Patent Abstract:
there is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional factor viii protein , wherein the portion of the nucleotide sequence encoding for the b domain of the factor viii protein is between 90 and 111 nucleotides in length and encodes for an amino acid sequence comprising a sequence having at least 85 % identity to seq id no : 4 and which comprises six asparagine residues . also provided is a functional factor viii protein , a vector comprising the above nucleic acid molecule , a host cell , a transgenic animal , a method of treating haemophilia , e . g . haemophilia a , and a method for the preparation of a parvoviral gene delivery vector .

Detailed Description:
in order to develop a safe and efficient gene transfer strategy for the treatment of haemophilia a ( ha ), the most common inherited bleeding disorder , the inventors have developed a new fviii variant called codop - hfviii - v3 ( fig1 ). this variant builds on a previous variant , a 5013 bp codon - optimised fviii called codop - hfviii - n6 . the inventors have further modified codop - hfviii - n6 to improve the efficiency with which it is packaged into raav without compromising its potency in vivo . the cdna in codop - hfviii - v3 has been modified to reduce its size to 4424 bp ( fig1 ) through the replacement of the 678 bp b domain spacer sequence with a 93 bp linker that codes for 31 amino acids of which 17 amino acids are unique , including the 6 asparagine moieties believed to be required for efficient cellular processing of fviii . the context in which these 6 asparagine moieties are brought together is important , raav vectors encoding codop - hfviii - v1 mediated fviii expression that was 16 and 10 fold lower than vectors encoding codop - hfviii - v3 and codop - hfviii - n6 , respectively , in cohorts of mice after a single tail vein injection of 1 × 10 11 vector genomes ( vg )/ mouse ( fig2 ). this difference was highly significant ( p = 0 . 0015 ). importantly , both codop - hfviii - v3 and codop - hfviii - n6 mediated significantly higher level of expression than codop - bdd - hfviii ( fig3 ). the inventors &# 39 ; data show that a raav expression cassette encoding the 5 . 2 kb codop - hfviii - v3 is packaged uniformly as a full length provirus as shown in fig4 . in contrast , the packaging of codop - hfviii - n6 expression cassette is heterogenous . this is due to the larger size of the codop - hfviii - n6 expression cassette , which at 5 . 7 kb significantly exceeds the packaging capacity of aav . packaging of heterogenous proviral dna raises safety concerns because of the potential to synthesis and express truncated forms of fviii , which could provoke an immunological response . by shortening the b domain of the codop - hfviii - n6 variant but retaining essential features of the b domain sequence , in particular the n - linked glycosylation consensus sequences , the inventors have been able to enable more efficient packaging of the transgene into aav . in the course of creating novel sequences for this purpose , one particular sequence n6v3 proved to be associated with highly efficient packaging into aav . this sequence also showed a remarkable and unpredicted further improvement of transgene expression in animal gene transfer studies . based on rational analysis of the structure of factor viii and on its known secretion pathway , requiring interaction with the chaperon protein lmann - 1 , the inventors have deduced that the expression improvement may be due to the following reasons . the interaction of factor viii b domain with the lectin lmann - 1 requires multiple n - linked carbohydrate side chains to be present and for them to adopt a specific conformation for binding between the nascent glycopeptide and the lectin . the wild type b domain is nearly 1000 amino acids long with no likely secondary structure . therefore , this lengthy peptide requires a considerable time for synthesis into the golgi and further time for the random coil to adopt a suitable structure stochastically to bring together the widely separated carbohydrate side chains into a conformation that would enable binding to the lectin ( lmann - 1 ). by shortening the sequence to the minimum length possible that still retains 6 potential n - glycosylation sites ( 17 mer ), the time required for synthesis is drastically reduced . furthermore only a very small number of conformations or possibly just one can occur in the glycosylated peptide amongst which is the required tertiary structure for binding the lectin . the inventors have calculated that the length of this peptide is only just long enough to span the distance between the c - terminal of the a2 