Patent Abstract:
inhibitors of the endoplasmic reticulum associated degradation pathway , particularly inhibitors of mannosidase i , are used for the preparation of a medicinal product intended to treat sarcoglycanopathies .

Detailed Description:
a 1075 bp dna fragment carrying exons 2 and 3 of the sgca gene obtained by bamhi - sfii digestion of a phage containing the sgca gene was amplified by pcr and then inserted into the psp72 plasmid vector ( promega ). the h77c mutation was generated in exon 3 by site - directed mutagenesis using the following primers : 5 ′- gcccaggtggctg tgc tacacacagcgca - 3 ′ ( seq id 1 ) and 5 ′- tgcgctgtgtgta gca cagccacctgggc - 3 ′ ( seq id 2 ). presence of the point mutation was confirmed by sequencing . a 5 ′ bglii - bamhi fragment of about 4 kb and a 2981 bp 3 ′ sfii - spei fragment were cloned into the psp72 vector on each side of the mutant insert . the loxp - neo r - loxp cassette from the pgem - neo r vector was inserted via the ecorv site in intron 3 and a thymidine kinase ( tk ) cassette was inserted downstream of the 3 ′ fragment to produce the final recombinant vector . the recombinant vector ( 25 μg ) was linearised by sali digestion and introduced into se 129sv cells by electroporation . the dna of g418 resistant colonies was then isolated and analysed by pcr or southern blot to check for recombination events . two distinct recombinant clones ( ib4 and viiicii ) were injected into c57b1 / 6 blastocysts and chimera mice were generated . chimeric males were crossed with c57b1 / 6 females to produce heterozygous mice . the neo r cassette was eliminated by crosses with the deleter strain ( 15 ). the resulting mice in which the neo r cassette had been excised were then bred to produce homozygous mutant mice . genotyping was performed by pcr on tail dna extracted using the qiagen dneasy tissue kit , using the upstream primers a - sarcoq5 ′: 5 ′- tataaccctggcttcctcta - 3 ′ ( seq id 3 ) and testneo 1 5 ′- cgaattcgccaatgacaagacgct - 3 ′ ( seq id 4 ) and the downstream primer a - sarcoq3 ′ 5 ′- tagtggctcatgcctttaat - 3 ′ ( seq id 5 ), generating a 639 bp product for the mutant allele carrying the neo r cassette and a 484 bp product for the wild - type allele , using the following pcr conditions : 94 ° c . for 3 minutes , then 30 cycles consisting of 94 ° c . for 30 s , 61 ° c . for 40 s and 72 ° c . for 1 minute , and then 3 minutes at 72 ° c . after excision of the neo r cassette , genotyping was performed with the a - sarcoq5 ′ ( seq id 3 ) and a - sarcoq3 ′ ( seq id 5 ) primers , generating a 575 bp product for the mutant allele . to check that the h77c mutation was present in the sgca gene of the model , a pcr was performed on tail dna with primers kigenoseq2 . s 5 ′- tgtgtttgggacttatgggg - 3 ′ ( seq id 6 ) and kigenoseq2 . as 5 ′- caatcagcagcagcagcctc - 3 ′ ( seq id 7 ) generating a 659 bp pcr product that was then sequenced . 8 μm cross - sections from frozen muscle were stained using haematoxylin and eosin ( h & amp ; e ). cross - sections from mice injected with evans blue were revealed by fluorescent excitation at 633 nm . cross - sections were dried , then rehydrated in pbs or fixed cells were treated for 20 min with 1 % triton in pbs , then incubated for 30 min at room temperature ( rt ) in pbs containing 15 % foetal calf serum . cross - sections were incubated with polyclonal anti - sarcoglycan antibodies ( α - sarcoglycan : dilution 1 / 1000 , targeting amino acids 366 - 379 of human α - sarcoglycan ; β - sarcoglycan : dilution 1 / 20 , ncl - b - sarc ( novocastra ); γ - sarcoglycan : dilution 1 / 20 , ncl - g - sarc ( novocastra ); dystrophin : dilution 1 / 20 , ncl - dys2 ( novocastra ); and calreticulin : dilution 1 / 70 , ab4109 ( abcam )) for 1 to 2 hours at rt then rinsed 3 times in pbs . primary antibodies were revealed after 1 hour incubation at rt with secondary antibodies conjugated with fluorochromes alexa488 ( a - 11032 , molecular probes ) or alexa594 ( a - 11037 , molecular probes ), diluted 1 : 1000 in pbs . cross - sections were then rinsed three times in pbs , mounted with fluoromount - g ( southern biotech 0100 - 01 ) and then observed with a confocal microscope ( leica ). immunohistochemical analysis of human biopsy specimens was performed as described in hackman et al . ( 9 ). plasmids paav_c5 - 12_α - sg , pcdna3_α - sg , pcdna3_β - sg , pcdna3_γ - sg and pcdna3_δ - sg were produced by pcrs on skeletal muscle cdna and clones using the topo ta cloning kit ( invitrogen ). sarcoglycans were then subcloned into the pcdna3 