Patent Application: US-25407799-A

Abstract:
the invention provides nucleic acids that encode a first glycosyltransferase that competes with a second enzyme for a substrate , thereby reducing the formation of a product of the second enzyme . the nucleic acids are useful in producing cells and organs with reduced antigenicity and which may be used for transplantation .

Description:
the invention will now be described by way of reference only to the following non - limiting figures and example . fig1 shows the nucleic acid sequence ( seq . id . no : 5 ) and corresponding amino acid sequence ( seq . id . no : 6 ) of a porcine secretor sequence . fig2 shows a comparison of the amino acid sequences of pig , human and rabbit fut1 and fut2 . the rows in each panel represent pig ( seq . id . no : 7 ), human ( seq . id . no : 8 ) and rabbit ( seq . id . no : 9 ) fut2 and pig ( seq . id . no : 10 ), human ( seq . id . no : 11 ) and rabbit ( seq . id . no : 12 ) fut1 from top to bottom . fig3 shows a typical facs profile of pig endothelial cells which express α ( 1 , 2 )- fucosyltransferase . fig4 is a dot blot showing the presence of α ( 1 , 2 )- fucosyltransferase in six offspring of mice injected with a transgenic construct . the work presented below is surprising in that the inventors had previously attempted to clone human secretor but were unsuccessful . a non - functional human pseudogene for secretor was cloned . this raised the question of whether other species such as pigs have a functional gene for secretor . the fact that the inventors were able to successfully clone the pig secretor gene and use it to down regulate unwanted epitopes was surprising . because of the differences in blood group antigens between pigs and humans , it was not known whether pigs have secretor antigens . the cloning of a functional gene indicates that pigs do have the epitope produced by the secretor glue . furthermore , although fut1 had been cloned , it did not permit the pig secretor gene to be isolated . fut1 and fut2 are sufficiently different in that probes based on the sequence of fut1 do not hybridise with that of fut2 . the gene encoding the sequence for the human secretor gene ( sec2 ) ( 27 ) was cloned from human genomic dna using a pcr strategy according to the published sequence , primers , and conditions . a pig genomic liver library in embl - 3 ( clonetech laboratories , palo alto , calif .) was screened using this human clone . nine clones were obtained after screening 5 × 10 5 plaques . two of these were randomly chosen for further examination . limited restriction mapping showed identical banding patterns for both clones , with a 3 . 3 kb psti fragment specifically hybridising with the human ( sec 2 α ( 1 , 2 )- fucosyltransferase ) probe . this fragment ( pse 16 . 1 ) was sequenced using the abi automated sequencer . for functional studies the coding segment of the genomic clone was subcloned into an expression vector . utilising the polymerase chain reaction ( pcr ), and the pig se sequence as obtained above , 1048 bp gene product was derived using primers : 5 ′ primer homologous to the 5 ′ utr : 5 ′ cag agactt atgctcagcatgcaggc ( seq . id . no : 1 ) in which the underlined sequence contains a unique hind iii site ; 3 ′ primer homologous to the 3 ′ utr : 5 ′- 5 ′- gtc ctgcag tgagtgcttaaggagtgg ( seq . id . no : 2 ) where the underlined sequence contains a psti site . this pcr product was purified as above , digested with hindii and psti , ligated with similarly digested pccna ( invitrogen corporation , san diego , calif . ), and then used to transform mc1061 / p3 . one clone , designated ppset , which encodes the cdna for the porcine α ( 1 , 3 )- galactosyltransferase ( 19 ), and ppht , which encodes the cdna for the porcine “ h ” α :( 1 , 2 )- fucosyltransferase ( 33 ). cos cells were maintained in dulbecco &# 39 ; s modified eagles medium ( dmem ) ( cytosystems pty . ltd ., castle hill , nsw , australia ). cos cells were transfected using the deae - dextran method , using dmem medium supplemented with foetal clone ii ( hy clone utah ), and 48 h later cells were examined for cell surface expression . direct fluorescence stainey of cell surface carbohydrate epitopes was performed with fitc or tritc conjugated lectins : ib4 lectin isolated from griffonia simplicifolia ( sigma , st . louis , mo .) detects gal - α ( 1 , 3 )- gal and ueai lectin isolated from ulex europaeus ( sigma , and ey laboratories , inc ., san mateo , calif .) detects h substance . h substance was also detected by indirect immunofluorescence using a monoclonal antibody ( mab ) specific for the h - epitope ( ash - 1952 ) developed at the ari , and fitc conjugated goat anti - mouse igg ( zymed laboratories , san francisco , calif .) used to detect murine antibody binding . cells were washed twice with phosphate buffered saline and lysed in either 1 % triton x100 / 100 mm tris ph7 . 0 or 1 % triton x100 / 100 mm sodium cacodylate ph 6 . 