Patent Application: US-4260108-A

Abstract:
there is disclosed an isolated , modified recombinant β - glucuronidase wherein the modification is having its carbohydrate moeties chemically modified so as to reduce its activity with respect to mannose and mannose 6 - phosphate cellular delivery system while retaining enzymatic activity also disclosed are methods for the treatment of lysosomal storage disease in mammals wherein the mammal is administered a therapeutically effective amount of isolated , modified recombinant β - glucuronidase whereby said storage diseased is relieved in the brain and visceral organs of the mammal . also disclosed are other lysosomal enzymes within the scope of the invention .

Description:
in summary , there has been discovered a means to successfully treat gus with periodate and borohydride without significantly reducing the enzymatic activity or stability . the treated protein has been shown to have modified carbohydrate that no longer has functional recognition signals for mannose and mannose 6 - phosphate receptors . because of this , the enzyme exhibits a vastly increased half - life in the circulation after intravenous infusion . this increased availability results in the improved delivery of the enzyme across the bbb by some unknown mechanism . whether it is increased opportunity for fluid phase pinocytosis or some other “ leakiness ”, the enzyme , once it has crossed the bbb , has increased access to cells in the brain . it is then able to use its enzymatic activity to clear accumulated storage material in the cells and hopefully reverse the progression of the disease mps vii . while not wishing to be bound by any particular theory , the use of periodate treated enzyme shows great promise for treating the brain in mps vii and any of the other lysosomal diseases where there is brain pathology . this method can reasonably be extended for use with other glycoproteins where rapid clearance from the circulation hinders their therapeutic effect . any number of lysosomal enzymes are included within the scope of this invention . examples of such enzymes are heparin n - sulfatase for treatment of mps iii ( sanfillipo a ), hexosaminidase a for treatment of tay - sachs disease , α - l - iduronidase for treatment of mps i hurler syndrome ), palmitoyl thiotransferase ( ppt1 ) for batten &# 39 ; s disease ( cln1 ), α - glucosidase for pompe disease , n - acetyl - galactosamine - 6 - sulfatase for mps iva and β - galactosidase for mps ivb ( morquio disease a and b ), and n - acetylgalactosamine 4 - sulfatase for mps vi ( maroteaux - lamy syndrome ). other enzymes can be easily envisioned by those of ordinary skill in view of this disclosure and are included within the scope of this invention . the enzymes disclosed herein when modified in accordance with this invention are therapeutically effective to treat various diseases . the effective amount of such modified enzymes can be easily determined by simple testing . however the term “ effective amount ” as used herein is intended to mean that amount which will be therapeutically effective to treat the disease . such amount is generally that which is known in the art for the use of such enzymes to therapeutically treat known diseases . using dna cloning techniques , the cdna sequence encoding the full length cdna for human β - glucuronidase was subcloned ( genbank accession # nm — 000181 ) ( fig1 ) into the mammalian expression vector pcxn ( 29 ). this expression vector contains an expression cassette consisting of the chicken beta - actin promoter coupled to the cmv intermediate - early ( cmv - ie ) enhancer . pcxn also contains a selectable marker for g418 allowing selection of stably expressing mammalian cells seq id no . 