plasmid ( invitrogen ) or paav_c5 - 12_mcs ( 3 ). plasmids pcdna3_α - sg - r77c and paav_c5 - 12_α - sg - r77c were obtained from pcdna3 - α - sg or paav_c5 - 12_α - sg by site - directed mutagenesis using the quickchange site - directed mutagenesis kit ( stratagene ) and the following primer : 5 ′- gccccggtggctctgctacacccagcgc - 3 ′ ( seq id 8 ). the constructions were checked by enzymatic digestion and sequencing . the adenovirus - free aav 2 / 1 viral preparations were produced by introducing recombinant aav2 - itr genomes into aav1 capsids using the tritransfection protocol ( 2 ). viral genomes were quantified by dot blot in comparison with a series of standard plasmid dilutions . nih3t3 or 911 cells were grown in dulbecco &# 39 ; s modified eagle medium supplemented with glutamine , gentamicin and 10 % foetal calf serum . cells were transfected using 6 μa fugene ( roche ) per 1 μg plasmid . 0 . 5 μg of each plasmid ( α - sg or α - sg - r77c and β - sg , γ - sg , δ - sg ) was used per well in 6 - well dishes . for treatment by inhibitors , 43 hours after transfection cells were incubated for 5 hours with either the mannosidase inhibitor ( kifunensin 5 μm , vwr ) or the proteasome inhibitor ( mg132 5 μm diluted in dmso , sigma ). cells were then rinsed in pbs and fixed with 3 . 7 % formaldehyde in pbs for 15 min at rt , and rinsed three more times in pbs before immunohistochemical labelling . sgca 77c / 77c mice were exercised for 30 minutes per day on a treadmill ( columbus treadmill instrument exer 6m ) on 3 consecutive days . mice were placed on a treadmill with a downward incline of 15 ° and the speed was set at 10 meters per minute . at the end of the 3 days , mice were injected intraperitoneally with evans blue dye ( 0 . 5 mg / g ). mice were sacrificed the day following injection and the deltoid , psoas , gastrocnemius , gluteus , extensor digitorum longus and quadriceps muscles were dissected and frozen rapidly in isopentane chilled with liquid nitrogen . the raav2 / 1 viral preparations were injected ( 10 10 viral genomes ( vg ) in 30 μl total volume ) in the left tibialis anterior muscle of sgca −/− mice . on days 20 , 22 , 25 and 27 after injection , 10 μm kifunensin or 20 μm mg132 were injected into the muscle ( i . e . either twice or 4 times the concentrations used in vitro to take into account diffusion within the muscle ). one day prior to sacrifice ( day 27 ), mice were injected intraperitoneally with evans blue dye . both left and right tibialis muscles were dissected and frozen rapidly in isopentane chilled with liquid nitrogen . the presence of mutation r77c in α - sarcoglycan in humans leads to destabilisation of the sarcoglycan complex , as shown by immunohistochemical labelling using antibodies against various distinct proteins of the complex ( fig1 ). 2 — in the mouse , the presence of a cysteine in position 77 does not prevent α - sarcoglycan membrane targeting and does not result in a pathological condition . in order to investigate the reasons for complex destabilisation , we produced , by homologous recombination , an animal model ( sgca 77c / 77c ) bearing a cysteine in position 77 . it should be noted that normal mice bear a histidine residue at this position and not an arginine residue . immunohistochemical analysis of muscles from these mice using antibodies against various dgc complex proteins demonstrated that this mutation did not prevent α - sarcoglycan membrane targeting or assembly of the sarcoglycan complex ( fig2 ). tissue cross - sections from wild - type and α - sarcoglycan deficient ( sgca −/−, 8 ) mice were used as controls . the histology of the deltoid , psoas , gastrocnemius and quadriceps muscles from 3 to 6 month old sgca 77c / 77c mice was examined by haematoxylin / eosin staining and compared to sgca −/− mouse tissue . although the latter display the signs of severe dystrophy , no anomalies were detected in sgca 77c / 77c muscles ( fig3 ). the absence of muscle anomalies was confirmed by functional analysis . sgca 77c / 77c mice were subjected to exercise enhancing eccentric muscle contractility and then injected intraperitoneally with evans blue dye , a dye that specifically stains necrotic cells . no evans blue dye penetration was observed in the muscles of sgca 77c / 77c mice ( fig4 ). to determine whether the differences observed between mice and humans when a cysteine residue is present in position 77 were related to the intrinsic properties of the human protein , we performed gene transfer experiments in muscles of α - sarcoglycan deficient mice using a viral vector derived from the adeno - associated virus ( aav ) carrying the human α - sarcoglycan gene mutated in position 77 . analysis of these injected muscles demonstrated that the mutant protein is localised in the membrane , although some protein is retained in the reticulum , that the sarcoglycan complex assembles and that the pathological phenotype is corrected ( fig5 and 6 ). it should be noted , as seen in fig5 , that α - sarcoglycan accumulates in the perinuclear space in some cells . 