domain and the n - terminal of the a3 domain in the crystal structure of b domain deleted factor viii at 53 angstroms . therefore , the n6v3 peptide is further constrained to an almost linear structure that would limit the number of sterically possible conformations and , provided the carbohydrate side chains are added in appropriate places , enable the chaperon to bind virtually co - translationally , thus optimizing to the maximum degree possible this essential step in the factor viii specific secretion pathway . the unique specificity of the novel n6v3 sequence is further supported by the fact that very minor deviation from this sequence , such as retaining a single extra amino acid between each n - glycosylation consensus sequence trimer , greatly reduces the synthesis and secretion efficiency of factor viii compared to that obtained with other versions of the truncated b domain . the inventors have modified codop - n6 - hfviii , to improve the efficiency with which it is packaged into aav virions as full length viral genome without compromising its potency in vivo . this involved the replacement of the 226 amino acid b domain spacer with a 31 amino acid ( 93 bp ) peptide , containing a 14 amino acid linker sequence as in b domain deleted fviii and 17 amino acids which are unique . this peptide contained the 6 asparagine residues present in codop - n6 - hfviii that are required for efficient intra - cellular processing . the peptide brings these residues in closer proximity . consequently , this new hfviii variant ( aav - hlp - codop - hfviii - v3 ) is 5 . 1 kb in size , 600 bp smaller than aav - hlp - codop - n6 - hfviii ( 5 . 7 kb ), and closer to the packaging capacity of aav of approximately 5 . 0 kb ( fig1 ). aav - hlp - codop - hfviii - v1 contains a 44 amino acid peptide that includes the same 6 asparagine residues instead of the 226 amino acid spacer in codop - n6 - hfviii . for comparison another aav vector ( aav - hlp - codop - bdd - hfviii , ˜ 5 . 0 kb in size ) was made which contains a codon optimised hfviii cdna from which the b domain has been deleted , retaining a small linker sequence of 14 amino acids . the yield of aav8 - hlp - codop - hfviii - v3 vector using the standard hek293 transient transfection method was comparable ( fig5 ) to that of aav - hlp - codop - n6 - fviii and aav8 - hlp - codop - bdd - hfviii . analysis of viral dna extracted from 2 . 5 × 10 10 particles of each vector preparation following separation on an alkaline agarose gel showed bands of ˜ 5 kb , the expected size for the hlp - codop - bdd - hfviii ( lane 1 , fig5 b ) and hlp - codop - hfviii - v3 ( lane 3 ). in comparison , a rather diffuse signal was observed for the genomes extracted from aav8 - hlp - codop - n6 - hfviii suggesting the packaging of a more heterogeneous proviral species ( fig5 b , lane 2 ). aav vectors containing the different codon optimised fviii variants , pseudotyped with serotype 8 capsid , were injected as a bolus into the tail vein of 4 - 6 week old males c57b1 / 6 mice ( n = 6 ) at a dose of 4 × 10 13 vg / kg to compare their potency in vivo . the highest level of hfviii expression was observed with aav - hlp - codop - fviii - v3 at 1 . 52 ± 0 . 15 iu / ml ( 152 ± 15 % of normal . fig6 ) 4 weeks after gene transfer . in contrast , aav8 - hlp - codop - n6 - hfviii and aav8 - hlp - codop - bdd - hfviii mediated hfviii expression at 0 . 86 ± 0 . 11 and 0 . 67 ± 0 . 12 iu / ml respectively . the difference in plasma fviii levels between the aav8 - hlp - codop - bdd - hfviii and aav - hlp - codop - hfviii - v3 cohorts of mice was highly significant ( p = 0 . 0015 , student t test ). the lowest level of hfvii expression was observed with aav - hlp - codop - hfviii - v1 ( 0 . 10 ± 0 . 01 u / ml ). this is significantly ( p & lt ; 0 . 0001 ) lower than fviii expression in the aav - hlp - codop - hfviii - v3 cohort of mice , which suggests that the context in which the 6 asparagine residues are brought together in the synthetic b domain amino acid peptide is important . tail vein administration of a higher dose of vector ( 2 × 10 13 vg / kg ) resulted in between 4 - 30 fold higher level of plasma hfviii in the cohort transduced with aav - hlp - codop - hfviii - v3 when compared to levels achieved in cohorts of mice transduced with aav - hlp - codop - n6 - hfviii and aav - hlp - bdd - hfviii following correction for transgene copy number ( fig6 b ) in the liver at 9 weeks . the difference in hfviii levels between aav - hlp - codop - hfviii - v3 and aav - hlp - bdd - hfviii was highly significant ( p = 0 . 