1 . this plasmid was introduced into the chinese hamster ovary cell line , cho - k1 ( 34 ) by electroporation ( 30 ). after selection in growth medium consisting of minimal essential medium + 35 μg / ml proline + 15 % fetal bovine serum ( fbs )+ 400 μg / ml g418 , colonies were picked and grown to confluency in 48 - well plates . high level expressing clones were identified by measuring gus activity secreted into the conditioned medium from these clones . the highest - producing clone was scaled up and secreted enzyme was collected in protein - free collection medium pf - cho . conditioned medium collected in this way was pooled , centrifuged at 5000 × g for 20 min and the supernatant was collected and frozen at 20 ° f . until sufficient quantities were accumulated for purification . gus activity was measured using the 10 mm 4 - methyl - umbelliferyl β - d - glucuronide as substrate in 0 . 1m sodium acetate buffer ph 4 . 8 , 1 mg / ml crystalline bsa as previously described ( 31 ). β - glucuronidase was purified by two different methods . the first method was by a multi - step procedure using conventional column chromatography . the second method utilized an anti - human β - glucuronidase monoclonal antibody affinity resin followed by a desalting step . the complete procedures for both methods are outlined below . a : ultrafiltration : ym - 100 membrane ; diafiltrate with 20 mm napo 4 + 150 mm nacl + 0 . 025 % nan 3 @ ph 5 . 5 ; ( 2 × 2 . 25 l ). b : blue sepharose ff ( ge healthcare ): equilibrate 10 × column volume column with 20 mm napo 4 @ ph 5 . 5 ; load concentrate from ultrafiltration ( don &# 39 ; t adjust ph , range : 5 . 5 - 5 . 7 ); wash 10 × column volume with 20 mm napo 4 + 150 mm nacl @ ph 5 . 5 ; elute column with 10 mm napo 4 + 800 mm nacl @ ph 7 . 5 ; regeneration : wash with 10 × column 20 mm napo 4 @ ph 5 . 5 + 2m nacl . c : phenyl sepharose ( high sub ff ): equilibrate 30 × column volume with 10 mm napo 4 + 1000 mm nacl @ ph 8 . 0 ; load pooled blue elute as is ( don &# 39 ; t adjust ph , range : 7 . 2 - 7 . 4 ); wash 10 × column volume with 10 mm napo 4 + 1000 mm nacl @ ph 8 . 0 ; elute column with 10 mm tris + 1 mm na - β - glycerophosphate @ ph 8 . 0 ; dialyze elution with 3 changes of 10 mm tris + 1 mm na - β - glycerophosphate @ ph 8 . 0 ; regeneration : wash with 0 . 5 m naoh , 30 min contact time ; wash with 30 column volumes of ddh 2 o . d : deae sephacel : equilibrate 10 × column volume with 10 mm tris + 1 mm na - β - glycerophosphate @ ph 8 . 0 ; load pooled dialyzed phenyl elute . wash 10 × column volume with 10 mm tris + 1 mm na - β - glycerophosphate @ ph 8 . 0 ; elute with 0 - 0 . 4m nacl gradient ; dialyze deae pooled eluate in 25 mm na acetate + 1 mm na - β - glycerophosphate ; + 0 . 025 % nan 3 @ ph 5 . 5 ; regeneration : wash with 20 × column volume 10 mm tris + 1 mm na - β - glycerophosphate @ ph 8 . 0 + 2 m nacl . e : cm sepharose : equilibrate lox column volume with 25 mm na acetate + 1 mm na - β - glycerophosphate + 0 . 025 % nan 3 @ ph 5 . 5 ; load dialyzed deae pooled eluate ; elute with 0 - 0 . 3m nacl gradient . regeneration : wash with 20 × column volume 25 mm na acetate + 1 mm na - β - glycerophosphate + 0 . 025 % nan 3 @ ph 5 . 5 + 2m nacl . affinity chromatography procedure was performed essentially as follows : conditioned medium from cho cells overexpressing the gus protein was filtered through a 0 . 22μ filter . sodium chloride ( crystalline ) was added to a final concentration of 0 . 5m , and sodium azide was added to a final concentration of 0 . 025 % by adding 1 / 400 volume of a 10 % stock solution . the medium was applied to a 5 ml column of anti - human β - glucuronidase - affigel 10 ( pre - equilibrated with antibody sepharose wash buffer : 10 mm tris ph 7 . 5 , 10 mm potassium phosphate , 0 . 5 m nacl , 0 . 025 % sodium azide ) at a rate of 25 ml / h at 4 ° c . the column was washed at 36 ml / h with 10 - 20 column volumes of antibody sepharose wash buffer . the column was eluted at 36 ml / hour with 50 ml of 10 mm sodium phosphate ph 5 . 0 + 3 . 5 m mgcl 2 . fractions of 4 ml each were collected and assayed for gus activity . fractions containing the purifed protein were pooled , diluted with an equal volume of p6 buffer ( 25 mm tris ph 7 . 5 , 1 mm β - glycerophosphate , 0 . 15 mm nacl , 0 . 