convincing evidence continues to emerge from clinical trials that gene therapy is 
 yielding therapeutic effects in patients suffering from a wide range of 
 diseases . in particular , liver - directed gene therapy is 
 becoming a promising modality to obtain sustained hepatocyte - specific expression 
 of secreted factors into the circulation . this has implications for other 
 liver - borne genetic and complex diseases . despite these successes , there have 
 been concerns regarding the efficacy and safety of some gene delivery 
 approaches . the major limiting factors are insufficient and/or transient 
 transgene expression levels and inappropriate expression of the transgene in 
 unwanted cell types . higher vector doses are typically used in gene therapy 
 clinical trials to improve therapeutic efficacy . however , this often triggers 
 t - cell  mediated immune responses against the vector capsid antigens 
 displayed by transduced cells , particularly hepatocytes , in the context of mhc 
 class i. this contributes to 
 the elimination of the gene - modified cells and liver toxicity , resulting in 
 short - term gene expression . moreover , inadvertent transgene expression in 
 antigen - presenting cells ( apcs ) , increases the risk of untoward immune responses 
 against the gene - modified hepatocytes and/or the therapeutic transgene 
 product . hence , there is a need to generate improved gene 
 therapy vectors allowing the use of lower and safer vector doses that enable 
 sustained hepatocyte - specific expression of the therapeutic gene . typically , conventional methods of vector design rely on haphazard 
 trial - and - error approaches whereby transcriptional enhancers are combined with 
 promoters to boost expression levels . though this can sometimes be 
 effective , it often results in non - productive combinations that 
 result in either modest or no increased expression levels of the gene of 
 interest in hepatocytes and/or loss of liver specificity . an intrinsic bias associated with the 
 design of gene therapy vectors is that it frequently relies on the in 
 vitro characteristics of its regulatory elements in cell lines , which 
 is usually not predictive of their in vivo performance . moreover , 
 these conventional approaches in vector design do not take into account the 
 importance of including evolutionary conserved regulatory motifs into the 
 expression modules , which is particularly relevant for clinical translation . in silico design offers unique opportunities to generate robust 
 liver - specific gene therapy vectors and overcome some of the limitations of 
 conventional gene therapy vector development . though data - mining has been used 
 to identify cis - regulatory modules ( crms ) and analyze 
 expression patterns in vivo , surprisingly 
 however , this area of research remains largely unexplored in the context of 
 vector development and gene therapy . to address this caveat and overcome some of 
 the limitations of conventional vector design , we have developed and validated 
 an alternative strategy to generate efficient , liver - specific gene therapy 
 vectors using computational methods . in this study , we relied on a genome - wide 
 in silico identification of crms that contained clusters 
 of evolutionarily conserved transcription factor binding site ( tfbs ) 
 motifs associated with robust hepatocyte - specific expression . we employed a 
 comprehensive computational strategy that takes into account not only the 
 over - representation of a given tfbs but also its context - dependent 
 co - occurrence with other tfbs . such an approach has not yet been used to improve the 
 performance of gene therapy vectors , which underscores the novelty of this 
 study . this computational approach has broad implications for liver - directed 
 gene therapy and allows for the use of lower and thus safer vector doses , while 
 maximizing therapeutic efficacy in pre - clinical mouse models and in non - human 
 primates . furthermore , the validation of these liver - specific crms 
 provides new insights into the molecular determinants underlying transcriptional 
 control in hepatocytes . rational de novo design of robust liver - specific gene therapy 
 vectors relies on the identification of tissue - specific crms 
