Patent Application: US-201515517718-A

Abstract:
a method for modifying the genome of a target phage is described . compositions comprising such modified phage are also described . the compositions may be formulated as a medicament , which are useful for human treatment and may treat various conditions , including bacterial infections .

Description:
this invention will now be described in more detail , by way of example only , and with reference to the accompanying drawings , in which : fig1 is a schematic diagram showing construction of plasmids containing laczδm15 and phi33 endolysin for genetic modification of p . aeruginosa to carry these genes in trans ; fig2 is a schematic diagram showing construction of plasmids to genetically modify phi33 to replace the endolysin gene with rpsb - sasp - c and laczα , and then to subsequently remove the laczα marker ; fig3 is a schematic diagram showing construction of a phi33 phage derivative which is a markerless , non - lytic phage that has endolysin replaced by rpsb - sasp - c , constructed via gain and then loss of a laczα genetic marker , according to the invention ; and fig4 shows plate a : recombinant 4 ) 33 with laczα sequence incorporated into the genome and plate b : wild type 4 ) 33 . in both plates , the phage were plagued on p . aeruginosa strain pao1 expressing laczδm15 , in the presence of s - gal and ammonium iron iii citrate . summary of the genetic modification of a lytic bacteriophage to render it non - lytic , and such that it carries sasp - c under the control of a promoter that usually controls expression of the 30s ribosomal subunit protein s2 gene ( rpsb ), utilising a laczα marker as a means of identifying genetically - modified phage . genes can be removed and added to the phage genome using homologous recombination . there are several ways in which phages carrying foreign genes and promoters can be constructed and the following is an example of such methods . for the construction of a phi33 derivative it is shown how , using an e . coli / p . aeruginosa broad host range vector , as an example only , how the phage may be rendered non - lytic , and how the sasp - c gene under the control of an rpsb promoter may be added to the bacteriophage genome via homologous recombination , utilising a laczα marker for the identification of recombinant phage . it is also shown how the laczα marker may be removed via a subsequent homologous recombination step , to yield a markerless , non - lytic phage that carries the sasp - c gene under the control of an rpsb promoter . since these bacteriophage to be modified are lytic ( rather than temperate ), a requirement for these described steps of bacteriophage construction is the construction of a suitable host p . aeruginosa strain that carries both the phi33 endolysin gene and the e . coli laczδm15 at a suitable location in the bacterial genome , to complement the δ endolysin , laczδ phenotypes of the desired recombinant bacteriophage . as an example , the construction of these p . aeruginosa strains may be achieved via homologous recombination using an e . coli vector that is unable to replicate in p . aeruginosa . the genomic location for insertion of the endolysin and laczδm15 transgenes should be chosen such that no essential genes are affected and no unwanted phenotypes are generated through polar effects on the expression of adjacent genes . as an example , one such location may be immediately downstream of the p . aeruginosa strain pao1 phoa homologue . the phi33 endolysin gene and the e . coli laczδm15 allele may be cloned into an e . coli vector that is unable to replicate in p . aeruginosa , between two regions of p . aeruginosa strain pa01 genomic dna that flank the 3 ′ end of phoa . this plasmid may be introduced into p . aeruginosa and isolates having undergone a single homologous recombination to integrate the whole plasmid into the genome selected according to the acquisition of tetracycline ( 50 μg / m1 ) resistance . isolates ( endolysin + , laczδm15 + ) which have undergone a second homologous recombination event may then be isolated on medium containing 10 % sucrose ( utilising the sacb counter - selectable marker present on the plasmid backbone ). homologous recombination may be used to replace the endolysin gene of phi33 , to simultaneously render it non - lytic , while introducing both the gene for sasp - c , under the control of a p . aeruginosa rpsb promoter , and the e . coli laczα genetic marker , under the control of the e . coli lac promoter . a region consisting of sasp - c controlled by the rpsb promoter , and the e . coli laczα genetic marker controlled by the lac promoter , may be cloned between two regions of phi33 that flank the endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by phi33 . progeny phage may be harvested and double recombinants identified