025 % sodium azide ) and desalted over a biogel p - 6 column ( pre - equilibrated with p6 buffer ) to remove the mgcl 2 and to change the buffer to p6 buffer for storage . gus protein was eluted with p6 buffer , fractions containing gus activity were pooled and the final pool assayed for gus activity and protein . purified gus was stored frozen at − 80 ° c . in p6 buffer for long - term stability . for mouse infusions , the enzymes were highly concentrated in centricon ym - 30 concentrators and the buffer was changed to p6 buffer without azide . these concentrates were frozen in small aliquots at − 80 ° c . until use . gus is a 300 kda protein that exists as a homotetramer consisting of four identical monomers of apparent molecular weight of 75 kda . the purified recombinant gus used in these experiments was similar to that described ( 11 , 19 ). the apparent molecular mass of the enzyme monomer was 75 kda on reducing sds - page . the tetrameric enzyme had a molecular mass of ≈ 300 kda when analyzed by sizing gel filtration chromatography ( data not shown ). the specific activity of the purified enzyme was 5 . 0 × 10 6 units / mg . the k uptake was 1 . 25 - 2 . 50 nm , calculated from uptake saturation curves by using human mps vii fibroblasts in which the uptake is almost entirely m6pr - dependent . to confirm molecular weight , 2 and 4 μg of purified gus were analyzed by sds - page under reducing conditions ( 35 ). the apparent molecular weight was 75 kda as expected . the following examples are presented to illustrate the instant invention and are not meant to limit the scope of the invention to these particular examples . the skilled artisan , in the practice of this invention , will readily and reasonably understand that the methods and compositions are applicable to any and all enzymes and proteins that gain entry into a cell via the mannose and mannose 6 - phosphate pathways . the mannose and manose 6 - phosphate recognition sites on gus are both located in the carbohydrate portion of gus enzyme . in order to inactivate this carbohydrate moiety , the enzyme was treated by a well established procedure utilizing reaction with sodium meta - periodate followed by sodium borohydride ( 17 , 18 ). approximately 10 mg of purified gus was treated with a final concentration of 20 mm sodium meta - periodate in 20 mm sodium phosphate , 100 mm nacl ph 6 . 0 for 6 . 5 h on ice in the dark . the reaction was quenched by the addition of 200 mm final concentration ethylene glycol and incubated for an additional 15 min on ice in the dark . afterwards , this mixture was dialyzed against 2 changes of 20 mm sodium phosphate , 100 mm nacl ph 6 . 0 at 4 ° c . the periodate treated , dialyzed enzyme was then treated with the addition of 100 mm final concentration sodium borohydride overnight on ice in the dark to reduce reactive aldehyde groups . after this treatment , the enzyme was dialyzed against two changes of 20 mm sodium phosphate , 100 mm nacl , ph 7 . 5 at 4 ° c . the final dialyzed enzyme was stored in this buffer at 4 ° c . where it was stable indefinitely . treatment of gus with periodate and borohydride resulted in only a slight inactivation of the enzymatic activity . the specific activity prior to treatment was 5 . 0 × 10 6 units / mg and following treatment was 4 . 5 × 10 6 units / mg . to assess the effectiveness of the periodate and borohydride treatment in inactivating the carbohydrate on the enzyme , the ability of the enzyme to be taken up by human β - glucuronidase deficient fibroblasts or by the permanent j774e mouse macrophage line was analyzed . m6pr - mediated uptake was determined by adding 4 , 000 units of gus or pb - gus ± 2 mm m6p in 1 ml of growth medium to 35 - mm dishes of confluent gm - 2784 gus - deficient fibroblasts . after incubation at 37 ° c . and 5 % co 2 for 2 h , the cells were cooled on ice , washed five times with cold pbs , then solubilized in 0 . 5 ml of 1 % sodium deoxycholate . extracts were assayed for gus activity and protein . values were expressed as units of enzyme taken up per mg of cell protein per hour of uptake . mr - mediated uptake was measured by adding 10 , 000 units of gus or pb - gus ± 1 . 