Patent Application: US-5103498-A

Abstract:
the invention relates to nucleic acids which encode glycosyltransferase and are useful in producing cells and organs from one species which may be used for transplantation into a recipient of another species . it also relates to the production of nucleic acids which , when present in cells of a transplanted organ , result in reduced levels of antibody recognition of the transplanted organ .

Description:
the nucleic acid sequences encoding the catalytic domain of a glycosyltransferase may be any nucleic acid sequence such as those described in pct / us95 / 07554 , which is herein incorporated by reference , provided that it encodes a functional catalytic domain with the desired glycosyltransferase activity . preferred catalytic domains from glycosyltransferase include h transferase and secretor . preferably these are based on human or porcine sequences . the nucleic acid sequences encoding the localisation signal of a second transglycosylase may be any nucleic acid sequence encoding a signal sequence such as signal sequences disclosed in p a gleeson , r d teasdale & amp ; j bourke , targeting of proteins to the golgi apparatus . glyconjugate j . ( 1994 ) 11 : 381 - 394 . preferably the localization sis is specific for the golgi apparatus , more preferably for that of the true golgi . still more preferably the localisation signal is based on that of gal transferase . even more preferably the localisation signal is based on porcine , sine or bovine sequences . even more preferably the nucleic acid encodes a signal sequence with following amino acid sequence ( in single letter code ): mnvkgr ( porcine ), mnvkgk ( mouse ) or mvvkgk ( bovine ). vectors for expression of the chimeric enzyme may be any suitable rector , including those disclosed in pct / us95 / 07554 . the nucleic acid of the invention can be used to produce cells and organs with the desired glycosylation pattern by standard techniques , such as those disclosed in pct / us95 / 07554 . for example , embryos may be transfected by standard techniques such as microinjection of the nucleic acid in a linear form into the embryo ( 22 ). the embryos are then used to produce live animals , the organs of which may be subsequently used as donor organs for implantation . cells , tissues and organs suitable for use in the invention will generally be mammalian cells . examples of suitable cells and tissues such as endothelial cells , hepatic cells , pancreatic cells and the like are provided in pct / us95 / 07554 . the invention will now be described with reference to the following non - limiting examples . the abbreviations used are bp , base pair ( s ); fitc , fluorescein isothiocyanate ; gt , galactosyltransferase ; h substance , α ( 1 , 2 ) fucosyl lactosamine ; ht , α ( 1 , 2 ) fucosyltransferase ; pcr , polymerase chain reaction ; cytoplasmic domains of glycosyltransferases play a central role in the temporal action of enzymes plasmids — the plasmids used were prepared using standard techniques ( 7 ); pgt encodes the cdna for the porcine α ( 1 , 3 ) galactosyltransferase ( 23 ), pht encodes the cdna for the α ( 1 , 2 ) fucosyltransferase ( human ) ( 25 ). chimeric glycosyltransferase cdnas were generated by polymerase chain reaction as follows : an 1105 bp product ht - gt was generated using primers corresponding to the 5 ′ end of ht - gt ( 5 ′- gcggatccatgtggctccggagcc atcgtcaggtggttctgtcaatgc tgcttg - 3 ′) coding for nucleotides 1 - 24 of ht ( 25 ) followed immediately by nucleotides 68 - 89 of gt ( 8 ) and containing a bamh1 site ( underlined ) and a primer corresponding to the 3 ′ end of ht - gt ( 5 ′- gctctagagcgtcagatgttatt tctaaccaaattatac - 3 ′) containing complementarity to nucleotides 1102 - 1127 of gt with an xbal site downstream of the translational stop site ( underlined ); an 1110 bp product gt - ht was generated using primers corresponding to the 5 ′ end of gt - ht ( 5 ′- gcggatccatgaatgtcaaaggaagactctgcctggcct tcctgc - 3 ′) coding for nucleotides 49 - 67 of gt followed immediately by nucleotides 25 - 43 of ht and containing a bamh1 site ( underlined ) and a primer corresponding to the 3 ′ end of gt - ht ( 5 ′- gctctagagcctcaaggcttag ccaatgtccagag - 3 ′) containing complementarity to nucleotides 1075 - 1099 of ht with a xba1 site downstream of the translational stop site ( underlined ). pcr products were restricted bamh1 / xba1 , gel - purified and ligated into a bamh1 / xba1 digested pcdna1 expression vector ( invitrogen ) and resulted in two plasmids pht - gt ( encoding