Patent Application: US-64779500-A

Abstract:
adenoviral particles are produced by incubating cells containing a helper adenovirus vector and a helper - dependent adenoviral vector including an adeno - associated virus rep gene , such as rep 78 . cells are provided containing a helper adenovirus vector . a helper - dependent adenoviral vector including an aav rep gene is introduced into the cells , for instance by infection with infective viral particles . the cells are incubated to produce adenoviral particles containing nucleic acid including the aav rep gene . advantageously , cells containing helper adenovirus vector are pre - incubated for 0 . 5 - 12 hours , preferably about 4 hours , to allow expression of viral proteins required for adenoviral genome replication before introducing the helper - dependent adenovirus vector including an aav rep gene .

Description:
in the construction of viral vector useful for gene delivery such as in gene therapy , i . e . the transfer of genetic information into cells or tissues , one of the advantages offered by the aav derived vectors is that the genetic material to be transfered can be directed to preferentially integrate in specific sites on the host chromosome . for this to occur the presence of at least one of the rep peptides is required , as discussed . the present invention is based in part on observation that when cells containing genes coding for the rep aav peptides are infected with ad viruses , the expression of the rep genes results in the suppression of replication of the ad viruses . this phenomenon is non - predictable , although in natural infection when ad acts as helper for the rescue and replication of aav a mechanism to down - regulate the replication of the helper is probably of advantage to the replicating aav . for the biotechnological development of recombinant aav / ad vectors the rep suppression poses a serious problem : amplification of any ad vector is suppressed if rep peptides are present in sufficient amount in the cell . as noted above , different approaches have been taken to reduce or silence the rep gene activity . with the development of helper dependent ad vectors it has been possible to separate the viral replication functions required for vector amplification from the vector itself . the present invention is based on the discovery that if a rep gene from aav is carried by a helper dependent ad vector , its expression can be suppressed or delayed during the helper replication stage thus bypassing the inhibitory effect due to the rep gene expression . the construction of the helper dependent ad vectors carrying a rep gene here described for particular embodiments of the present invention , is based on the exploitation of the helper dependent ad system described by f . graham and coworkers ( parks et al . 1996 ). in brief , the helper dependent ad system is based on the development of recombinant helper adenovirus that , when they are replicating in suitable cells , are able to produce all the proteins and rna necessary to perform helper functions , but whose genome cannot be packaged in infective viral particles . this can be achieved by inserting artificial sites for specific recombinases ( e . g . loxp sites target of cre recombinase ) on both sides of the helper packaging signals sequences . for example , the infection of a cre expressing cell with an ad virion containing the proper loxp sites , results in the derivation of cells expressing all the ad helper function , but where the packaging of the helper genome is selectively imparted . superinfection or transfection of such cells with an adeno helper dependent vector results in the replication and packaging of the helper dependent vector with minimal helper virus contamination . construction and amplification of a first generation adenoviral vector carrying the integration cassette a plasmid denoted “ plbg40 ” contains the ad5 genome deleted in e1 and e3 regions and is fully infectious when transfected in 293 cells . the e1 - e3 deletions allow a theoretical insertion up to 6 kb of foreign dna by direct cloning into unique restriction sites present in both e1 and e3 regions . ( a ) the aav2 - itrgfp - hygror cassette was constructed by inserting the humanized version of green fluorescent protein ( gfp ) gene driven by hcmv promoter from the pgreen lantern plasmid ( gibco brl ) and the hygromicin b resistance gene fused to the tk promoter between aav2 itrs in the context of plasmid plitmus28 ( new england biolabs ) generating pitrgfp hygro . ( b ) the aav2 - itrgfp / hygror cassette was excised from pitrgfphygro by xbai - bglii digestion and cloned into xbai - bamhi digested pabs . 