7 mg / ml yeast mannan ( sigma - aldrich ) in 1 ml of growth medium to 35 - mm dishes of confluent j774e mouse macrophages ( 33 ). after incubation at 37 ° c . and 5 % co 2 for 4 h , the cells were washed as above and then solubilized in 1 ml of 1 % sodium desoxycholate and assayed for gus activity . table 1 below shows the m6p - receptor mediated uptake of untreated or mock - treated gus by the human fibroblast cell line . gus is taken up by this line at the rate of 377 units / mg cell protein / 1 h of uptake . two mm m6p completely inhibits this uptake . in contrast , the uptake of the periodate and borohydride treated gus ( pbgus ) has been completely destroyed . table 2 below shows that untreated gus is taken up by the mouse macrophage line at a rate of 316 u / mg cell protein / 1 h of uptake and the uptake is inhibited by the presence of 1 . 69 mg / ml yeast mannan . in contrast , three separate batches of periodate and borohydride treated gus ( pbgus ) have essentially no uptake by this cell line . since both mannose 6 - phosphate and mannose receptor mediated uptake are dependent on functional mannose 6 - phosphate or mannose residues , respectively , these results indicate that the periodate and borohydride treatment of gus ( pb - gus ) has inactivated the carbohydrate structures on the enzyme . the carbohydrates on glycoproteins often confer enhanced thermal stability , and removal of oligosaccharide chains often destabilizes glycoproteins ( 21 ). human gus has been shown to be relatively stable to thermal inactivation at 65 ° c . ( 22 - 26 ). purified gus or pb - gus was diluted in equal volumes of heat inactivation buffer [ 40 mm tris - hcl ( ph 7 . 5 ), 150 mm nacl , 10 mg / ml bsa ], and aliquots were incubated for 0 , 0 . 5 , 1 , 2 , or 3 h at 65 ° c . after treatment , aliquots were cooled on ice and then assayed for gus activity . results were expressed as the percentage of original units of gus activity remaining at the indicated times . as shown in fig2 , recombinant gus retained 90 % of initial activity after 3 h at 65 ° c ., whereas pb - gus retained 40 % of its activity under these conditions ( fig2 ). to compare the stability of gus and pb - gus in lysosomes of living cells at 37 ° c ., a study was conducted to determine their half - life after uptake by mps vii fibroblasts . the low rate of endocytosis of pb - gus by fibroblasts required exposure to 100 , 000 units / ml pb - gus per plate for 48 h to accumulate sufficient enzyme by fluid phase pinocytosis ( 28 units per plate ) to allow measurement of its half - life . by contrast , fibroblasts exposed to 500 units / ml m6p containing native gus for 48 h contained 228 units per plate . tissue culture dishes ( 35 mm ) of confluent gm - 2784 gus - deficient fibroblasts were incubated with 500 units of gus or 100 , 000 units of pb - gus in 1 ml of growth medium at 37 ° c . and 5 % co 2 for 48 h under sterile conditions . the plates were washed twice with sterile growth medium and then fed with 2 ml of the same . duplicate plates were taken off at 0 , 2 , 5 , 7 , 14 , and 21 days , washed five times with pbs and frozen at − 20 ° c . remaining plates were fed twice weekly with 2 ml of growth medium . after all plates had been collected , the cells were solubilized in 0 . 5 ml of 1 % desoxycholate and assayed for gus activity . values were expressed as percentage of zero time cell - associated gus activity remaining at the indicated time points . fig3 shows the half - life for the two enzymes in fibroblasts upon subsequent incubation at 37 ° c . the t 1 / 2 of gus was 18 . 9 days . the t 1 / 2 of pb - gus was shorter ( 12 . 