5 / 25 mm mncl 2 at 4 ° c . for 30 min , lysates centrifuged and the supernatant collected and stored at − 70 ° c . protein concentration was determined by the bradford test , using bovine serum albumin as a standard ; 5 - 20 μg of cell extract was used per transferase assay . the assay for α - 1 , 2 fucosyltransferase involved mixing cell extracts and acceptor ( 75 mm pheny - β - dgalactoside ( sigma )) in 50 μl 50 mm mops ( 3 -[ n - morpholino ] propanesulphonic acid ) ph 6 . 5 ; 20 mm mncl 2 ; 5 mm atp ; 3 μm gdp [ 14 c ]- fuc ( specific activity 287 mci / mmol , amersham international plc , amersham , uk ) and incubation for 2 h at 37 ° c . the reaction was terminated by the addition of ethanol , and the incorporated 14 c - fuc determined by liquid scintillation counting after separation in sep - pak c18 cartridges ( waters - millipore , millford , mass .). in all cases the parallel reactions were performed in the absence of added acceptor molecules , to allow for the calculation of specific incorporation . two clones were obtained after screening 5 × 10 5 plaques of a pig genomic liver library in embl - 3 ( clonetech laboratories , palo alto , calif .) with the cdna fragment encoding the full length human fut2 ( 27 ). limited restriction mapping showed identical banding patterns for both clones , with a 3 . 3 kb pst i fragment specifically hybridising with the human fut2 probe . this fragment was subcloned to generate the clone psel6 . 1 , which was sequenced . the complete nucleotide sequence of the pig fut2 dna contains 1269 bp of nucleotide sequence ( fig1 ): a 8 bp 5 ′ untranslated ( ut ) region , an open reading frame of 1060 bp encoding a 340 amino acid protein with the initiation codon being nucleotide 9 , succeeded by 156 bp of 3 ′ ut . the predicted protein sequence of the pig fut2 suggests a type ii integral membrane protein , typical of other glycosyltransferases . there are three distinct structural features of the predicted protein : ( i ) a short ( 4 amino acid ) amino - terminal cytoplasmic tail ; ( ii ) a putative transmembrane region composed of 21 hydrophobic amino acids ( residues 5 - 26 ), flanked on either side by charged amino acid residues ; ( iii ) a 314 amino acid carboxyl - terminal domain which contains three potential n - linked glycosylation sites . comparison of the amino acid sequences of pig fut2 with the human ( 22 , 27 ) and rabbit ( 29 ) α ( 1 , 2 )- fucosyltransferases shows the highest identity with the se transferase rather than the h transferase ( fig2 ): the pig fut2 shows 83 . 2 % identity with human fut2 , 74 . 1 % identity with rabbit fut2 , 58 . 5 % identity with pig fut1 , 57 . 1 % identity with human fut1 , and 58 . 8 % identity with rabbit fut1 . we note that the highest sequence identity is in the carboxyl portion of the molecule , which contains the catalytic domain ( 30 ). the pig fut2 nucleotide sequence shows about 36 % homology with human fut1 . the 1 . 3 kb pst i fragment containing the coding sequence was subcloned into the cos cell expression vector pcdna - 1 ( invitrogen corporation san diego , calif .). cos cells transfected with the cloned genomic dna encoding the pig fut2 expressed h substance , as indicated by staining with fluoresceinated uea i lectin , which detects h substance ( 31 ) (˜ 65 % positive as shown in table 1 ). after transfection with the pig fut1 cdna clone similar staining was observed while no staining was seen with the reagent on cos cells transfected with the cdna for the pig α ( 1 , 3 )- galactosyltransferase ( 19 ). in contrast , staining with fluoresceinated ib4 lectin , which detects galα ( 1 , 3 ) gal ( 32 ), was detected on cos cells transfected with pig α ( 1 , 3 )- galactosyltransferase cdna but not with the pig fut1 or fut2 dna . cell lysates prepared from cos cells transfected with pfut2 and pfut1 were assayed for α ( 1 , 2 )- fucosyltransferase activity . using mock - transfected cos cells to show baseline activity ( 1 . 1 nmol hr − 1 mg − 1 ), significant α ( 1 , 2 )- fucosyltransferase activity was observed in lysates from both pfut2 ( 151 . 1 nmol hr − 1 mg − 1 ) and pfut1 ( 140 . 0 nmol hr − 1 mg − 1 ) transfected cos cells , but not in ppgt transfected cos cells ( 6 . 7 nmol hr − 1 mg − 1 ). the enzyme activity measured in these lysates reflects the expression of h substance on the cell surface as shown in example 2 . cos cells transfected with the pig α ( 1 , 3 )- galactosyltransferase cdna clone expressed gal - α ( 1 , 3 )- gal as indicated by reactivity with the ib4 lectin ( 65 % of cells reactive ) ( table 1 ). cos cells was also able to express h substance , as after transfection with either the pig fut2 or fut1 clones they stained with the ueai lectin ( 68 and 75 % of cells respectively reactive , table 1 ). however , when the cos cells were simultaneously transfected with the pig α ( 1 , 3 )- galactosyltransferase cdna clone and either pig fut2 or pig fut1 , and examined for cell surface staining of either carbohydrate , the cells predominantly expressed h substance ( 72 % of cells positive , table 1 ), compared with 8 % of cells expressing galα ( 1 , 3 )- gal ( table 1 ). when both pig fut2 and pig fut1 were cotransfected together with the pig α ( 1 , 3 )- galactosyltransferase cdna , only one h