the chimeric glycosyltransferase ht - gt ) and pgt - ht ( encoding the chimeric glycosyltransferase gt - ht ) which were characterised by restriction mapping , southern blotting and dna sequencing . transfection and serology — cos cells were maintained in dubecco &# 39 ; s modified eagles medium ( dmem ) ( trace biosciences pty . ltd . , castle hill , nsw , australia ) and were transfected ( 1 - 10 μg dna / 5 × 105 cells ) using deae - dextrau ( 26 ); 48 h later cells were examined for cell surface expression of h substance or gal - α ( b 1 , 3 )- gal using fitc - conjugated lectins : ib4 lectin isolated from griffonia simplicifolia ( sigma , st . louis , mo .) detects gal - α ( 1 , 3 )- gal ( 27 ); ueai lectin isolated from ulex europaeus ( sigma , st . louis , mo .) detects h substance ( 28 ). h substance was also detected by indirect immunofluorescence using a monoclonal antibody ( mab ) specific for the h substance ( ash - 1952 ) developed at the austin research institute , using fitc - conjugated goat anti - mouse igg ( zymed laboratories , san francisco , calif .) to detect mab binding . fluorescence was detected by microscopy . rna analyses — cytoplasmic rna was prepared from transfected cos cells using rnazol ( biotecx laboratories , houston , tex . ), and total rna was electrophoresed in a 1 % agarose gel containing formaldehyde , the gel blotted onto a nylon membrane and probed with random primed gt or ht cdna . glycosyltransferase assays — forty - eight hours after transfection , cells were washed twice with phosphate buffered saline and lysed in 1 % triton x - 100 / 100 mm cacodylate ph 6 . 5 / 25 mm mncl2 , at 4 ° c . for 30 min ; lysates were centrifuged and the supernatant collected and stored at − 70 ° c . protein concentration was determined by the bradford method using bovine serum allumin as standard ( 29 ). assays for ht activity ( 30 ) were performed in 25 μl containing 3 mm [ gdp - 14 c ] fucose ( specific activity 287 mci / mmol , amersham international ), 5 mm atp , 50 mm mps ph 6 . 5 , 20 mm mncl2 , using 2 - 10 μl of cell extract ( approximately 15 - 20μg of protein ) and a range of concentrations ( 7 . 5 - 75 mm ) of the acceptor phenyl - b - d - galactoside ( sigma ). samples were incubated for 2 h at 37 ° c . and reactions terminated by the addition of ethanol and water . the amount of 14 c - fucose incorporated was counted after separation from unincorporated label using sep - pak c18 cartridges ( waters - millipore , millford , mass .). gt assays ( 31 ) were performed in a volume of 25 μl using 3 mm udp [ 3 h ]- gal ( specific activity 189 mci / mmol , amersham international ), 5 mm atp , 100 mm cacodylate ph 6 . 5 , 20 mm mncl 2 and various concentrations ( 1 - 10 mm ) of the acceptor n - acetyl lactosamine ( sigma ). samples were incubated for 2 h at 37 ° c . and the reactions terminated by the addition of ethanol and water . 3 h - gal incorporation was counted after separation from non - incorporated udp [ 3 h ]- gal using dowex i anion exchange columns ( bdh ltd ., poole , uk ) or sep - pak accell plus qma anion exchange cartridges ( waters - millipore , millford , mass .). all assays were performed in duplicate and additional reactions were performed in the absence of added acceptor molecules , to allow for the calculation of specific incorporation of radioactivity . we had previously shown that when cdnas encoding α ( 1 , 3 ) galactosyltransferase ( gt ) and α ( 1 , 2 ) fucosyltransferase ( ht ) were transfected separately they could both function efficiently leading to expression of the appropriate carbohydrates : gal - α ( 1 , 3 )- gal for gt and h substance for ht ( 32 ). however when the cdnas for gt and ht were transfected together , the ht appeared to “ dominate ” over the gt in that h substance expression was normal , but gal - α ( 1 , 3 )- gal was reduced . we excluded trivial reasons for this effect and considered that the localisation of the enzymes may be the reason . thus , if the ht localisation signal placed the enzyme in an earlier temporal compartment than gt , it would have “ first use ” of the n - acetyl lactosamine substrate . however , such a “ first use ” if it occurred , was not sufficient to adequately reduce gt . two chimeric glycosyltransferases were constructed using pcr wherein the cytoplasmic tails of gt and rt were switched . the two chimeras constructed are shown in fig1 : ht - gt which consisted of the nh 2 terminal cytoplasmic tail of ht attached to the transmembrane , stem and catalytic domain of gt ; and gt - ht