4 ( bett et al ., 1994 ) generating pab - itrgfp / hygro . ( c ) the itrgfp / hygro cassette obtained from pab - itrgfp / hygro by paci digestion was inserted in the unique paci restriction site of plbg40 , an ad5 genome deleted of the e1 and e3 regions and fully infectious when transfected in 293 cells , generating plb - itrgfp / hy . ( a ) 60 mm dishes of semiconfluent monolayer of 293 cells were transfected with 3 , 5 , or 10 μg of plb - itrgfp / hy using a standard calcium - phosphate technique ( graham f . l . et al . 1973 ). ( b ) the cell monolayer was incubated overnight at 37 ° c ., the medium was removed and 10 ml of medium - agarose overlay was added . ( c ) plaques were visible after 10 days of incubation at 37 ° c . picking , screening and amplification of the isolated plaques were performed as described in hitt , m . et al . 1995 . purification of viral dna from infected cells and purified virions was performed by digestion at 37 ° c . in proteinasek / sds buffer ( 10 mm tris - hcl , ph7 . 5 , 1 mm edta / 1 % sds / 1 mg / ml proteinasek ) followed by phenol extraction and ethanol precipitation . episomal dna was isolated from chromosomal dna following the hirt protocol ( hirt b ., 1967 ). the rep gene of aav ( srivastava et al ., 1983 ) was modified by point mutations to encode only the large polypeptide rep78 under the transcriptional control of the α1 - anti trypsin or t7 promoter ( horer , m ., et al ., 1995 ; de simone , v ., et al ., 1987 ). a hd ad / rep virus was constructed by insertion of the appropriately modified rep gene into a hd ad genome by restriction digestion . plasmids were constructed using standard protocols ( j . sambrook , et al . 1989 ). plasmid prp1030 was used as vector for the insertion of rep expression cassette . it was derived from prp1001 ( parks , et al . 1996 ) by deleting all ad5 coding sequence and substituting it with a lambda phage dna stuffer . ( a ) the helper dependent plasmid prs1032 was derived from prp1030 . it contains the insertion of a cassette constituted by rep 78 atg deletion mutant fused to t7 promoter . rep 78 gene was obtained as described in horer et al . ( 1995 ) by mutating the atg start codon for rep 52 / 40 to gga ( methionine & gt ; glycine , amino acid 225 ) and the g of the splice donor site for expression of spliced versions rep 68 / 40 ( nucleotide 1907 ) to a . rep78δatg was obtained by pcr designed to delete the first atg of the rep open reading frame and fused to the t7 promoter in the context of the shuttle plasmid pabs - 4 ( bett et al ., 1994 ) generating pabt7 - rep78δatg . ( b ) pabt7rep78δatg : a pvuii fragment containing the t7rep78δatg cassette was ligated into the unique stui site of prp1030 generating the ad helper dependent plasmid prs1032 . ( a ) the t7rep cassette was constructed following the strategy reproted for t7rep78δatg and inserted into the unique bamhi cloning site of pabs . 4 , generating pabt7rep . ( b ) pabt7rep was converted in a rep 78 expression vector by substituting the sfii - xbai dna fragment with the sfii - xbai restriction fragment obtained from pciiirep78 plasmid . this fragment contains a mutation of the first atg of rep 52 / 40 ( atg - gga ) and a g & gt ; a mutation in the splice donor sequence present in the rep orf . ( c ) the t7rep78 cassette was than excised from pabt7rep78 by pvuii digestion and inserted into the stui site of prp1030 generating prs1033 . the helper dependent plasmid pra1034 was derived from prp1030 . pra1034 contains rep 78 gene fused to aδ137α1 - antitrypsin promoter . ( a ) α1 - antitrypsin ( α1 - at )- rep78 cassette was constructed by fusing rep78 gene to the δ137 α1 - at minimal promoter in the context of pabs - 4 shuttle plasmid generating pra1034 . ( b ) the α1at - rep78 cassette was then excised by pvuii digestion from pabα1at - rep78 and cloned into prp1030 generating pra1034 . although the experiments directed to rescue of the t7rep cassette into a first generation vector , e1 deleted adenoviral vectors were completely unsuccessful , an adenoviral vector carrying the aav2 rep78 gene were positively rescued and amplified following the helper dependent strategy . amplification of a hd ad / rep virus was obtained with a modification of the established protocol for the amplification of hd ad viruses ( parks , r ., et al ., 1996 ). semiconfluent monolayers of 293cre cells ( chen l , et al 1996 ) in 60 mm dishes were infected with adlc8cluc at a multiplicity of infection ( m . o . i .) of 5 plaque - forming units ( pfu )/ cell . 