substance was detected ( 76 %) and & lt ; 1 % galα ( 1 , 3 )- gal ( table 1 ). this reduction observed using fut1 and fut2 was specific and not due to amount of dna used for transfection , because using twice the amount of dna for either fut1 or fut2 alone had no effect on the expression of galα ( 1 , 3 )- gal . thus expression of both fut2 and fut1 resulted in a major decrease in expression of galα ( 1 , 3 )- gal . cell lysates prepared from cos cells transfected in the manner described in example 1 with pfut2 ( pig se ), pfut1 ( pig h transferase ), or with vector alone were assayed for α ( 1 , 2 )- fucosyltransferase activity , and the kinetic values were calculated . the km values ( reflecting the affinity for substrate ) obtained for pfut1 , and pfut2 are shown in table 2 . these values were compatible with those of human and rabbit homologues that have been reported . the respective km values obtained for pfut1 , and pfut2 with various substrates were : ( a ) galβ ( 1 , 3 ) glcnac ( type i ): 6 . 0 mm for pfut1 and 1 . 3 mm for pfut2 . the km values reported for rabbit fut1 and rabbit fut2 were 3 . 1 mm and 1 . 5 mm respectively ( 34 ) and 2 mm and 1 mm for human fut1 and human fut2 respectively ( 35 ). ( b ) galβ ( 1 , 4 ) glcnac ( type ii ): 3 . 7 mm for pfut1 and 4 . 4 mm for pfut2 . the km values reported for rabbit fut1 and rabbit fut2 were 4 . 2 mm and 6 . 7 mm respectively ( 34 ) and 1 . 9 mm and 5 . 7 mm for human fut1 and human fut2 respectively ( 37 ). ( c ) galβ ( 1 , 3 ) galnac ( type iii ): 14 mm for pfut1 and for pfut2 0 . 2 mm . the km values reported for rabbit fut1 , and rabbit fut2 were 5 . 8 mm and 1 mm respectively ( 34 ). thus , pfut1 can be distinguished from pfut2 on the basis of substrate preference ; pfut1 is relatively specific for type ii and type iv substrates , while pfut2 ( and other secretor homologues ), although having greater affinity for type i and iii acceptors , will use other substrates . the pig endothelial cell line piec expressing the secretor type α ( 1 , 2 )- fucosyltransferase were produced by lipofectamine transfection of pfut2 plasmid dna ( 20 μg ) and psv2neo ( 2 μg ). cells with stable integration were selected by growing the transfected piec in media containing g418 ( 500 ug / 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 : fig3 shows a typical facs profile obtained for these clones . a 1023 bp nrui / notl dna fragment , encoding the full length pfut2 was generated utilizing the polymerase chain reaction and the phht plasmid ( 36 ) using the primers : the dna was purified on gels before being electroeluted and subcloned into a nrui / noti cut genomic h - 2k b containing vector ( 38 ), resulting in the plasmid clone ( ph - 2k b - pfut2 ) encoding the pfut2 gene directionally cloned into exon 1 of the murine h - 2k b gene . this produced a transcript that commences at the h - 2k b transcriptional start site , continuing through the pfut2 cdna insert . the construct was engineered such that translation would begin at the initiation codon ( atg ) of the pfut2 cdna and terminate at the stop codon ( tga ) 1023 bp downstream . dna was prepared for microinjection by digesting ph - 2k b - pfut2 with xhoi and purification of the h - 2k b - pfut2 dna from the vector by electrophoretic separation in agarose gels , followed by extraction with chloroform , and precipitation in ethanol to decontaminate the dna . infections were performed on the pronuclear membrane of ( c57bl / 6 × sjl ) f 1 zygotes at concentrations between 2 - 5 ng / μl , and the zygotes were then transferred to pseudopregnant ( c57bl / 6 × sjl ) f 1 females . the presence of the transgene in live offspring was detected by dot blotting . 5 μg of genomic dna was transferred to nylon filters and hybridized with the insert from pfut2 , using a final wash comprising 0 . 1 × ssc / 1 % sds at 68 ° c . fig4 shows the results of testing 16 live offspring , of which six were found to have the transgenic construct integrated into the genome . expression of transgenic protein is examined by haemagglutination and fucosyltransferase activity . 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 . 1 . leventhal , j r et al . complement depletion prolongs discordant cardiac xenograft survival in rodents and non - human primates . transplantion proc . 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 preformed natural antibody in primates for discordant xenotransplantation by continuous donor organ plasma perfusion . transplantation proc . 25 , 385 - 386 ( 1993 ). 5 . mccurry , k r et al . human complement regulatory proteins protect swine - to - primate cardiac xenografts from humoral injury . nature med . 1 , 423 - 427 ( 1995 ). 6 . fodor , w l et al . expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection . proc . natl . acad . sci usa 91 , 11153 - 11157 ( 1994 ). 7 . rosengard , a m et al . tissue expression of the human complement inhibitor decay accelerating factor in transgenic pigs . transplantation 59 , 1325 - 1333 ( 1995 ). 8 . sandrin , m s , vaughan , h a , dabkowski , p l & amp ; 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