by plaquing on p . aeruginosa ( endolysin + laczδm15 + ), looking for acquisition of the laczα reporter on medium containing a chromogenic substrate that detects the action of f3 - galactosidase . in a subsequent step , a similar homologous recombination may be used to remove the laczα marker from the previously described , ( laczα + ) phi33 derivative that has been modified to replace the endolysin gene with the gene for sasp - c , under the control of a p . aeruginosa rpsb promoter . a region consisting of sasp - c controlled by the rpsb promoter , may be cloned between two regions of phi33 that flank the endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 k ) strain , and the resulting strain infected by the previously described ( laczα + ) phi33 derivative that has been modified to replace the endolysin gene with the gene for sasp - c , under the control of a p . aeruginosa rpsb promoter . progeny phage may be harvested and double recombinants identified by plaquing on p . aeruginosa ( endolysin + laczδm15 + ), looking for loss of the laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . pcr reactions to generate dna for cloning purposes may be carried out using herculase ii fusion dna polymerase ( agilent technologies ), depending upon the melting temperatures ( t m ) of the primers , according to manufacturers instructions . pcr reactions for screening purposes may be carried out using taq dna polymerase ( neb ), depending upon the t m of the primers , according to manufacturers instructions . unless otherwise stated , general molecular biology techniques , such as restriction enzyme digestion , agarose gel electrophoresis , t4 dna ligase - dependent ligations , competent cell preparation and transformation may be based upon methods described in sambrook et al ., ( 1989 ). enzymes may be purchased from new england biolabs or thermo scientific . dna may be purified from enzyme reactions and prepared from cells using qiagen dna purification kits . plasmids may be transferred from e . coli strains to p . aeruginosa strains by conjugation , mediated by the conjugation helper strain e . coli hb101 ( prk2013 ). a chromogenic substrate for β - galactosidase , s - gal , that upon digestion by β - galactosidase forms a black precipitate when chelated with ferric iron , may be purchased from sigma ( s9811 ). primers may be obtained from sigma life science . where primers include recognition sequences for restriction enzymes , additional 2 - 6 nucleotides may be added at the 5 ′ end to ensure digestion of the pcr - amplified dna . all clonings , unless otherwise stated , may be achieved by ligating dnas overnight with t4 dna ligase and then transforming them into e . coli cloning strains , such as dh5α or top10 , with isolation on selective medium , as described elsewhere ( sambrook et al ., 1989 ). an e . coli / p . aeruginosa broad host range vector , such as psm1080 , may be used to transfer genes between e . coli and p . aeruginosa . psm1080 was previously produced by combining a broad host - range origin of replication , from a pseudomonas plasmid , orit from prk2 , the tetar selectable marker for use in both e . coli and p . aeruginosa , from plasmid prk415 , and the high - copy - number , e . coli origin of replication , oriv , from plasmid puc19 . an e . coli vector that is unable to replicate in p . aeruginosa , psm1104 , may be used to generate p . aeruginosa mutants by allelic exchange . psm1104 was previously produced by combining orit from prk2 , the tetar selectable marker for use in both e . coli and p . aeruginosa , from plasmid prk415 , the high - copy - number , e . coli origin of replication , oriv , from plasmid puc19 , and the sacb gene from bacillus subtilis strain 168 , under the control of a strong promoter , for use as a counter - selectable marker . construction of plasmids to generate a pseudomonas aeruginosa strain that carries both the phi33 endolysin gene and the escherichia coli laczδm15 gene , immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx600 ( fig1 ), comprising psm1104 carrying dna flanking the 3 ′ end of the p . aeruginosa pao1 phoa homologue , may be constructed as follows . a region comprising the terminal approximately 1 kb of the phoa gene from p . aeruginosa may be amplified by pcr using primers b4600 and b4601 ( fig1 ). the pcr product may then be cleaned and digested with spei and bglii . a second region comprising approximately 1 kb downstream of the phoa gene from p . aeruginosa , including the 3 ′ end of the pa3297 open reading frame , may be amplified by pcr using primers b4602 and b4603 ( fig1 ). this second pcr product may then be cleaned and digested with bglii and xhoi . the two digests may be cleaned again and ligated to psm1104 that has been digested with spei and xhoi , in a 3 - way ligation , to yield plasmid psmx600 ( fig1 ). primer b4600 consists of a 5 ′ spei restriction site ( underlined ), followed by sequence located approximately 1 kb upstream of the stop codon of phoa from p . aeruginosa strain pao1 ( fig1 ). primer b4601 consists of 5 ′ bglii and aflii restriction sites ( underlined ), followed by sequence complementary to the end of the phoa gene from p . aeruginosa strain pao1 ( the stop codon is in lower case ; fig1 ). primer b4602 consists of 5 ′ bglii and nhei restriction sites ( underlined ), followed by sequence immediately downstream of the stop codon of the phoa gene from p . aeruginosa strain pao1 ( fig1 ). primer b4603 consists of a 5 ′ xhoi restriction site ( underlined ), followed by sequence within the pa3297 open reading frame , approximately 1 kb downstream of the phoa gene from p . aeruginosa strain pao1 ( fig1 ). 2 . plasmid psmx601 ( fig1 ), comprising psmx600 carrying the phi33 endolysin gene under the control of an endolysin promoter , may be constructed as follows . the endolysin promoter may be amplified by pcr from phi33 using primers b4604 and b4605 ( fig1 ). the endolysin gene itself may be amplified by pcr from phi33 using primers b4606 and b4607 ( fig1 ). the two pcr products may then be joined together by splicing by overlap extension ( soeing ) pcr , using the two outer primers , b4604 and b4607 . the resulting pcr product may then be digested with aflii and bglii , and ligated to psmx600 that has also been digested with aflii and bglii , to yield plasmid psmx601 ( fig1 ). primer b4604 consists of a 5 ′ aflii restriction site ( underlined ), followed by a bi - directional transcriptional terminator ( soxr terminator , 60 - 96 bases of genbank accession number dq058714 ), and sequence of the beginning of the phi33 endolysin promoter region ( underlined , in bold ) ( fig1 ). primer b4605 consists of a 5 ′ region of sequence that is complementary to the region overlapping the start codon of the endolysin gene from phi33 , followed by sequence that is complementary to the end of the endolysin promoter region ( underlined , in bold ; fig1 ). primer b4606 is the reverse complement of primer b4605 ( see also fig1 ). primer b4607 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence complementary to the end of the phi33 endolysin gene ( fig1 ). 3 . plasmid psmx602 ( fig1 ), comprising psmx601 carrying laczam15 under the control of a lac promoter , may be constructed as follows . the laczδm15 gene under the control of a lac promoter may be amplified by pcr from escherichia coli strain dh10b using primers b4608 and b4609 ( fig1 ). the resulting pcr product may then be digested with bglii and nhei , and ligated to psmx601 that has also been digested with bglii and nhei , to yield plasmid psmx602 ( fig1 ). primer b4608 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). primer b4609 consists of a 5 ′ nhei restriction site ( underlined ), followed by a bi - directional transcriptional terminator and sequence complementary to the 3 ′ end of laczδm15 ( underlined , in bold ; fig1 ). genetic modification of pseudomonas aeruginosa to introduce the phi33 endolysin gene and escherichia coli laczδm15 immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx602 ( fig1 ) may be transferred to p . aeruginosa by conjugation , selecting for primary recombinants by acquisition of resistance to tetracycline ( 50 μg / m1 ). 2 . double recombinants may then be selected via sacb - mediated counter - selection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that the endolysin gene and laczδm15 have been introduced downstream of the p . aeruginosa phoa gene . 4 . following verification of an isolate ( pax60 ), this strain may then be used as a host for further modification of phi33 , or similar bacteriophage , where complementation of both an endolysin mutation and a laczα reporter are required . construction of a plasmid to replace the endolysin gene of phi33 and similar phage , by rpsb - sasp - c and laczα 1 . plasmid psmx603 ( fig2 ), comprising psm1080 containing regions of phi33 flanking the endolysin gene , may be constructed as follows . the region of phi33 sequence immediately downstream of the endolysin gene may be amplified by pcr using primers b4665 and b4666 ( fig2 ). this pcr product may then be cleaned and digested with ndei and nhei . the region of phi33 sequence immediately upstream of the endolysin gene may be amplified by pcr using primers b4667 and b4668 ( fig2 ). this second pcr product may then be cleaned and digested with ndei and nhei . the two pcr product digests may then be cleaned again and ligated to psm1080 that has been digested with nhei and treated with alkaline phosphatase prior to ligation . clones carrying one insert of each of the two pcr products may be identified by pcr using primers b4665 and b4668 , and ndei restriction digest analysis of the