0062 , student t test ). a direct comparison of the biologic potency of codop - n6 - fviii , codop - bdd - fviii and codop - fviii - v3 was performed in f8 −/− mice . vector encoding each of these fviii variants , pseudotyped with serotype 8 capsid was administered into the tail vein of male f8 −/− mice at a dose of 4 × 10 12 ( low - dose cohort , n = 5 / 6 ) or 4 × 10 13 ( high - dose cohort , n = 5 / 6 ) vg / kg . for all three constructs the kinetics of expression was broadly similar with plasma hfviii levels reaching peak levels between 2 - 6 weeks after gene transfer . for a given construct , hfviii levels were roughly two fold higher in animals transduced with the high dose of vector when compared to the low dose ( fig7 ). irrespective of the vector dose , peak hfviii expression in the cohorts of mice transduced with codop - bdd - hfviii was approximately two fold lower than observed in animals transduced with codop - n6 - hfviii or codop - hfviii - v3 . at the high dose level the difference in hfviii expression between the codop - bdd - hfviii cohort and codop - hfviii - v3 between weeks 4 - 8 post gene transfer was highly significant ( p & lt ; 0 . 001 2 way anova ). the average ratio of hfviii coagulation activity ( hfviii : c ) to hfviii antigen was slightly above 1 . 0 , suggesting the transgenic hfviii molecules were biologically active . to establish if the fviii activity correlated with phenotypic correction in aav - treated mice , blood loss was analysed by tail clip assay at 8 week after gene transfer ( fig8 ). the amount of blood loss in the aav - codop - hfviii - injected mice was almost similar for the 3 codop - hfviii variants and the two dose levels but substantially lower than observed in fviii −/− mice treated with vehicle instead of aav . this difference between aav and vehicle treated f8 −/− mice was highly significant ( p & lt ; 0 . 001 one - way anova test ). the amount of blood loss in the aav treated animals was comparable to that observed in f8 −/− mice treated with recombinant human fviii ( rfviii ) suggesting that raav - mediated expression of fviii restores haemostasis to levels observed with recombinant fviii . anti - hfviii antibodies were detected over time in all aav transduced animals with the highest levels being observed in the high dose aav - hlp - codop - hfviii - v3 cohort . when compared to the response observed after administration of recombinant hfviii protein ( 2 u fviii per week for 6 weeks ) the response in the aav - codop - hfviii transduced animals was at least 400 fold lower and insufficient to completely neutralise fviii activity as illustrated by the tail clip assay ( fig9 ). consistent with this inhibition of coagulation was not observed when two murine samples with the highest anti - fviii igg level were assessed in a bethesda assay , suggesting that these antibodies do not have neutralising activity . biodistribution studies ( fig1 ) using a sensitive qpcr based assay demonstrated that the aav8 - hlp - codop - hfviii - v3 proviral dna was found predominantly in liver with a mean of 56 ± 15 proviral copies / cell in the 4 × 10 13 vg / kg cohort of mice at 8 weeks after gene transfer , followed by 2 . 1 ± 1 copies / cell in the heart , 0 . 5 ± 0 . 2 copies / cell in the spleen and kidney and 0 . 2 ± 0 . 0 . 05 in the lungs . the detection limit of qpcr is 0 . 0003 copy / diploid genome . the bdd deleted and n6 ( kindly provided by professor steven pipe ( miao et al , 2004 ))- human fviii variants containing the wild type dna sequences were cloned downstream of the previously described liver specific lp1 promoter ( nathwani et al , 2006 ). a 5012 bp codon optimized human n6 fviii ( codop - n6 - hfviii ) was generated using codons most frequently found in highly expressed eukaryotic genes , ( haas et al , 1996 ) synthesized and also cloned downstream of the lp1 promoter . the smaller hlp enhancer / promoter was constructed by synthesizing a 251 bp fragment containing a 34 bp core enhancer from the human apolipoprotein hepatic control region ( hcr ) upstream of a modified 217 bp alpha - 1 - antitrypsin ( haat ) gene promoter consisting only of the distal x and the proximal a + b regulatory domains . aav - hlp - codop - n6 - hfviii was generated by cloning the codop - n6 - hfviii cdna downstream of the hlp promoter but upstream of a 60 bp synthetic polyadenylation signal . the aav - hlp - codop - fviii variants 1 and 3 were made by synthesis of a 1485 and a 1446 bp fragment , respectively . hlp - codop - n6 - fviii was cut with kpni and the 2028 bp fragment was replaced with the synthesised fragments cut with kpni . aav vectors were made by the adenovirus free transient transfection method described before ( davidoff et at , 2004 ). aav5 pseudotyped vector particles were