Patent Application: US-68799603-A

Abstract:
the invention relates to a recombinant lactococcus strain , with environmentally limited growth and viability . more particularly , it relates to a recombinant lactococcus that can only survive in a medium , where well - defined medium compounds are present . a preferred embodiment is a lactococcus that may only survive in a host organism , where such medium compounds are present , but cannot survive outside the host organism in the absence of such medium compounds .

Description:
as previously identified , the invention includes a suitable biological containment system for lactococcus . in one aspect , the invention is an isolated strain of lactococcus sp . comprising a defective thymidylate synthase gene . preferably , the defective thymidylate synthase gene is inactivated by gene disruption . even more preferably , the lactococcus sp . is lactococcus lactis . a special embodiment is a lactococcus sp . strain , preferably lactococcus lactis , more preferably a lactococcus lactis mg1363 derivative , wherein the thymidylate synthase gene has been disrupted and replaced by an interleukin - 10 expression unit . the interleukin - 10 expression unit is preferably , but not limited to , a human interleukin - 10 expression unit or gene encoding for human interleukin - 10 . the lactococcus lactis subsp . lactis thymidylate synthase gene ( thya ) has been cloned by ross et al . ( 1990a ). its sequence is comprised in seq id no : 3 and seq id no : 5 . european patent application publication number 0406003 discloses a vector devoid of antibiotic resistance and bearing a thymidylate synthase gene as a selection marker ; the same vector has been described by ross et al . ( 1990b ). however , this vector could not be used in a lactococcus lactis strain due to the lack of a suitable thya mutant that has never been described . the present invention discloses how to construct such a mutant by gene disruption using homologous recombination in lactococcus . in a preferred embodiment , the thya gene is disrupted by a functional human interleukin - 10 expression cassette . however , it is clear that any construct can be used for gene disruption , as long as it results in an inactivation of the thya gene or in an inactive thymidylate synthase . as a nonlimiting example , the homologous recombination may result in a deletion of the gene , in one or more amino acid substitutions that lead to an inactive form of the thymidylate synthase , or in a frame shift mutation resulting in a truncated form of the protein . such a lactococcus sp . thya mutant is very useful as a host strain for transformation in situations where more severe containment than purely physical containment is needed . indeed , thya mutants cannot survive in an environment without or with only a limited concentration of thymidine and / or thymine . when such a strain is transformed with a plasmid that does not comprise an intact thya gene and cannot complement the mutation , the transformed strain will become suicidal in a thymidine / thymine - poor environment . such a strain can be used in a fermentor as an additional protection for the physical containment . moreover , the present invention discloses that such a strain is especially useful in cases where the strain is used as a delivery vehicle in an animal body . indeed , when such a transformed strain is given , for example , orally to an animal — including humans — it survives in the gut , provided that a sufficiently high concentration of thymidine / thymine is present , and produces homologous and / or heterologous proteins , such as human interleukin - 10 , that may be beneficial for the animal . the invention further demonstrates that the transformed strains surprisingly pass the gut at the same speed as the control strains , showing that their loss of viability indeed is not different from that of the control strains . however , once the strain is secreted in the environment , for example , in the feces , it is not able to survive any longer . the transforming plasmid can be any plasmid , as long as it does not complement the thya mutation . it may be a self - replicating plasmid that preferably carries one or more genes of interest and one or more resistance markers , or it may be an integrative plasmid . in the latter case , the integrative plasmid itself may be used to create the mutation by causing integration at the thya site , whereby the thya gene is inactivated . preferably , the active thya gene is replaced by double homologous recombination by a cassette comprising the gene or genes of interest , flanked by targeting sequences that target the insertion to the thya target site . it is of extreme importance that these sequences are sufficiently long and sufficiently homologous to integrate the sequence into the target site . preferably , the targeting sequences include at least 100 contiguous nucleotides of seq id no : 1 at one side of the gene of interest and at least 100 contiguous nucleotides of seq id no : 2 at the other side . more preferably , the targeting sequences consist of at least 500 contiguous nucleotides of seq id no : 1 at one side of the gene of interest and at least 500 contiguous nucleotides of seq id no : 2 at the other side . most preferably , the targeting sequences consist of seq id no : 1 at one side of the