chem 274, 5861-7 (1999).

8. Spiro, R. G. Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation. Cell MoI Life Sd 61, 1025-41 (2004).

9. Platt, F. M., Neises, G. R., Karlsson, G. B., Dwek, R. A. & Butters, T. D. N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing. J Biol Chem 269, 27108-14 (1994).

10. Tsuda, H., Tokunaga, F., Nagamitsu, H. & Koide, T. Characterization of endoplasmic reticulum-associated degradation of a protein S mutant identified in a family of quantitative protein S deficiency. Thromb Res 117, 323-31 (2006).

11. Tropea, J. E. et al Mannostatin A, a new glycoprotein-processing inhibitor. Biochemistry 29, 10062-9 (1990).

12. Isa, X., Tran, T., Simsek, E. & Block, T. M. The alkylated imino sugar, n-(n-nonyl)-deoxygalactonojirimycin, reduces the amount of hepatitis B virus nucleocapsid in tissue ►— culture. / Virol 11, 11933-40 (2003).

13. Kanfer, J. N., Legler, G., Sullivan, J., Raghavan, S. S. & Mumford, R. A. The Gaucher mouse. Biochem Biophys Res Commun 67, 85-90 (1975).

14. Datta, S. C. & Radin, N. S. Normalization of liver glucosylceramide levels in the "Gaucher" mouse by phosphatidylserine injection. Biochem Biophys Res Commun 152, 155-60 (1988).

15. Woynarowska, B. et al. Inhibition of human ovarian carcinoma cell- and hexosaminidase-mediated degradation of extracellular matrix by sugar analogs. Anticancer Res 12, 161-6 (1992).

16. Shinozaki, K. et al. Improvement of insulin sensitivity and dyslipidemia with a new alpha-glucosidase inhibitor, voglibose, in nondiabetic hyperinsuhnemic subjects. Metabolism 45, 731-7 (1996).

17. A glucosidase inhibitor (Toronto Research Chemicals Inc.)

18. An inhibitor of glucosidase I (Toronto Research Chemicals Inc.)

19. A potent inhibitor of alpha-mannosidase (Toronto Research Chemicals Inc.)

20. A selective and very strong inhibitor of beta-glucosidase (Toronto Research Chemicals Inc.)

21. A glucosidase inhibitor (Toronto Research Chemicals Inc.)

22. An inhibitor of yeast α-glucosidase (Toronto Research Chemicals Inc.) •

Table 2: Observation of the effects of pairs of chemical compounds

SV-40-transfoπned fibroblasts from Pl, bearing mutation 382-387dup were incubated for 16 hours in complete culture medium supplemented with IFNγ (2.4 x 104 IU/ml). The following compounds were added, in pairs, to the culture medium: (1) NB-DMJ (0.375 mM), (2) NB-DNJ (0.375 mM), (3) castanospermine (0.5 mM), (4) swainsonine (0.962 mM) (5) kifunensine (0.125 μM), (6) DMJ (0.483 mM), (7) australine (0.338 mM), (8) NB-DGJ (1.75 mM), (9) DNJ (2.36 mM), (10) DIM (3.86 mM), (11) mannnostatin A (0.15 mM), (12) miglitol (0.93 mM), (13) NJl-S (0.37 mM), (14) NM-DNJ (1.4 mM), (15) bromoconduritol (mixture of isomers) (0.92 mM) and (H) distilled water. The surface expression of EDLA-DR molecules was determined by flow cytometry, using a specific antibody, 72 hours later.

Methods

Affected individuals

We studied three children from three unrelated families with severe mycobacterial disease. All three families were consanguineous. Patient 1 (Pl) developed disseminated M. avium disease at the age of two years. She tested negative for HIV, CMV and EBV in serological tests. Despite anti- mycobacterial treatment with rifamycin, moxifloxacine, hydroxychloroquine, clofazimine and ethambutol, Pl died at the age of five years, due to dissemination and uncontrolled M. avium infection. Neither of her two brothers has ever developed mycobacterial infection. Patient 2 (P2) developed disseminated M. avium infection at the age of three years, which responded to antibiotics. He has received continuous treatment with these antibiotics for the last seven years. Patient 3 (P3) developed disseminated BCG vaccine infection at the age of one month; he is now 2.5 years old and is still on antibiotic treatment. P2 and P3 have been described elsewhere 6. This study was approved by the local institutional review committee (CCPPRB), and informed consent was obtained from all families.