9 days ), but nearly one - third of the initial activity was still present at 21 days . clearance of the periodate and borohydride treated gus from the circulation after iv infusion as stated previously , the purpose of treating gus with periodate and borohydride , was to drastically slow its clearance time from the circulation after infusion . to test this , the tail veins of mps vii mice were infused with gus or pb - gus at a dose of 4 mg / kg body weight in a total volume of 125 μl of pbs . after infusion , blood samples were taken by supraorbital puncture at 2 , 5 , 10 , 20 , 60 , 90 , and 120 min for gus and 4 , 240 , 1 , 440 , and 2 , 880 min for pb - gus into heparinized capillary tubes . plasma was collected after centrifugation and assayed for gus activity . values were expressed as a percentage of gus activity remaining compared with the first time point . fig4 and table 3 below show the results of that clearance study . as can be seen , the clearance of untreated gus is very rapid with a t 1 / 2 of 11 . 7 min . in contrast , the clearance of pb - gus in four separate mice was drastically slower with a t 1 / 2 of 18 . 5 ± 1 . 0 h . this would indicate that the rapid clearance of this enzyme due to the mannose and mannose 6 - phosphate receptor ( 15 ) has been abrogated . previously , the plasma clearance of the enzyme was observed to be slowed when treating mps vii mice with high - dose gus and facilitated enzyme delivery to the brain ( 11 ). in these experiments , it was not clear whether it was the higher dose of enzyme itself or the delayed plasma clearance of the enzyme that accounted for improved delivery to brain . to address this question , comparative measurements were made of the distribution of gus and pb - gus in brain and other tissues 48 h after infusion into mps vii mice . mice were perfused with tris - buffered saline before collection of tissues to ensure that tissue was not contaminated with residual plasma enzyme . mps vii mice were infused via tail vein with gus or pb - gus at a dose of 4 mg / kg in a total volume of 125 μl of pbs . at 48 h after infusion , the mice were perfused with 30 ml of 25 mm tris ( ph 7 . 2 ), 140 mm nacl . perfused tissues were collected and flash frozen in liquid nitrogen until further processing . tissues were thawed , weighed , and homogenized for 30 s with a polytron homogenizer in 10 - 20 volumes of 25 mm tris ( ph 7 . 2 ), 140 mm nacl , 1 mm phenylmethylsulfonyl fluoride . total homogenates were frozen at − 80 ° c ., thawed , and then sonicated for 20 s to produce a homogeneous extract . extracts were assayed for gus activity and protein , and the results were expressed as units / milligrams of tissue protein . the results of these measurements appear in table 4 below . as is evident from the data in table 4 , delivery of native gus to brain at 48 h was minimal . however , native gus was delivered to other tissues at levels similar to those previously reported . pb - gus was delivered to heart , kidney , muscle , lung , and eye at levels higher than those seen with native gus . the levels in liver and spleen were nearly 4 - fold lower after pb - gus infusion than after gus infusion . this result undoubtedly reflects the curtailment of receptor - mediated uptake by the mpr and m6pr that are highly expressed in these two tissues . by contrast , brain levels were greatly increased ( 7 . 8 % of wild - type ) in pb - gus - infused animals . this result suggests that the long circulating pb - gus has an advantage in crossing the bbb . thus , it was of great interest to study its effectiveness in clearing storage from cells in the cns . comparison of the efficacy of periodate / borohydride treated gus for ert in clearing neuronal storage as stated previously , it was believed that slowing the clearance of gus from the circulation might facilitate the delivery to the brain . it has been shown above that the periodate and borohydride treatment accomplished this producing an enzyme with a much reduced rate of clearance from the circulation after iv infusion . the effectiveness of the treated enzyme in clearing the storage material from the lysosomes of the mps vii mouse after a typical ert regimen was tested . mps vii mice were treated with 12 weekly infusions , one group with untreated gus at doses of 2 or 4 mg / kg