gene of interest and seq id no : 2 at the other side , or the targeting sequences consist of at least 100 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 1 at one side of the gene of interest and at least 100 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 2 at the other side of the gene of interest . preferably , the targeting sequences consist of at least 500 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 1 at one side of the gene of interest and at least 500 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 2 at the other side of the gene of interest . most preferably , the targeting sequences consist of at least 1000 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 1 at one side of the gene of interest and at least 1000 nucleotides that are at least 80 % identical , preferably 90 % identical to a region of seq id no : 2 at the other side of the gene of interest . the percentage identity is measured with blast , according to altschul et al . ( 1997 ). a preferred example of a sequence homologous to seq id no : 1 is given in seq id no : 7 . for the purpose of the invention , seq id no : 1 and seq id no : 7 are interchangeable . transformation methods of lactococcus are known to the person skilled in the art and include , but are not limited to , protoplast transformation and electroporation . a transformed lactococcus sp . strain according to the invention is useful for the delivery of prophylactic and / or therapeutic molecules and can be used in a pharmaceutical composition . the delivery of such molecules has been disclosed , as a nonlimiting example , in pct international publication numbers wo 97 / 14806 and wo 98 / 31786 . prophylactic and / or therapeutic molecules include , but are not limited to , polypeptides such as insulin , growth hormone , prolactin , calcitonin , group 1 cytokines , group 2 cytokines and group 3 cytokines and polysaccharides such as polysaccharide antigens from pathogenic bacteria . a preferred embodiment is the use of a lactococcus sp . strain according to the invention to deliver human interleukin - 10 . this strain can be used in the manufacture of a medicament to treat crohn &# 39 ; s disease as indicated herein . the invention is further explained with the use of the following illustrative examples . from l . lactis mg1363 ( gasson , 1983 ) regions flanking the sequence according to ross et al . ( 1 990a ) have been cloned . the knowledge of these sequences is of critical importance for the genetic engineering of any lactococcus strain in a way as described below , as the strategy will employ double homologous recombination in the areas of 1000 bp at the 5 ′ end ( seq id no : 1 ) and 1000 bp at the 3 ′ end ( seq id no : 2 ) of thya , the “ thya target .” these sequences are not available from any public source to date . these flanking dna fragments have been cloned and their sequence has been identified . the sequence of the whole locus is shown in seq id no : 3 ; a mutant version of this sequence is shown in seq id no : 5 . both the 5 ′ and 3 ′ sequences are different from the sequence at genbank ae006385 describing the l . lactis il1403 sequence . ( bolotin , in press ) or at af336368 describing the l . lactis subsp . lactis chcc373 sequence . from the literature , it is apparent that homologous recombination by use of the published sequences adjacent to thya ( ross et al ., 1990a ) ( 86 bp at the 5 ′ end and 31 bp at the 3 ′ end ) is virtually impossible due to the shortness of the sequences . indeed , biswas et al . ( 1993 ) describe a logarithmically decreasing correlation between the length of the homologous sequences and the frequency of integration . the sequences of l . lactis thy 11 , thy 12 , thy 15 and thy 16 at the thya locus as determined in the present invention are given by seq id nos : 19 , 20 , 21 , 22 respectively . the thya replacement is performed by making suitable replacements in a plasmid - borne version of the thya target , as described below . the carrier plasmid is a derivative of pori19 ( law et al ., 1995 ), a replication - defective plasmid , which only transfers the erythromycin resistance to a given strain when a first homologous recombination , at either the 5 ′ 1000 bp or at the 3 ′ 1000 bp of the thya target . a second homologous recombination at the 3 ′ 1000 bp or at the 5 ′ 1000 bp of the thya target yields the desired strain . the thya gene is replaced by a synthetic gene encoding a protein which has the l . lactis usp45 secretion leader ( van asseldonk et al ., 1990 ) fused to a protein of an identical amino - acid sequence when : ( a ) the mature part of human - interleukin 10 ( hil - 10 ) or ( b ) the mature part of hil - 10 in which proline at position 2 has been replaced with alanine or ( c ) the mature part of hil - 10 in which the first two amino acids have been deleted ; ( a ), ( b ) and ( c ) are called hil - 10 analogs , the fusion products are called usp45 - hil - 10 . the thya gene is replaced by an expression unit comprising the lactococcal p1 promoter ( waterfield et al ., 1995 ), the e . coli bacteriophaget7 expression signals , putative rna stabilizing sequence and modified gene10 ribosomal binding site ( wells and schofield , 1996 ). at the 5 ′ end , the insertion is performed in such way that the atg of thya is fused to the p1 - t7usp45 - hil - 10 expression unit . 