 associated with high hepatocyte - specific expression . to identify these 
 critical hepatocyte - specific crms ( i.e. , 
 hs - crms ) this 
 bio - informatics approach was originally used to identify crms 
 associated with differential gene expression in response to in 
 vitro stimuli . in this 
 study , we extended this computational approach to identify evolutionary 
 conserved crms associated with highly expressed liver - specific 
 promoters . one of the unique features of this computational strategy is that 
 it takes into account the over - representation of a given tfbs and 
 its context - dependent co - occurrence with other tfbs on a 
 genome - wide scale . a total of 14 different hs - crms were identified , ranging in size 
 from 41  bp to 551  bp ( figure 
 1 and table 1 and 
 supplementary figure s1 and table s2 ) . the hs - crm 
 comprised putative binding sites for eight different tfs : hnf1 , 
 c / ebp , lef1 , fox , irf , lef1/tcf , tal1/e47 , and myod . these 
 hs - crms contain a  molecular signature  that 
 represents a hallmark of highly expressed genes in the liver . we observed 
 that most hs - crms contain multiple tfbs that are similar 
 but the uniqueness of each hs - crm is dictated by its specific 
 arrangement of the tfbs . since all hs - crms exhibited a high degree 
 of phylogenetic conservation among 44 divergent species , this suggests a 
 strong evolutionary selective pressure to maintain these particular 
 tfbs combinations for high tissue - specific expression . phylogenetic conservation also increases the likelihood that the performance 
 of the hs - crms is preserved across species in the process of 
 clinical translation . we subsequently assessed the efficiency and specificity of these novel 
 hs - crms in liver . we first expressed human clotting factor ix 
 ( hfix ) from a chimeric promoter , composed of a potent liver - specific minimal 
 transthyretin promoter ( ttr ) in conjunction with the 
 different hs - crm ( figure 2a ) . we 
 choose hydrodynamic delivery to screen the 14 different hepatocyte - specific 
 cis - regulatory modules ( hs - crms ) since it allows for a rapid and 
 reliable assessment of their in vivo performance , without having to 
 produce aav vectors for each of them . only the best hs - crm was then 
 selected for a subsequent comprehensive characterization in an aav vector 
 context ( see below ) . semi - high throughput hydrodynamic transfection of these 
 expression plasmids into c57bl/6 mice revealed that the large majority 
 ( i.e. , 85% ) of the de novo designed hs - crms 
 resulted in a significant increase in hfix expression ( figure 3a ) . in particular , the most potent 
 hs - crm8 resulted in a robust improvement in hfix expression , 
 yielding up to 2.5 g / ml . we subsequently validated the 
 hs - crm8 element in aav9-based vectors in which the 
 hfix gene was driven from either a moderately active 
 ( palm ) ( figure 3b ) or potent 
 liver promoter ( ttr ) ( figure 
 3c ) . the de novo designed hs - crm8 element in 
 the aav vector resulted in a robust and sustained 100-fold increase in hfix 
 expression from the moderately active palm promoter ( p 
 < 0.0001 ) ( 5    10 vg , figure 3b ) . moreover , this hs - crm8 
 element significantly improved hfix expression seven- to tenfold ( p 
 < 0.00005 ) , even when a potent liver - specific promoter was used like 
 ttr ( figure 3c ) , consistent 
 with the increased hfix expression after hydrodynamic transfection 
 ( figure 3a ) . this resulted in 
 stable therapeutic hfix expression levels of about 100% , even at relatively 
 low vector doses ( 5    10 vg ) . the 
 hs - crm8 element increased transcription from both ttr 
 and palm promoters ( supplementary figure s2a , b ) . the 
 increased hfix antigen levels correlated strongly with the fix mrna 
 levels ( supplementary figure s2c ) . there was no significant 
 difference in vector copy number in mice injected with the vectors with or 
 without the hs - crm8 element ( supplementary figure s2d ) . the 
 vector copy number correlated with the vector doses . the potency of the hs - crm8 element was subsequently evaluated 
 through assessment of phenotypic correction in hemophilic fix - deficient mice 
 following a tail - clip injury . the treated mice survived the injury in 
 contrast to naive controls , consistent with a significant dose - dependent 