generated using a chimeric aav2 rep - 5cap packaging plasmid called plt - rco3 which is based on xx2 ( xiao et al , 1998 ) and paav5 - 2 ( chiorini et al , 1999 ) and similar in configuration to that described before ( rabinowitz et al , 2002 ). aav8 pseudotyped vectors were also made using the packaging plasmid paav8 - 2 ( gao et al , 2002 ). aav2 / 5 and 2 / 8 vectors were purified by the previously described ion exchange chromatography method ( davidoff et al , 2004 ). vector genome ( vg ) titers were determined by previously described quantitative pcr and gel based methods ( nathwani et al , 2001 ), ( fagone et al , 2012 ). to determine the size of the packaged genome , vector stocks were run on an alkaline gel as previously described in fagone et al , 2012 . all procedures were performed in accordance with institutional guidelines under protocols approved by the institutional and / or national committees for the care and use of animals in the united states and europe . fviii - deficient mice ( mixed c57b16 / j - 129 sv background with a deletion in exon 16 ) were bred in - house and used for experiments between 8 and 10 weeks of age . tail vein administration of raav vector particles was performed in 7 - 10 week old male mice as described before ( nathwani et al . 2001 ). human fviii antigen levels in murine samples were determined by elisa using a paired fviii elisa kit ( affinity biologicals , quadratech , dorking , uk ). flat - bottomed 96 - well plates ( maxisorp , nunc , fisher scientific . loughborough , uk ) were coated with a combination of two mouse monocloncal antibodies ( esh2 ( sekisui diagnostica , axis - shield , dundee , uk ), and n77110m ( biodesign international , ams biotechnology , abingdon , uk )) 50 μl of a 100 μg / ml in 50 mm carbonate buffer ph9 . 6 at 4 ° c . overnight , washed with pbs containing 0 . 05 % tween 20 (= pbst ), and blocked with 200 μl / well of 6 % bovine serum albumin ( bsa , sigma , pool , uk ) in pbst during a 1 hour incubation at 37 ° c . standards were made by serial dilutions of murine plasma spiked with recombinant human fviii , starting concentration 4 iu / ml ( 11 th bs 95 / 608 6 . 9 iu / ml , nibsc , south mimms ). murine samples and standards were diluted 1 : 10 in kit buffer with 50 μl in duplicates . following a 2 hour incubation at 37 ° c ., the plates were washed and incubated for a further hour with 100 μl of horseradish peroxidase conjugated goat anti - human fviii polyclonal secondary antibody . after a final wash step , plates were developed with o - phenylenediamine dihydrochloride peroxidase substrate ( sigma ) and the optical density was assessed spectrophotometrically at 492 nm . probability of statistical difference between experimental groups was determined by one - way anova and paired student t test using graphpad prizm version 4 . 0 software ( graphpad , san diego , calif .). fviii activity was measured in a two - stage coagulation assay , using human plasma as a standard . mice were anaesthetized with tribromoethanol ( 0 . 15 ml / 10 g bodyweight ) and 3 mm of the distal tail was cut with a scalpel . the tail was immersed immediately in 50 ml saline buffer at 37 ° c . and blood was collected for 30 min . two parameters were monitored : first , time to arrest of bleeding was measured from the moment of transection . second , collected erythrocytes were pelleted at 1500 g and lysed in h 2 o . the amount of released haemoglobin was determined by measuring the optical density at 416 nm and using a standard curve prepared upon lysis of 20 - 100 microliter of mouse blood . genomic dna was extracted from murine tissues using the dneasy blood and tissue kit ( qiagen , crawley , uk ), 37 ng of genomic dna extracted from various murine tissues was subjected to quantitativereal - time pcr using primers which amplified a 299 bp region of codop - hfviii ( 5 ′ primer : 5 ′ aaggacttccccatcctgcctgg 3 ′ and 3 ′ primer : 5 ′ gggttgggcaggaacctctgg 3 ′) as described previously ( nathwani et al , 2011 ). plasma samples from mice were screened for the presence of antibodies against hfviii using an elisa . a 96 well maxisorp plate ( nunc ) was coated with 50 μl of 2 iu / ml recombinant fviii in 50 m carbonate buffer ph 9 . 6 at 4 ° c . overnight . plates were washed with pbs - t and blocked with 3 % bsa / tbs - t ( 25 mm tris , 150 mm nacl , 5 mm cacl 2 , 0 . 01 % tween , ph7 . 5 ). 50 μl of serial dilutions of the plasma samples were prepared in 3 % bsa / tbs - t . following a 2 hour incubation at 37 ° c ., the plates were washed and incubated for a further hour with 100 μl of horseradish peroxidase conjugated goat anti - 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