which consisted of the nh 2 terminal cytoplasmic tail of gt attached to the transmembrane , stem and catalytic domains of ht . the chimeric cdnas were subcloned into the eukaryotic expression vector pcdnai and used in transfection experiments . the chimeric cdnas encoding ht - gt and gt - ht were initially evaluated for their ability to induce glycosyltransferase expression in cos cells , as measured by the surface expression of the appropriate sugar using lectins . forty - eight hours after transfection cos cells were tested by immunofluorescence for their expression of gal - α ( 1 , 3 )- gal or h substance ( table 1 & amp ; fig2 ). the staining with ib4 ( lectin specific for gal - α ( 1 , 3 )- gal ) in cells expressing the chimera ht - gt ( 30 % of cells stained positive ) was indistinguishable from that of the normal gt staining ( 30 %) ( table 1 & amp ; fig2 ). similarly the intense cell surface fluorescence seen with ueai staining ( the lectin specific for h substance ) in cells each expressing gt - ht ( 50 %) was similar to that seen in cells expressing wild - type pht ( 50 %) ( table 1 & amp ; fig2 ). furthermore , similar levels of mrna expression of the glycosyltransferases gt and ht and chimeric glycosyltransferases ht - gt and gt - ht were seen in northern blots of total rna isolated from transfected cells ( fig3 ). thus both chimeric glycosyltransferases are efficiently expressed in cos cells and are functional indeed there was no detectable difference between the chimeric and normal glycosyltransferases . glycosyltransferase activity in cells transfected with chimeric cdnas encoding ht - gt and gt - ht to determine whether switching the cytoplasmic tails of gt and ht altered the kinetics of enzyme function , we compared the enzymatic activity of the chimeric glycosyltransferases with those of the normal enzymes in cos cells after transfection of the relevant cdnas . by making extracts from transfected cos cells and performing gt or ht enzyme assays we found that n - acetyl lactosamine was galactosylated by both gt and the chimeric enzyme ht - gt ( fig4 . panel a ) over a the 1 - 5 mm range of substrate concentrations . lineweaver - burk plots showed that both gt and ht - gt have a similar apparent michealis - menten constant of km 2 . 6 mm for n - acetyl lactosamine ( fig4 . panel b ). further ht , and the chimeric enzyme gt - ht were both able to fucosylate phenyl - b - d - galactoside over a range of concentrations ( 7 . 5 - 25 mm ) ( fig4 panel c ) with a similar km of 2 . 3 mm ( fig4 panel d ), in agreement with the reported km of 2 . 4 mm for ht ( 25 ). therefore the chimeric glycosyltransferases ht - gt and gt - ht are able to utilise n - acetyl lactosamine ( ht - gt ) and phenyl - b - d - galactoside ( gt - ht ) in the same way as the normal glycosyltransferases , thus switching the cytoplasmic domains of gt and ht does not alter the function of these glycosyltransferases and if indeed the cytoplasmic tail is the localisation signal then both enzymes function as well with the gt signal as with the ht signal . switching cytoplasmic domains of gt and ht results in a reversal of the “ dominance ” of the glycosyltransferases the cdnas encoding the chimeric transferases or normal transferases were simultaneously co - transfected into cos cells and after 48 h the cells were stained with either ib4 or uea1 lectin to detect gal - α ( 1 , 3 )- gal and h substance respectively on the cell surface ( table 1 & amp ; fig5 ). cos cells co - transfected with cdnas for ht - gt + gt - ht ( fig5 panel c ) showed 30 % cells staining positive with ib4 ( table 1 ) but no staining on cells co - transfected with cdnas for gt + ht ( 3 %) ( fig5 panel a ). furthermore staining for h substance on the surface of ht - gt + gt - h co - transfectants gave very few cells staining positive ( 5 %) ( fig5 panel d ) compared to the staining seen in cells co - transfected with cdnas for the normal transferases gt + ht ( 50 %) ( fig5 panel b ), ie . the expression of gal - α ( 1 , 3 )- gal now dominates over that of h . clearly , switching the cytoplasmic tails of gt and ht led to a complete reversal in the glycosylation pattern seen with the normal transferases i . e . the cytoplasmic tail sequences dictate the pattern of carbohydrate expression observed . that exchanging the cytoplasmic tails of gt and ht reverses the dominance of the carbohydrate epitopes points to the glycosyltransferases being relocalized within the golgi . to address this question , experiments were performed with cdnas encoding