4 hr after infection 293cre cells were transfected using standard calcium - phosphate technique with 5 μg of helper - dependent vector ( prs1032 , prs1033 , pra1034 ) for 6 hr at 37 ° c . the medium was replaced and the cells incubated until the monolayer showed complete cytopathic effect ( cpe ). the cells were scraped into the medium and the virus released by freezing and thawing . the resulting crude lysate was serially passaged on 60 - mm dishes of 293cre cells as follows . during each round of amplification of the three different helper - dependent vectors , the 293cre monolayers were infected with adlc8cluc at an m . o . i . of 1 pfu / cell and incubated at 37 ° c . for 4 hr before the medium was removed and cells were incubated at 30 ° c . with an aliquot ( 500 μl ) of the crude lysate obtained from previous passage . amplification was monitored at each passage by infecting 293 cells and counting lac - z positive cells ( blue forming unit ( bfu ), as described in parks et al . ( 1996 ) ( supra ). fig1 b shows the amplification results of the three rep containing helper - dependent vectors in comparison with the non - recombinant vector rp1030 . the presence of rep gene did not affect the amplification of the adhd vector up to 1 . 8 × 10 7 b . f . u ./ ml ( corresponding to 60 - 100 rep78 expressing viral particles per cells ). time course of cpe was not affected by rep virus infection and full cpe was usually achieved after 48 - 72 hours post - infection even during late passages in the presence of an increasing titre of rep expressing viruses . the inhibition of ad replication is mediated by rep expression and dependent on the ratio of the multiplicity of infection of the two viruses and the temporal order of addition ( berns , k . i , 1996 ). to reduce the rep expression levels in the packaging cell line , and as direct consequence the detrimental effect on the vector production , the rep gene was fused to δ - 137 dna fragment of α1 - antitrypsin promoter or to the t7 promoter . although a residual rep enzymatic activity was detected with both constructs by transfecting 293 cells , the protein was not detectable using a standard western blot . furthermore , to rescue the ad viruses 293cre cells were first infected with the helper virus and than transfected with rep plasmids 3 - 5 hr post - infection when the adenovirus early gene expression has already reached the peak ( sharp p . a , 1984 ). the same protocol was followed during the serial passages of the hd viruses . to detect the presence of rearrangements involving the rep expression cassette of the amplified hd vectors , the episomal dna extracted from cell lysate at different passages was digested with drai and analyzed by southern blot using a rep dna probe . the detection of a single band of the expected size ( 6 . 3 kb for adhdra1034 , about 2 . 1 kb for adhdrs1032 and adhdrs1033 ) demonstrated the integrity of the rep cassette . no minor bands were detected after 48 hr exposure indicating that vector rearrangement did not occur during the serial passages . furthermore , the restriction pattern of the viral dna analysed by agarose gel confirmed the structural integrity of the amplified vectors . rep 78 expression was evaluated by western blot , infecting hep3b and as control hela cells with 50 b . f . u ./ cell of hdra1034 or hdrs1032 . cells were harvested 36 hours post - infection and a western blot analysis was performed on proteins extracted using a rabbit antiserum . pciii - rep78 containing the same gene fused hcmv promoter was transfected as positive control . rep 78 expression was detected in hep3b but not in hela cells infected with hdra1034 as expected . this result confirms the tissue specificity of the rep78 gene fused to the liver specific α1at promoter ( de simone et al ., 1987 ). large scale virus preparation was obtained by infecting 6 large 150 mm dishes with 500 ml of crude lysate approximately containing 10 7 vector transducing particles . 