 reduction in blood loss ( naive mice : 1,167    215 
 l ; 5    10 vg / mouse : 
 596    92 l ( p vg / mouse : 
 495    82 l ( p < 0.00005 ) . hfix 
 mrna expression was restricted to the liver ( figure 3d , e ) , even 
 upon administration of extremely high vector doses 
 ( 3    10 vg ) , despite the detection of 
 viral genomes in non - hepatic tissues ( i.e. , heart , spleen , muscle , 
 etc . ) ( data not shown ) . to further enhance hfix expression from the hs - crm8-ttr - fix 
 construct , we subsequently replaced hfix intron a with an mvm 
 intron upstream of the hfix transgene ( figure 2c ) or its codon - optimized equivalent 
 ( hfixco ) ( figure 2d ) . each of these 
 changes led to a significant increase in hfix expression ( figure 4a , b ) . this optimized hs - crm8-ttr - mvm - fixco 
 construct was then packaged into scaav for subsequent validation in a 
 non - human primate model . the scaav9-hs - crm8-ttr - mvm - hfixco vector was injected into two cynomolgus 
 macaques ( oug6 and obev7 ) at a dose of 
 9    10 vg / kg . there was a steady 
 increase in the circulating hfix protein in both animals that reached a peak 
 by week 4 , corresponding to 1,718  ng / ml and 885  ng / ml for oug6 
 and obev7 , respectively ( figure 5a ) . these levels correspond to therapeutic fix levels in the 2035% range 
 of normal fix levels . after a few weeks , the hfix transgene expression began 
 to decline ( figure 5a ) , which could be 
 ascribed to the induction of anti - hfix antibodies ( figure 5c , d ) , often 
 seen in non - human primates following aav liver gene transfer , 
 despite the use of liver - specific promoters . the aav9 vector administration 
 also triggered anti - aav9 neutralizing antibodies ( figure 5b ) . both animals had consistent coagulation 
 parameters despite the occurrence of hfix inhibitory antibodies 
 ( supplementary figure s3 ) . this indicates that the anti - hfix 
 antibodies did not cross - react with the macaque fix . the animals did not 
 develop any adverse events or toxicity ( supplementary figures s4 and 
 s5 ) . human fix mrna expression was restricted to the liver 
 whereas only background levels were apparent in any other organ or tissue 
 tested ( figure 6a ) . this confirms the 
 liver - specificity of the synthetic in silico designed 
 hs - crm8-ttr promoter , consistent with the mouse data 
 ( figure 3d , e ) . the aav9 vector resulted predominately in liver 
 transduction though significant gene transfer was also apparent in heart , 
 lungs , spleen , kidney and brain ( figure 
 6b ) . one of our animals ( oug6 ) was euthanized due to unexpected complications 
 unrelated to the gene therapy , which occurred after a surgical procedure to 
 obtain a liver biopsy . we subsequently tried to eliminate the anti - hfix 
 inhibitors in the remaining animal ( obev7 ) following , transient 
 immunosuppression with cyclosporin ( csa ) and rituximab ( rtx ) . this led to a 
 modest increase in circulating fix levels ( figure 
 5a ) , consistent with a decrease in anti - hfix antibodies 
 ( figure 5c , d ) . this was consistent with the profound decrease in 
 cd20 b cells ( figure 5e ) , 
 whereas the percentage of cd3 t cells remained unaffected 
 ( figure 5f ) . unfortunately , only 
 partial and transient immunosuppression could be achieved , since the animal 
 refused to ingest csa after the initial treatment . transient 
 immunosuppression also led to transient and partial decline in anti - aav9 
 neutralizing antibodies ( figure 5b ) . rational de novo design of robust liver - specific gene therapy 
 vectors relies on the identification of tissue - specific crms 
 associated with high hepatocyte - specific expression . to identify these 
 critical hepatocyte - specific crms ( i.e. , 
 hs - crms ) this 
 bio - informatics approach was originally used to identify crms 
 associated with differential gene expression in response to in 
 vitro stimuli . in this 
 study , we extended this computational approach to identify evolutionary 
 conserved crms associated with highly expressed liver - specific 
 promoters . one of the unique features of this computational strategy is that 
 it takes into account the over - representation of a given tfbs and 
 its context - dependent co - occurrence with other tfbs on a 