glycosyltransferases with the same cytoplasmic tail : cos cells transfecterases with cdnas encoding ht + ht - gt stained strongly with both ueai ( 50 %) and ib4 ( 30 %) ( table 1 & amp ; fig5 panels e , f ) the difference in staining reflecting differences in transfection efficiency of the cdnas . similarly cells transfected with cdnas encoding gt + gt - ht also stained positive with ueai ( 50 %) and ib4 ( 30 %) ( table 1 & amp ; fig5 panel g , h ). thus , glycosyltransferases with the same cytoplasmic tail leads to equal cell surface expression of the carbohydrate epitopes , with no “ dominance ” of one glycosyltransferase over the other observed , and presumably the glycosyltransferases localised at the same site appear to compete equally for the substrate . in cos cells the levels of transcription of the cdnas of chimeric and normal glycosyltransferases were essentially the same ( fig3 ) and the immunofluorescence pattern of cos cells expressing the chimeric glycosyltransferases : ht - gt and gt - ht showed the typical staining pattern of the cell space gal - α ( 1 , 3 )- gal and h substance respectively ( table 1 & amp ; fig2 ), the pattern being indistinguishable from that of cos cells expressing normal gt and ht . our studies showed that the km of ht - gt for n - acetyl lactosamine was identical to the km of gt for this substrate , similarly the km of gt - ht for phenylbdgalactoside was approximately the same as the km of ht for phenylbdgalactoside ( fig3 ). these findings indicate that the chimeric enzymes are functioning in a cytoplasmic tail - independent manner , such that the catalytic domains are entirely functional , and are in agreement with those of henion et al ( 23 ), who showed that an nh 2 terminal truncated marmoset gt ( including truncation of the cytoplasmic and transmembrane domains ) maintained catalytic activity and confirmed that gt activity is indeed independent of the cytoplasmic domain sequence . if the golgi localisation signal for gt and ht is contained entirely within the cytoplasmic domains of the enzymes , then switching the cytoplasmic tails between the two transferases should allow a reversal of the order of glycosylation . co - transfection of cos cells with cdna encoding the chimeric glycosyltransferases ht - gt and gt - ht caused a reversal of staining observed with the wild type glycosyltransferases ( fig5 ), demonstrating that the order of glycosylation has been altered by exchanging the cytoplasmic tails . furthermore , co - transfection with cdna encoding glycosyltransferases with the same cytoplasmic tails ( i . e . ht + ht - gt and gt + gt - ht ) gave rise to equal expression of both gal - α ( 1 , 3 )- gal and h substance ( fig5 ). the results imply that the cytoplasmic tails of gt and ht are sufficient for the localisation and retention of these two enzymes within the golgi . to date only twenty or so of at least one hundred predicted glycosyltransferases have been cloned and few of these have been studied with respect to their golgi localisation and retention signals ( 34 ). studies using the elongation transferase n - acetylglucosaminyltransferase i ( 33 - 37 ), the terminal transferases α ( 2 , 6 ) sialyltransferase ( 24 - 26 ) and β ( 1 , 4 ) galactosyltransferase ( 38 - 40 ) point to residues contained within the cytoplasmic tail , transmembranes and flanking stem regions as being critical for golgi localisation and retention . there are several examples of localization signals existing within cytoplasmic tail domains of proteins including the kdel and kkxx motifs in protein resident within the endoplasmic reticulum ( 41 , 42 ) the latter motif also have been identified in the cis golgi resident protein ergic - 53 ( 43 ) and a di - leucine containing peptide motif in the mannose - 6 - phosphate receptor which directs the receptor from the trans - golgi network to endosomes ( 44 ). these motifs are not present within the cytoplastic tail sequences of ht or gt or in any other reported glycosyltransferase . to date a localisation signal in golgi resident glycosyltransferases has not been identified and while there is consensus that transmembrane domains are important in golgi localisation , it is apparent that this is not essential for the localisation of all glycosyltransferases , as shown by the study of munro ( 45 ) where replacement of the transmembrane domain of α ( 2 , 6 ) sialyltransferase in a hybrid protein with poly - leucine tract resulted in normal golgi retention . dahdal and colley ( 46 ) also showed that sequences in