4 hr before the incubation with the crude lysate the 293cre cells were infected with 10 7 pfu of helper virus per 150 mm dish . after complete cytopathic effect the virus was purified as described ( hitt , m ., et al . 1995 ) the total yield was 3 × 10 9 bfu from 5 × 10 7 cells , providing indication that 50 - 100 of rep expressing virus per cell can be produced without interference problems . the rep78 protein is able to support in vitro the replication of aav dna in presence of an adenovirus - infected cell extract ( ni th , et al . 1994 ). an assay of rep 78 expression based on the rescue and replication of an aav - itrs flanked dna in cells infected by adenovirus was used to demonstrate the functionality of hd rep vectors amplified by serial passages in 293cre cells . 293 and 293cre monolayer were infected with about 0 . 5 × 10 6 b . f . u . using a crude lysate obtained from 293cre cells after 8 serial passages of vectors hdrp1030 , hdrs1032 , hdrs1033 , hdra1034 . 4 hr later the cells were infected with the first generation vector adlbgitr - gfp / hy carrying the aav itr flanked transgene . as positive control , a dish of 293 cells infected with adlbgitr - gfp / hy was transfected with pciii - rep 78 , a plasmid containing an human cytomegalovirus ( hcmv ) promoter driven rep 78 gene expression . at 48 hr post infection , episomal dna was recovered and analyzed by southern blotting using a dna probe specific for the transgene . the low molecular weight dna bands observed in the southern blot corresponding to the size of aav itr / transgene cassette monomer and dimer demonstrated that 293 and 293cre cell lines were successfully infected by hdrs1033 and hdrs1034 and that rep 78 protein was correctly expressed . the same signal was visible in the pciii - rep 78 transfection experiment , while in presence of the two control vectors hdrp1030 and hdrs1032 the rescue and replication did not occur — as expected . a nested pcr based assay on genomic dna was set up to detect junctions between the aavs1 and aav itrs . two pairs of primers specific for aavs1 and aav itrs have been used . hepg2 or huh7 cells were infected first at an moi of 1 bfu / cell with hdrs1032 or hdra1034 and 3 - 4 hours later with moi 1 pfu / cell of adlb - itrgfp / hy . cells were harvested by scraping 48 hours after infection and total dna extracted by standard techniques ( sambrook j . et al . t . 1989 ) nested pcr was performed on 1 μg of cellular dna in a reaction mixture containing 1 . 25 u of amplitaq gold ( perkin helmer ), 1 × amplitaq gold buffer , 2 . 5 mm mgcl 2 , 200 mm of deoxynucleoside triphosphates and 50 pmol of each primer . amplification conditions were : 10 min at 94 ° c . followed by 25 cycles of 94 ° c . for 1 min , 60 ° c . for 1 min and 72 ° c . for 2 min . the second step of amplification was done using 10 μl of the first reaction following the reported reaction conditions . primers 16s ( 5 ′- gtagcatggcgggttaatca - 3 ′; seq . id . no . 3 ) and 17s long ( 5 ′- ttaactacaaggaacccctagtgatgg - 3 ′; seq . id . no . 4 ) { fraction ( 1 / 10 )} of the amplified dna was loaded in duplicate on 1 . 3 % agarose gel , transferred hybondn + membrane ( amersham ) and hybridized with an aavs1 or aav itr probes . aavs1 dna probe was labelled by random priming with 32 p ( multiprime , amersham ). aavitr probe was obtained with two primers derived from psub201 ( nts 1 - 119 ) labelled with 32 p by fill - in reaction in presence of 5 u of klenow fragment of dna polymerase and 50 μci of 32pdatp and 32pdctp ( nen ). cell transfections with plasmids demonstrated that rep expressed from a cassette located outside viral itrs can act in trans promoting site - specific integration of an aav - itrs - flanked transgene . the results demonstrate that rep 78 expressed from hdra1034 mediates integration of the rescued transgene in aavs1 locus . hepg2 and huh7 cells were coinfected with hdra1034 and ad lbitr - gfp / hy . 48 hours post - infection cells were harvested and genomic dna extracted . a nested pcr - based assay on genomic dna was set up to detect junctions between aavs1 and viral itrs . two pairs of primers specific for aavs1 and aavitrs were used . aavs1 primer sequences was chosen in the dna sequence located downstream the 100 - bp aavs1 region identified as “ hot spot ” for site specific integration ( samulski r . j . et al . 