 genome - wide scale . a total of 14 different hs - crms were identified , ranging in size 
 from 41  bp to 551  bp ( figure 
 1 and table 1 and 
 supplementary figure s1 and table s2 ) . the hs - crm 
 comprised putative binding sites for eight different tfs : hnf1 , 
 c / ebp , lef1 , fox , irf , lef1/tcf , tal1/e47 , and myod . these 
 hs - crms contain a  molecular signature  that 
 represents a hallmark of highly expressed genes in the liver . we observed 
 that most hs - crms contain multiple tfbs that are similar 
 but the uniqueness of each hs - crm is dictated by its specific 
 arrangement of the tfbs . since all hs - crms exhibited a high degree 
 of phylogenetic conservation among 44 divergent species , this suggests a 
 strong evolutionary selective pressure to maintain these particular 
 tfbs combinations for high tissue - specific expression . phylogenetic conservation also increases the likelihood that the performance 
 of the hs - crms is preserved across species in the process of 
 clinical translation . we subsequently assessed the efficiency and specificity of these novel 
 hs - crms in liver . we first expressed human clotting factor ix 
 ( hfix ) from a chimeric promoter , composed of a potent liver - specific minimal 
 transthyretin promoter ( ttr ) in conjunction with the 
 different hs - crm ( figure 2a ) . we 
 choose hydrodynamic delivery to screen the 14 different hepatocyte - specific 
 cis - regulatory modules ( hs - crms ) since it allows for a rapid and 
 reliable assessment of their in vivo performance , without having to 
 produce aav vectors for each of them . only the best hs - crm was then 
 selected for a subsequent comprehensive characterization in an aav vector 
 context ( see below ) . semi - high throughput hydrodynamic transfection of these 
 expression plasmids into c57bl/6 mice revealed that the large majority 
 ( i.e. , 85% ) of the de novo designed hs - crms 
 resulted in a significant increase in hfix expression ( figure 3a ) . in particular , the most potent 
 hs - crm8 resulted in a robust improvement in hfix expression , 
 yielding up to 2.5 g / ml . we subsequently validated the 
 hs - crm8 element in aav9-based vectors in which the 
 hfix gene was driven from either a moderately active 
 ( palm ) ( figure 3b ) or potent 
 liver promoter ( ttr ) ( figure 
 3c ) . the de novo designed hs - crm8 element in 
 the aav vector resulted in a robust and sustained 100-fold increase in hfix 
 expression from the moderately active palm promoter ( p 
 < 0.0001 ) ( 5    10 vg , figure 3b ) . moreover , this hs - crm8 
 element significantly improved hfix expression seven- to tenfold ( p 
 < 0.00005 ) , even when a potent liver - specific promoter was used like 
 ttr ( figure 3c ) , consistent 
 with the increased hfix expression after hydrodynamic transfection 
 ( figure 3a ) . this resulted in 
 stable therapeutic hfix expression levels of about 100% , even at relatively 
 low vector doses ( 5    10 vg ) . the 
 hs - crm8 element increased transcription from both ttr 
 and palm promoters ( supplementary figure s2a , b ) . the 
 increased hfix antigen levels correlated strongly with the fix mrna 
 levels ( supplementary figure s2c ) . there was no significant 
 difference in vector copy number in mice injected with the vectors with or 
 without the hs - crm8 element ( supplementary figure s2d ) . the potency of the hs - crm8 element was subsequently evaluated 
 through assessment of phenotypic correction in hemophilic fix - deficient mice 
 following a tail - clip injury . the treated mice survived the injury in 
 contrast to naive controls , consistent with a significant dose - dependent 
 reduction in blood loss ( naive mice : 1,167    215 
 l ; 5    10 vg / mouse : 
 596    92 l ( p < 0.0005 ) ; 
 2    10 vg / mouse : 
 495    82 l ( p < 0.00005 ) . hfix 
 mrna expression was restricted to the liver ( figure 3d , e ) , even 
 upon administration of extremely high vector doses 
 ( 3    10 vg ) , despite the detection of 
 viral genomes in non - hepatic tissues ( i.e. , heart , spleen , muscle , 
 etc . ) ( data not shown ) . to further enhance hfix expression from the hs - crm8-ttr - fix 
 construct , we subsequently replaced hfix intron a with an mvm 
 intron upstream of the hfix transgene ( figure 2c ) or its codon - optimized equivalent 