body weight and a second group with pb - gus at doses of 2 or 4 mg / kg body weight . two other groups of mps vii mice were infused two times daily for 1 week with a total of 48 mg / kg , one group with gus and one group with pb - gus . one week after the last infusion , tissues from the group receiving untreated gus ( n = 3 ), 2 mg / kg ( n = 3 ) or 4 mg / kg gus ( n = 2 ), and pb - gus , 2 mg / kg ( n = 2 ) or 4 mg / kg ( n = 3 ) were obtained at necropsy after tris - buffered saline perfusion , fixed in 2 % paraformaldehyde and 4 % glutaraldehyde , post fixed in osmium tetroxide , and embedded in spurr &# 39 ; s resin . for evaluation of lysosomal storage by light microscopy , toluidine blue - stained 0 . 5 - μm - thick sections of liver , spleen , kidney , brain , heart , rib , and bone marrow were assessed blind . to evaluate storage in cortical neurons , 500 contiguous parietal neocortical neurons were scored for the number of lucent cytoplasmic vacuoles , indicating lysosomal storage . a maximum of seven vacuoles were counted per cell , and results were evaluated by anova or student &# 39 ; s t test . also evaluated were the hippocampal neurons by counting the number of vacuoles in 100 neurons in ca2 sector . other tissues were examined by using a semiquantitative scale , as described in ref . 11 . as can be seen in fig5 , gus results in a slight reduction of the storage material in the brain whereas pb - gus results in almost complete reversal of the storage . this would indicate that the periodate and borohydride treated gus was vastly more effective in treating the brain storage in this disease . in fig5 , reduction in neuronal and meningeal storage with ert with gus and pb - gus is shown as follows : ( a ) neocortical neurons from an untreated mps vii mouse have abundant lysosomal storage in the cytoplasm ( arrow ). ( b ) after treatment with 4 mg / kg gus , there is still a moderate amount of cytoplasmic storage ( arrow ) despite the therapy . ( c ) after 4 mg / kg pb - gus , there is a marked reduction in the amount of storage in the neocortical neurons ( arrow ). ( d ) the ca2 sector hippocampal neurons have abundant storage ( arrow ) in untreated mps vii mice . ( e ) after treatment with gus , the amount of storage in neurons ( arrow ) the same area of the hippocampus is similar to that of the untreated mouse . ( f ) after treatment with pb - gus , there is a remarkable reduction in the amount of storage in neurons ( arrow ) in the ca2 sector of the hippocampus . ( g ) the meninges of an untreated mps vii mouse has abundant storage in fibroblasts around vessels ( arrow ). ( h ) storage ( arrow ) is moderately decreased after treatment with gus . ( 1 ) treatment with pb - gus also produces moderate reduction in storage ( arrow ) in the meninges . [ scale bars : 10 μm ( a - c , uranyl acetate - lead citrate ) and 30 μm ( d - i , toluidine blue ).] two of the problems associated in the analysis of micrographs for the clearance of storage material in these types of experiments are : 1 ) that there is some inconsistency from field to field i . e . the clearance varies from one microscopic field to another ; and 2 ) the procedure is somewhat subjective from person to person as to the amount of storage present . to address these problems , a new method was developed to quantify the storage material by counting the number of vacuoles ( distended lysosomes filled with storage material ) present in a total of 500 cells counted . fig6 shows the results of such an analysis of the mice treated with gus or pb - gus . gus at 2 mg / kg is not very effective at reducing the number of vacuoles , though somewhat better at the higher dose of 4 mg / kg . however , pb - gus appears to be almost completely effective at both 2 and 4 mg / kg . this analysis agrees with the conclusion drawn from the visual analysis of the images in fig5 . table 5 below summarizes the results of assessment of storage in neocortical and hippocampal neurons of untreated gus and pb - gus in mps vii mice . ert with gus over 12 weeks with both 2 mg / kg and 4 mg / kg gus reduced storage in neocortical neurons compared with untreated mps vii mice ( p = 0 . 