3 — the mutation causes α - sarcoglycan retention and its degradation by the proteasome . we have established a cellular model reproducing the phenomena observed in humans by quadritransfecting plasmids coding the four different sarcoglycans . in this model , the complex assembles correctly at the membrane when normal α - sarcoglycan is co - transfected together with the three other sarcoglycans . however , correct assembly is not observed at the membrane when the r77c α - sarcoglycan is transfected . this was demonstrated by immunohistochemical labelling using an antibody against the extracellular segment of α - sarcoglycan on non - permeabilised cells ( fig7 ). double - labelling of α - sarcoglycan and an endoplasmic reticulum marker ( calreticulum ) in permeabilised cells showed that mutant protein was retained in the secretion pathway ( fig8 ). we postulated that mutant α - sarcoglycan was recognised as abnormal by the protein quality control system of the reticulum and then degraded by the proteasome . this was confirmed by use of the proteasome inhibitor mg132 which restored membrane targeting ( fig9 ). the protein quality control system of the reticulum leads to degradation of incorrectly folded proteins . the mannosidase i enzyme plays an important role in this process by modifying the oligosaccharide chains of glycosylated proteins such as sarcoglycans . bearing in mind these facts and our results , we postulated that use of an inhibitor of this enzyme might restore membrane targeting of the mutant α - sarcoglycan protein . this hypothesis was validated in our cellular model in cells quadritransfected with mutant α - sarcoglycan and then treated by kifunensin ( fig1 ). the differences observed between humans and mice meant that we did not have an in vivo mouse model corresponding to the molecular events observed in humans . however , a decrease in the partial retention of the mutant protein after gene transfer would suggest that mannosidase i inhibition could have a beneficial effect on α - sarcoglycan membrane targeting . to determine whether use of the inhibitor might be effective in vivo , sgca −/− mice first received an injection of the aav - sgca77c vector and then ( 15 days later ) received three intramuscular injections of the inhibitor over a one week period . muscles that were injected with the inhibitor displayed a nearly complete absence of intracellular aggregates , and notably an absence of accumulation in the perinuclear space . these results validated our working hypothesis ( fig1 ). the same convincing results were obtained with kifunensin for two other point mutations on two other sub - units from the sarcoglycan complex : mutation e262k on sub - unit δ ( fig1 a ) and mutation q11e on sub - unit β ( fig1 b ). experiments similar to those performed with kifunensin on mutant r77c α - sarcoglycan were performed using 1 - deoxymannojirimycin ( dmj ), another class i mannosidase inhibitor . results are shown in fig1 a and demonstrate the efficacy of this substance for restoring membrane targeting of mutant α - sarcoglycan ( r77c mutation ). in addition , similar experiments confirmed our working hypothesis for other mutations on other sub - units of the sarcoglycan complex : mutation e262k on sub - unit δ ( fig1 b ) and mutation q11e on sub - unit β ( fig1 c ). to conclude , we have shown that mutant α - sarcoglycan bearing a cysteine in position 77 is managed quite differently in mouse and human cells , and that this mutant protein is functional if it is located correctly and can remedy the pathological condition related to sarcoglycan deficiency . we have demonstrated , in a cellular model , that mannosidase i inhibition prevents degradation of mutant sarcoglycan and restores membrane targeting . use of this substance in vivo also seems to produce the same results . 1 — allamand , v ., k . m . donahue , v . straub , r . l . davisson , b . l . davidson , and k . p . campbell . 2000 . early adenovirus - 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