purified putative clones , to identify plasmid psmx603 ( fig2 ). primer b4665 consists of a 5 ′ nhei restriction site ( underlined ), followed by phi33 sequence located approximately 340bp downstream of the phi33 endolysin gene ( fig2 ). primer b4666 consists of 5 ′ ndei and kpni restriction sites ( underlined ), followed by sequence of phi33 that is located immediately downstream of the endolysin gene ( fig2 ). primer b4667 consists of a 5 ′ ndei restriction site ( underlined ), followed by sequence that is complementary to sequence located immediately upstream of the phi33 endolysin gene ( fig2 ). primer b4668 consists of a 5 ′ nhei site ( underlined ), followed by phi33 sequence that is located approximately 340 bp upstream of the endolysin gene ( fig2 ). 2 . plasmid psmx604 ( fig2 ), comprising psmx603 containing sasp - c under the control of an rpsb promoter , may be constructed as follows . the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ) may be amplified by pcr using primers b4669 and b4670 ( fig2 ). the resulting pcr product may then be digested with kpni and ncoi . the rpsb promoter may be amplified by pcr from p . aeruginosa using primers b4671 and b4672 ( fig2 ). the resulting pcr product may then be digested with ncoi and ndei . the two digested pcr products may then be cleaned and ligated to psmx603 that has been digested with kpni and ndei , yielding plasmid psmx604 ( fig2 ). primer b4669 comprises a 5 ′ kpni restriction site , followed by 5 bases , and then a bi - directional transcriptional terminator , and then sequence complementary to the 3 ′ end of the sasp - c gene from b . megaterium strain km ( atcc 13632 ) ( underlined , in bold ; fig2 ). primer b4670 comprises a 5 ′ ncoi restriction site ( underlined ), followed by sequence of the 5 ′ end of the sasp - c gene from b . megaterium strain km ( atcc 13632 ) ( fig2 ). primer b4671 comprises a 5 ′ ncoi restriction site ( underlined ), followed by sequence complementary to the end of the rpsb promoter from p . aeruginosa pao1 ( fig2 ). primer b4672 comprises a 5 ′ ndei restriction site ( underlined ), followed by sequence of the beginning of the rpsb promoter from p . aeruginosa pao1 ( fig2 ). 3 . psmx605 ( fig2 ), comprising psmx604 containing laczα , may be constructed as follows . laczα may be pcr amplified using primers b4673 and b4674 ( fig2 ). the resulting pcr product may then be digested with kpni and ligated to psmx604 that has also been digested with kpni and treated with alkaline phosphatase prior to ligation , to yield psmx605 ( fig2 ). primer b4673 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence complementary to the 3 ′ end of laczα ( fig2 ). primer b4674 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence of the lac promoter driving expression of laczα ( fig2 ). genetic modification of phi33 and similar phage , to replace the endolysin gene with rpsb - sasp - c and laczα 1 . plasmid psmx605 ( fig2 ) may be introduced into p . aeruginosa strain pax60 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / m1 ), yielding strain pta60 . 2 . strain pta60 may be infected in individual experiments with phage phi33 , or similar phage , and the progeny phage harvested . 3 . recombinant phage , in which the endolysin gene has been replaced by rpsb - sasp - c and laczα , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax60 , onto medium containing s - gal , looking for black plaques , which are indicative of β - galactosidase activity . 4 . pcr may be carried out to check that the endolysin gene has been replaced , and that rpsb - sasp - c and laczα are present . 5 . following identification of a verified isolate ( ptpx60 ; fig3 ), the isolates may be plaque purified twice more on p . aeruginosa strain pax60 , prior to further use . genetic modification to remove the laczα marker from ptpx60 , to generate a markerless version of phi33 , which has been rendered non - lytic , and which carries sasp - c under the control of an rpsb promoter 1 . plasmid psmx604 ( fig2 ) may be introduced into p . aeruginosa strain pax60 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta61 . 2 . strain pta61 may be infected in individual experiments with phage ptpx60 ( fig3 ) and the progeny phage harvested . 3 . recombinant phage , in which laczα marker has been removed , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax60 , onto medium containing s - gal , looking for clear plaques , which are indicative of loss of β - galactosidase activity . 4 . pcr may be carried out to confirm removal of the laczα marker , while ensuring that rpsb - sasp - c is still present . 5 . following identification of a verified isolate ( ptpx61 ; fig3 ), the isolate may be plaque purified twice more on p . aeruginosa strain pax60 , prior to further use . abedon s t . 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