DNA and RNA extraction, cDNA synthesis, and PCR amplification

Genomic DNA and total RNA were extracted from Epstein-Barr virus- transformed lymphoblastoid cell with Trizol (Gibco-BRL). RNA was reverse transcribed in the presence of oligo (dT), with Superscript II reverse transcriptase (Invitrogen Corporation, Paisley, UK). The IFNGRl and IFNGR2 cDNAs were amplified using appropriate pairs of primers (PCR conditions available upon request).

Cell culture and transf ection

EBV-transformed B cells and SV40-transformed fibroblasts were cultured in RPMI 1640 supplemented with 10% heat-inactivated bovine fetal serum (Gibco-BRL) (complete medium). Two days before transf ection, 3x1 O^

SV40-transformed fibroblasts were plated in 35 mm dishes (Nunc). SV40- transformed fibroblasts and 293T/17 cells were transformed by lipof ection

(Lipofectamine Plus Reagent; Invitrogen) according to the manufacturer's instructions. Aliquots of IO^ EBV-transformed B cells were transf ected by electroporation with a pulse at 300 V, 900 μF, R ∞, in 400 μL of complete medium, with 30 μg of plasmid. Electroporated cells were tested by EMSA the following day. Transfected cells were used for flow cytometry, western blotting or confocal microscopy within two days.

Cytokines, enzymes, inhibitors and antibodies

We used recombinant non-glycosylated human IFNγ (Imukin), recombinant IFNα2b (R&D Systems), anti-V5-antibody (1/5000; Invitrogen), Endo-H (New England Biolabs), PNGase-F (New England Biolabs), NBDNJ (Sigma), castanospermine (Sigma), mousse antibody to human IL2RG conjugated to phycoerythrin (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjugated mouse antibody against human CD18 (Immunotech), streptavidin-agarose (Invitrogen), phycoerythrin-conjugated mouse anti- HLA-DR antibody (Becton Dickinson) and horseradish peroxidase (HRP)- labeled anti-mouse Ig antibody (1/10000; Amersham Biosciences), NBDNJ and australine (Alexis Biochemical). All other drugs were obtained from Toronto Research Chemicals Inc. All chemical compounds were dissolved or suspended in water.

Expression vectors

We used AmpliTaq DNA polymerase (Applied Biosystems) to amplify cDNA fragments encoding human IFNγ-R2 and other genes. PCR products were digested with BglH/Xmal and inserted into peGFP-Nl (Clontech) or the pcDNA3 (Invitrogen) vector with or without a V5/His Tag, by a directional topoisomerase-based method. We used the Stratagene kit, according to the manufacturers' instructions, for direct mutagenesis.

Cell-surface biotinylation

Two days after transfection, SV40-transformed fibroblasts were labeled by incubation for 30 minutes at 4°C in PBS pH 8.0, with or without sulfo-NHS-LC-Biotin (Pierce). They were then washed twice in PBS. The cross-linking reaction was stopped by adding 50 mM NH4CI in PBS. Cells were harvested by centrifugation in an Eppendorf tube (1.5 ml) and washed three times with PBS. Biotinylated SV40-transformed fibroblasts were lysed with the appropriate buffer for immunoprecipitation (as recommended by the manufacturer) and incubated overnight with streptavidin-agarose. Immunoprecipitates were analyzed by SDS-PAGE, using an anti-V5- antibody and the enhanced chemiluminescence system for detection.

Electrophoretic mobility shift assay

EBV-transformed B cells were analyzed by EMSA, as previously described 6.

Endoglycosidase H digestions Endoglycosidase-H (Endo-H) specifically cleave N-linked carbohydrates. Endo-H cleaves the immature oligosaccharide core added in the endoplasmic reticulum, but not the carbohydrate moieties matured in the Golgi apparatus. Two days after transfection, SV40-transformed fibroblasts were lysed and digested with Endo-H in the appropriate buffer (according to the manufacturers' instructions). Lysates were digested for three hours or overnight and the products analyzed by SDS-PAGE and chemiluminescence analysis.