the transmembrane domain were not essential to golgi retention . this study is the first to identify sequence requirements for the localisation of α ( 1 , 2 ) fucosyltransferase and α ( 1 , 3 ) galactosyltransferase within the golgi . it is anticipated that other glycosyltransferases will have similar localisation mechanisms . a construct is made using pcr and subcloning as described in example 1 , such that amino acids # 1 to # 6 of the pig α ( 1 , 3 )- galactosyltransferase ( mnvkgr ) replace amino acids # 1 to 5 of the pig secretor ( fig6 ). constructs are tested as described in example 1 . a construct is made using pcr and subcloning as described in example 1 , such that amino acids # 1 to # 6 of the pig α ( 1 , 3 )- galactosyltransferase ( mnvkgr ) replace amino acids # 1 to 8 of the pig h transferase ( fig7 ). constructs are tested as described in example 1 . the pig endothelial cell line piec expressing the chimeric α1 , 2fucosyltransferase was produced by lipofectamine transfection of pgtht plasmid dna ( 20 μg ) and psv2neo ( 2 μg ) and selecting for stable integration by growing the transfected piec in media containing g418 ( 500 μg / ml ; gibco - brl , gaithersburg , md .). fourteen independant clones were examined for cell surface expression of h substance by staining with uea - 1 lectin . & gt ; 95 % of cells of each of these clones were found to be positive . fig8 shows a typical facs profile obtained for these clones . a nrui / noti dna fragment , encoding the full length chimeric α1 , 2fucosyltransferase , was generated utilising the polymerase chain reaction and the phht plasmid using the primers : 5 ′- ttcgcgaatgaatgtcaaaggaagactctg , in which the underlined sequence contains a unique nrui site ; the dna was purified on gels , electroeluted and subcloned into a nrui / noti cut genomic h - 2kb containing vector resulting in the plasmid clone ( ph - 2kb - gtht ) encoding thee chimeric α ( 1 , 2 )- fucosyltransferase gene directionally cloned into exon 1 of the murine h - 2kb gene , resulting in a transcript that commences at the h - 2kb transcriptional start site , continuing through the gtht cdna insert . the construct was engineered such that translation would begin at the initiation condon ( atg ) of the hht cdna and terminate at the in - phase stop codon ( tga ). dna was prepared for microinjection by digesting ph - 2kb - hht with xhoi and purification of the h - 2kb - hrt dna from vector by electrophoretic separation in agarose gels , followed by extraction with chloroform , and precipitation in ethanol to decontaminate the dna . injections were performed into the pronuclear membrane of ( c57bl / 6xsjl ) f1 zygotes at concentrations between 2 - 5 ng / ml , and the zygotes transferred to pseudopregnant ( c57bl / 6xsjl ) f1 females . the presence of the transgene in the live offspring was detected by dot blotting . 5 mg of genomic dna was transferred to nylon filters and hybridized with the insert from gtht , using a final wash at 68 ° c . in 0 . 1xssc / 1 % sds . fig9 thaws the results of testing 12 live offspring , with two mice having the transgenic construct integrated into the genome . expression of transgenic protein is examined by estimating the amount of ueai lectin ( specific for h substance ) or anti - h mab required to haemagglutinate red blood cells from transgenic mice . hemagglutination in this assay demonstrates transgene expression . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . transfected cos cells were stained with fitc - labelled ib4 ( lectin specific for gal - α ( 1 , 3 ) gal or ueai ( lectin specific for h substance ) and positive staining cells were visualized and counted by fluorescence microscopy . results are from at least three replicates . 1 . leventhal , j r et al . complement depletion prolongs discordant cardiac xenograft survival in rodents and non - human primates . transplant prod . 25 , 398 - 399 ( 1993 ). 2 . pruitt , s et al . the effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts . transplantation 57 , 363 - 370 ( 1994 ). 3 . leventhal , j r et al . removal of baboon and human antiporcine igg and igm natural antibodies by immunoabsorption . transplantation 59 , 294 - 300 ( 1995 ). 4 . brewer , r j et al . depletion of performed natural antibody in primates for discordant xenotransplantation by continuous donor organ plasma perfusion . transplantation proac 25 , 385 - 386 ( 1993 ). 5 . mccurry , k r et al . human complement regulatory proteins protect swine - 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