 ( hfixco ) ( figure 2d ) . each of these 
 changes led to a significant increase in hfix expression ( figure 4a , b ) . this optimized hs - crm8-ttr - mvm - fixco 
 construct was then packaged into scaav for subsequent validation in a 
 non - human primate model . the scaav9-hs - crm8-ttr - mvm - hfixco vector was injected into two cynomolgus 
 macaques ( oug6 and obev7 ) at a dose of 
 9    10 vg / kg . there was a steady 
 increase in the circulating hfix protein in both animals that reached a peak 
 by week 4 , corresponding to 1,718  ng / ml and 885  ng / ml for oug6 
 and obev7 , respectively ( figure 5a ) . these levels correspond to therapeutic fix levels in the 2035% range 
 of normal fix levels . after a few weeks , the hfix transgene expression began 
 to decline ( figure 5a ) , which could be 
 ascribed to the induction of anti - hfix antibodies ( figure 5c , d ) , often 
 seen in non - human primates following aav liver gene transfer , 
 despite the use of liver - specific promoters . the aav9 vector administration 
 also triggered anti - aav9 neutralizing antibodies ( figure 5b ) . both animals had consistent coagulation 
 parameters despite the occurrence of hfix inhibitory antibodies 
 ( supplementary figure s3 ) . this indicates that the anti - hfix 
 antibodies did not cross - react with the macaque fix . the animals did not 
 develop any adverse events or toxicity ( supplementary figures s4 and 
 s5 ) . human fix mrna expression was restricted to the liver 
 whereas only background levels were apparent in any other organ or tissue 
 tested ( figure 6a ) . this confirms the 
 liver - specificity of the synthetic in silico designed 
 hs - crm8-ttr promoter , consistent with the mouse data 
 ( figure 3d , e ) . the aav9 vector resulted predominately in liver 
 transduction though significant gene transfer was also apparent in heart , 
 lungs , spleen , kidney and brain ( figure 
 6b ) . one of our animals ( oug6 ) was euthanized due to unexpected complications 
 unrelated to the gene therapy , which occurred after a surgical procedure to 
 obtain a liver biopsy . we subsequently tried to eliminate the anti - hfix 
 inhibitors in the remaining animal ( obev7 ) following , transient 
 immunosuppression with cyclosporin ( csa ) and rituximab ( rtx ) . this led to a 
 modest increase in circulating fix levels ( figure 
 5a ) , consistent with a decrease in anti - hfix antibodies 
 ( figure 5c , d ) . this was consistent with the profound decrease in 
 cd20 b cells ( figure 5e ) , 
 whereas the percentage of cd3 t cells remained unaffected 
 ( figure 5f ) . unfortunately , only 
 partial and transient immunosuppression could be achieved , since the animal 
 refused to ingest csa after the initial treatment . transient 
 immunosuppression also led to transient and partial decline in anti - aav9 
 neutralizing antibodies ( figure 5b ) . in this study , we have validated a computational approach to improve the 
 performance of current gene therapy vectors . our data provide evidence that 
 evolutionary conserved clusters of tfbs motifs corresponding to 
 hepatocyte - specific crms could be identified in silico that 
 are strongly associated with robust liver - specific expression in vivo . we discovered that the selected hs - crms identified contain a 
  molecular signature  composed of tfbs clusters that 
 represent a hallmark of highly expressed genes in the liver . we demonstrated 
 that these hs - crms resulted in a substantial increase in transgene 
 expression in the liver after gene therapy , while maintaining a high degree of 
 tissue - selectivity . in particular , a significant 10- to 100-fold increase in 
 gene expression could be obtained depending on the type of hs - crm and 
 promoter used . we subsequently validated the potential of 
 these hepatocyte - specific hs - crms in a clinically relevant therapeutic 
 setting and demonstrated robust and sustained supra - physiologic expression 
 levels of coagulation fix following hepatic transduction with aav vectors . the 
 use of these potent hs - crms therefore allows for the use of lower and 
 thus safer vector doses in gene therapy clinical trials , that may in turn reduce 
 the risk of developing t - cell  mediated immune responses against the vector 
 antigens displayed on the transduced target cells . 