Flow cytometry The HLA-DR expression profile of SV40-transformed fibroblasts was investigated by flow cytometry, as previously described 6.

Electrophoresis and western blotting

SDS-PAGE was carried out with 10% polyacrylamide gels (separating gel buffer: 0.375 M Tris pH 8.8, 0.01% SDS; stacking gel buffer: 0.5 M Tris, pH

6.8, 0.01% SDS; Laemmli buffer: 25.5 mM Tris pH 8.3, 0.18 M glycine, 0.13%

SDS). Samples were washed in PBS, boiled for 5 min at 1000C in stacking gel buffer with 20% glycerol, 4% SDS, 1.4 M β-mercaptoethanol and bromophenol blue. They were then subjected to SDS-PAGE and the resulting bands transferred to PVDF membranes (0.2 μm Bio-Rad) by electroblotting for two hours at 1.5 mA/cm^ in transfer buffer (25.5 mM Tris, 0.18 M glycine and 20% methanol). The membrane was blocked by incubation for one hour in 5% BSA in PBS, rapidly washed in 0.033% Tween in PBS, and incubated overnight with an appropriate primary antibody diluted in 0.027% Tween, 1% BSA in PBS. The membrane was then washed five times in 0.035% Tween in PBS and incubated for 30 minutes with horseradish peroxidase-conjugated secondary antibody in 0.027% Tween, 1% BSA in PBS. Bound antibody was detected with ECL detection reagents (Amersham Biosciences), on BioMax MR film (Kodak).

PBMC cultures and activation by BCG

Peripheral blood mononuclear cells were obtained from heparin- treated whole blood from the proband and control, by centrifugal separation through a Ficoll gradient and washing with RMPI 1640 (GIBCOTM, Carsbad, CA, USA). We cultured 2xlO5 cells in triplicate in 200 ul of RMPI 1640 supplemented with 10% FCS (GIBCOTM) per well, in 96-well plates (Nunc®, Roskilde, Denmark). The plate Was incubated for 48 hours at 370C under an atmosphere of 5% CO2/95% air; and was treated with NBDNJ or castanospermine (200 μg/ml) at 370C, with medium alone, with live BCG (M. bovis BCG, Pasteur substrain) at a multiplicity of infection (MOI) of 20, with BCG/leukocytes and with BCG plus IFNγ (5,000 IU/ml) (Imukin, Boehringer Ingelheim), as described elsewhere 6 . Supernatants were collected after 48 h of activation and were stored frozen at -800C. Finals results were standardized per million PBMC, and are expressed as pg/ml/ IOSPBMC

Cytokine ELISA

Cytokine concentrations were analyzed by ELISA, using the human Quantikine IL-12ρ40 and IL-12ρ70 kit from R&D Systems and the human Pelipair IFNγ kit from Sanquin, according to the manufacturers' instructions. Optical density was determined with an automated MR5000 ELISA Reader (Thermolab Systems). A non-linear four-parameter logistic (4PL) calibration model was used for quantitative analysis. Intermediate results for each cytokine are expressed in pg/ml, as previously described 6. References

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28. Torres, G. BUCAST: another new antiviral. GMHC Treat Issues 9, 4-5 (1995). 29. Tierney, M. et al. The tolerability and pharmacokinetics of N-butyl- deoxynojirimycin in patients with advanced HTV disease (ACTG 100).

The AIDS Clinical Trials Group (ACTG) of the National Institute of

Allergy and Infectious Diseases. / Acquir Immune Defic Syndr Hum

Retroυirol 10, 549-53 (1995). 30. Cox, T. et al. Novel oral treatment of Gaucher's disease with N- butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis.

Lancet 355, 1481-5 (2000). 31. Dwek, R. A., Butters, T. D., Platt, F. M. & Zitzmann, N. Targeting glycosylation as a therapeutic approach. Nat Rev Drug Discov 1, 65-75 (2002).