1991 ). amplified dna was loaded on agarose gel in duplicate and analyzed by southern blot using either aavs1 or aavitr specific probes . similar results were observed upon infection of huh7 and hepg2 cells . positive signals were detected only in cells infected with rep expressing virus . no aavs1 - aavitr junctions were amplified from cells infected with hdra1032 carrying the rep78δatg mutant or with adlbgitr - gfp / hy alone . the hybridization patterns appeared to be identical with both probes suggesting that different aavs1 - aavitr junctions were actually amplified . three major bands ranging in size between 500 bp and 100 bp superimposed on a smear were detected , with the major species being approximately 100 bp . amplified dna was eluted from the gel , cloned and sequenced . the sequence data confirm that the amplified dna contain the junction between the aavs1 genomic region and the aav itr . a 3 . 7 kb dna fragment corresponding to the transgene cassette and a 80 kb aavs1 dna fragment isolated by screening a genomic dna library were labelled using nick translation kit ( boehringer mannheim ) according to the manufacture &# 39 ; s instruction and used as probes in chromosome analysis . the chromosome spreads from a pool of hygromicin resistant hepg2 cells were prepared by standard cytogenetic techniques ( lawrence et al ., 1988 ). cytogenetic preparations were pre - treated with rnase a solution ( 10 mg / ml in 2 × ssc ), with pepsin solution ( 0 . 005 % in 10 mm hcl ) and dehydrated through 70 , 90 and 100 % ethanol . the preparations were then denatured using a 70 % deionized formamide solution and dehydrated again through cold ethanol at increasing concentration . for each sample , 300 ng of probe specific for the transgene cassette and 100 ng of probe specific for the aavs1 sequence , respectively biotin and digoxigenin labelled , were precipitated with 10 × excess of cot1 human dna ( gibco ) and 10 × excess of sonicated salmon sperm dna and then resuspended in hybridization solution ( 50 % formamide , 2 × ssc and 10 % dextran sulphate ). probes were denatured 5 minutes at 75 ° c . and subsequently incubated for 10 minutes at 37 ° c . to allow preannealing of repeated sequences . finally the hybridization solution was placed on the samples , covered with coverslips and incubated overnight at 37 ° c . in a moist chamber . the samples were then washed three times in 50 % formamide / 2 × ssc and three times in 2 × ssc in a water bath at 37 ° c . visualization of biotin - labelled probe was carried out by an incubation with cy3 - avidin ( amersham ). the digoxigenin - labelled probe was detected using a sheep anti - digoxigenin antibody fitc - labelled ( boehringer mannheim ). after immunodetection , slides were counterstained with 100 ng / ml of 4 ′, 6 ′- diamidino - 2 - phenylindole ( dapi ). ultraviolet excitation was used to locate metaphases and photographic images were taken by a ccd camera ( princeton instruments , inc .) using green ( fitc signal ) or blue violet ( cy3 signal ) illumination . images were processed using adobe photoshop on a apple power macintosh computer . since fish analysis permit the direct study of chromosomal localization of a single copy gene , this technique was used to confirm aav - itr transgene integration in chromosome 19 . for this purpose , adlbgitr - gfp / hy was substituted with an helper dependent vector , hdfb1 , derived from the pstk120 helper dependent plasmid carrying the same transgene . a system based on two helper dependent vector allowed the elimination of the toxic effect of leaky expression and replication of δe1 ad in hepg2 cells ( sprengel r . et al . 1991 ). since rep expression exerts a cytostatic activity , to develop stable clones with a system based on double infection a strategy was chosen aimed to maximize the probability of cell co - infection avoiding high rep gene copy number . hepg2 cells were co - infected with 5 transducing unit / cell of hdra1034 or hdrs1032 , the lowest moi compatible with 80 / 90 % cell infection , in combination with the same moi of hdfb1 . fish analysis was performed after several passaging of cells in presence of hygromicin b . aavitr transgene was detected in 6 out of 16 different metaphase spreads on one allele of chromosome 19 . in summary , the present inventors have succeeded in providing an approach for generation of novel hybrid ad / aav vectors encoding the aav rep78 gene , which are able to mediate site specific integration of a itr - flanked dna . ausubel et al . 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