002 and p = 0 . 003 , respectively ), although hippocampal neurons failed to show a morphological response to this therapy . pb - gus at 2 mg / kg also reduced neocortical neuronal storage ( p = 0 . 001 ). at 4 mg / kg , the therapeutic effect of pb - gus was even more striking ( p = 0 . 003 for 2 vs . 4 mg of pb - gus and p & lt ; 0 . 001 compared with untreated ). in addition , there was virtually no storage in the hippocampal neurons in the three pb - gus - treated mice available for quantitation ( the ca2 region was not present in the section and was therefore not available for quantitation in two of the five pb - gus - treated mice ). these results indicate that ert with pb - gus is remarkably more effective than traditional gus at clearing storage in the neocortical and especially hippocampal neurons in the mps vii mouse . as a group , the pb - gus - treated mice also had slightly less storage in glial and perivascular cells than the gus - treated mice . however , the dose - dependent reduction in storage in meninges , which was moderate at 4 mg / kg , was equivalent in the pb - gus - and the gus - treated animals . from the above results it is reasonable to expect that treatment of mammalian species in accordance with this invention will provide relief of lysosomal storage disease , particularly in humans particularly in the brain of humans . the following references are cited throughout this disclosure and are herein incorporated by reference . they are meant to illustrate and support the invention . applicants reserve the right to challenge the veracity of any statements made therein . 1 . neufeld e f , muenzer j ( 2001 ) in the metabolic and molecular bases of inherited disease , eds scriver c r , beaudet a l , sly w s , valle d ( mcgraw - hill , new york ), pp 3421 - 3451 . 2 . desnick r j ( 2004 ) enzyme replacement and enhancement therapies for lysosomal diseases . j inherit metab dis 27 : 385 - 410 . 3 . brady r o , barton n w ( 1996 ) enzyme replacement and gene therapy for gaucher &# 39 ; s disease . lipids 31 : s137 - s139 . 4 . kakkis e d , et al . 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( 1994 ) over expression rescues the mutant phenotype of l176f mutation causing p - glucuronidase deficiency mucopolysaccharidosis in two mennonite siblings . j biol chem 269 : 23681 - 23688 . 26 . natowicz m r , chi m m , lowry o h , sly w s ( 1979 ) enzymatic identification of mannose 6 - phosphate on the recognition marker for receptor - mediated pinocytosis of p - glucuronidase by human fibroblasts . proc natl acad sci usa 76 : 4322 - 4326 . 27 . schlesinger p h , et al . ( 1980 ) the role of extrahepatictissues in the receptor - mediated plasma clearance of glycoproteins terminated by mannose or n - acetylglucosamine . biochem j 192 : 597 - 606 . 28 . banks w a ( 2004 ) are the extracellular [ correction of extracelluar ] pathways a conduit for the delivery of therapeutics to the brain ? curr pharm des 10 : 1365 - 1370 . 29 . niwa h , yamamura k , miyazaki j ( 1991 ) efficient selection for high - expression transfectants with a novel eukaryotic vector . gene 108 : 193 - 199 . 30 . ulmasov b , et al . ( 2000 ) purification and kinetic analysis of recombinant ca xii , a membrane carbonic anhydrase over expressed in certain cancers . proc natl acad sci usa 97 : 14212 - 14217 . 31 . glaser j h , sly w s ( 1973 ) p - glucuronidase deficiency mucopolysaccharidosis : methods for enzymatic diagnosis . j lab clin med 82 : 969 - 977 . 32 . lowry o h , rosebrough n j , farr a l , randall r j ( 1951 ) protein measurement with the folin phenol reagent . j biol chem 193 : 265 - 275 . 33 . diment s , leech m s , stahl pd ( 1987 ) generation of macrophage variants with 5 - aza - cytidine : selection for mannose receptor expression . j leukocyte biol 42 : 485 - 490 . 34 . chinese hamster ovary cell line american type culture collection , atcc crl 9618 .