Claims

1. A use of a therapeutically effective amount of at least one chemical compound for the preparation of a pharmaceutical composition for the diagnosis, prevention and /or treatment diseases associated with genomic or mitochondrial, germline or somatic mutations in animals, including human beings, in plants and in fungi, wherein said at least one chemical compound is a molecule inhibiting any step of the glycosylation process as an inhibitor selected from the group consisting of inhibitors of glucosidase and inhibitors of mannosidase.
2. The use according to claim 1, for the preparation of a pharmaceutical composition for the prevention and/or treatment of genetic defects or inherited diseases in such a living matter, in vitro or ex vivo, whereas said at least one chemical compound is at least one chemical compound capable of effecting a substantially complete chemical complementation of misfolding mutations in polyclonal cells.
3. The use according to claim 1, wherein the said at least one chemical compound is a molecule capable of complementing a IFNGR2 defect in animals, including human beings, as well as in plants and in fungi.
4. The use of claim 1, wherein the said at least one chemical compound is an inhibitor of the synthetic pathway of the N-linked carbohydrates and/or of any other pathway encoding enzymes selected from the group consisting of glucosidases and mannosidases
5. The use according to claim 2 or 3, wherein the said at least one chemical compound is a chemical compound inhibiting enzymatic targets selected from the group consisting of glucosidase I, glucosidase II, endoplasmic reticulum mannosidase I and II, and/or Golgi mannosidase.
6. The use according to claims 1 to 5, wherein the said at least one chemical compound is selected from the group consisting of: N-butyldeoxynojirimycin, castanospermine, kifunensine, N-butyl- deoxymannojirimycin, deoxynojirimycin, deoxynojirimycin, 1,4- dideoxy-l,4-immo-D-mannitol, N-methyl-deoxynojMmycin, nojirimycin-1 -sulfonic acid, N-(n-nonyl)deoxynojirimycin, and australine, or mixtures thereof.
7. The use according to claim 1, wherein said use is for the preparation of a pharmaceutical composition for the detection and /or treatment of genetic defects or inherited disorders in a living matter.
8. The use according to claim 5, wherein the pharmaceutical composition is for preventing or treating at least one mutation selected from the group consisting of 663del27, 382-387dup, IL2RG, TGB2 (CD18), T168N, and analog mutations in the IFNGR2 gene.
9. An inhibitor of the glycosylation process in a living matter, for use in a medicament for the prevention or the treatment of genetic defects or inherited diseases in animals, including human beings, in plants and /or in fungi.
10. The inhibitor according to claim 9, wherein the said inhibitor is selected from the group consisting of: N-butyldeoxynojirimycin, castanospermine, kifunensine, N-butyl-deoxymannojirimycin, deoxynojirimycin, deoxynojirimycin, l,4-dideoxy-l,4-imino-D- mannitol, N-methyl-deoxynojirimycin, nojirimycin-1-sulfonic acid, N-
(n-nonyl)deoxynojirimycin, and australine, or mixtures thereof.
11. A pharmaceutical composition, which comprises the inhibitor according to claim 9 or 10.
12. The pharmaceutical composition according to claim 11, which further comprises a pharmaceutically acceptable carrier.
13. The pharmaceutical composition according to claim 11, which comprises as a dose unit appropriate for providing approximately 80 mg active compound or combination of active compounds per liter of serum.
14. The use of an inhibitor according to claim 9, or of a pharmaceutical composition according to anyone of claims 11-13 for the preparation of a pharmaceutical composition for the prevention and/or treatment of genetic defects or inherited diseases in a living matter.
PCT/IB2006/004074 2006-12-08 2006-12-08 Use of inhibitors of the glycosylation process for the prevention and treatment of genetic diseases WO2008068548A1 (en)

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US8450345B2 (en) 2009-02-23 2013-05-28 The Chancellor, Masters And Scholars Of The University Of Oxford Iminosugars and methods of treating viral diseases
US9044470B2 (en) 2009-02-23 2015-06-02 United Therapeutics Corporation Iminosugars and methods of treating viral diseases
US8426445B2 (en) 2009-06-12 2013-04-23 United Therapeutics Corporation Iminosugars and methods of treating bunyaviral and togaviral diseases
US8748460B2 (en) 2009-06-12 2014-06-10 United Therapeutics Corporation Iminosugars and methods of treating togaviral diseases
CN102417515B (en) 2010-09-28 2014-03-19 河北大学 Thiazole (piperazine) azululanone azasugar derivative and synthesis method and application thereof to medicinal preparation
CN102417515A (en) * 2010-09-28 2012-04-18 河北大学 Thiazole (piperazine) azululanone azasugar derivative and synthesis method and application thereof to medicinal preparation

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