5 ′ agataggaaaatttc atg acttacgcagatcaagttttt ... thya wild - type ( seq id no : 27 ) gattaagtcatcttacctctt ... p1 - t7 - usp45 - hil10 ( seq id no : 14 ) 5 ′ agataggaaaatttc atg gattaagtcatcttacctctt ... thya − , p1 - t7 - usp45 - hil10 ( seq id no : 15 ) alternatively , at the 5 ′ end , the insertion is performed in such a way that the thya atg is not included : 5 ′ agataggaaaatttcacttacgcagatcaagttttt ... thya wild - type ( seq id no : 28 ) gattaagtcatcttacctctt ... p1 - t7 - usp45 - hil10 ( seq id no : 14 ) 5 ′ agataggaaaatttcgattaagtcatcttacctctt ... thya − , p1 - t7 - usp45 - hil10 ( seq id no : 16 ) alternatively , at the 5 ′ end , the insertion is performed in such a way that the thya promoter ( ross , 1990 a ) is not included : 5 ′ tctgagaggttattttgggaaatacta ttgaac catatcgaggtgtgtgg tataat gaagggaattaaaaaa ( seq id no : 29 ) gata ggaa aatttc atg ... thya wild - type gattaagtcatcttacctctt ... p1 - t7 - usp45hil10 ( seq id no : 29 ) 5 ′ tctgagaggttattttgggaaatactagattaagtcatcttacctctt ... thya − , p1 - t7 - usp45 - hil10 ( seq id no : 14 ) at the 3 ′ end , an actagt spel restriction site was engineered immediately adjacent to the taa stop codon of the usp45 - hil - 10 sequence . this was ligated in a tctaga xbai restriction site , which was engineered immediately following the thya stop codon aaaatccgtaac taa ctagt 3 ′... usp45 - hil10 ( seq id no : 30 ) gatttagcaatt taa attaaattaatctataagtt 3 ′... thya - wild - type ( seq id no : 31 ) tctaga attaatctataagttactga 3 ′... engineered thya target ( seq id no : 32 ) aaaatccgtaac taa ctaga attaatctataagttactga 3 ′... thya − , usp45 - hil10 ( seq id no : 18 ) these constructs are depicted in fig2 . the sequences of pothy 11 , pothy12 pothy15 and pothy16 are given by seq id nos : 23 , 24 , 25 , and 26 respectively . the resulting strains are thya deficient , a mutant not yet described for l . lactis . it is strictly dependent upon the addition of thymine or thymidine for growth . the map of the deletion , as well as the pcr analysis of all the isolates / mutants of the present invention , is shown in fig3 a - 4b . the presence of the thymidylate synthase and the interleukin 10 ( il - 10 ) gene in the wild - type strain and in the independent isolates / mutant was analyzed by southern analysis as shown in fig5 a - 6b . the region around the inserted hil - 10 gene was isolated by pcr and the dna sequence was verified . the structure is identical to the predicted sequence . human interleukin - 10 ( hil - 10 ) production in the mutants was checked by western blot analysis and compared with the parental strain , transformed with ptrex1 as negative control , and the parental strain , transformed with the il10 - producing plasmid pt1hil10apxa as a positive control ( fig7 a ). the concentration in the culture supernatant was quantified using elisa . as shown in fig7 b , both isolates of the mutant produce a comparable , significant amount of hil - 10 , be it far less than the strain , transformed with the nonintegrative plasmid pt1hil10apxa . fig8 a and 8b further demonstrate that all mutants produce a significant amount of hil - 10 . fig9 shows the production of hil - 10 by the l . lactis strains ll108 carrying pothy11 , pothy12 , or pothy16 . quantification ( by elisa ) of hil - 10 present in the culture supernatant of the indicated strains is shown . the n - terminal protein sequence of the recombinant hil - 10 was determined by edman degradation and was shown to be identical to the structure as predicted for the mature , recombinant hil - 10 . the protein showed full biological activity . ll108 is an l . lactis strain carrying a genomic integration of the repa gene , required for replication ofpori19 derived plasmids such as pothy11 , pothy12 , pothy15 or pothy16 . this strain was kindly donated by dr . jan kok , university of groningen , the netherlands . the plasmids pothy11 , pothy12 , pothy15 and pothy16 carry the synthetic human il - 10 gene in different promoter configurations ( see fig2 ), flanked by approximately 1 kb of genomic dna derived from the thya locus , upstream and downstream from thya . these plasmids were used for the construction of the genomic integration as described . the effect of the thymidylate synthase deletion on the growth in thymidine less and thymidine - supplemented media was tested ; the results are summarized in fig1 and 11 . an absence of thymidine in the medium strongly limits the growth of the mutant and even results in a decrease of colony - forming units after four hours of cultivation . the addition of thymidine to the medium results in an identical growth curve and amount of colony - forming units , compared to the wild - type strain , indicating that the mutant does not affect the growth or viability in thymidine - supplemented medium . fig1 clearly demonstrates that thy12 viability is severely impaired in the absence of thymidine . fig1 finally shows that l . lactis thy12 passes through the intestine of the mice at the same speed as mg1363 . loss of viability does not appear to differ between thy12 and mg1363 . thy12 appears fully dependent on thymidine for growth , indicating that no thy12 bacteria had taken up a foreign thya gene . altschul , s . f ., madden , t . l ., schaffer , a . a ., zhang , j ., zhang , z ., miller , w . and lipman d . j . 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