 in silico vector design may therefore pave the way towards a more 
 rational and comprehensive strategy for vector optimization with broad 
 implications to improve the efficacy and safety of gene therapy indeed , we have 
 confirmed the superiority of the hs - crm8 expression module for 
 liver - directed gene therapy using other transgenes ( i.e. , factor viii , 
 phenylalanine hydroxylase ; ref . 15 and data 
 not shown ) another advantage of these crms is their small size so that 
 they can be readily accommodated into viral vectors in conjunction with 
 relatively large therapeutic transgenes . the use of hyper - active fix transgenes 
 can further enhance vector performance . we subsequently validated the efficacy and safety of the aav - fix vectors 
 containing the most potent hs - crm8 expression module in a non - human 
 primate model . systemic vector administration resulted in robust therapeutic fix 
 levels in the 2035% range at clinically relevant vector doses 
 ( 9    10 the performance of our vector compared 
 favorably with that of one of the most potent aav vectors that was used in a 
 clinical trial in patients suffering from severe hemophila b. subjects injected with 
 6    10 vg / kg , comparable to the dose used 
 in our primate study , resulted in ~13% fix . in those subjects injected 
 with higher vectors doses ( i.e. , 
 2    10 previously , we had 
 assessed the performance of the same clinical trial vector in cynomolgus 
 macaques . when calibrating 
 the vector doses , it would appear that our current vector design is about 
 tenfold more efficient . since a human fix transgene was employed , it is known 
 that this could potentially evoke neutralizing and non - neutralizing anti - hfix 
 antibodies in non - human primate models . this is consistent with our observations that anti - hfix 
 antibodies were detected in the recipients which accounted for the subsequent 
 decline in fix expression . this provided us with an opportunity to test whether 
 we could restore fix by transient immunosuppression using rituximab and csa . fix 
 could be detected after immune - suppression , even up to nearly 2 years after gene 
 transfer . this suggests that the fix - transduced hepatocytes persisted long - term 
 and that the short - term fix expression was primarily due to a humoral immune 
 response . our results caution that rituximab by itself may not suffice to treat 
 patients that develop inhibitory antibodies after gene therapy and that more 
 complex combination therapies may be required to achieve optimal immune 
 suppression . in this study , we choose to employ aav9 instead of aav8 . previously , gao et 
 al . evaluated the relationship of phylogenetic relatedness of distinct 
 aav serotypes and serologic activity . polyclonal antisera generated against aav2 cross - reacted 
 to a lesser extent with aav9 than with aav8 . conversely , polyclonal antisera 
 generated against aav9 cross - reacted to a lesser extent with aav2 than when the 
 polyclonal antisera were generated against aav8 . these experiments suggest that 
 it may be desirable to select aav9 over aav8 to overcome neutralization by 
 pre - existing anti - aav2 antibodies in human subjects , which represents an 
 important bottleneck in clinical trials . moreover , we choose to explore aav9 
 since the bio - distribution had not systematically been analyzed in non - human 
 primates . finally , we had previously conducted a side - by - side comparison between 
 aav8 and aav9 and found that aav9 transduced the liver just as efficiently as 
 aav8 in mice . the advantage of this particular computational approach used to identify the 
 tissue - specific crms compared to other in silico approaches 
 was discussed previously . one 
 particularly distinctive feature is that it identifies not only the 
 tfbs that are over - represented in liver - specific crms but 
 also allows for the identification of frequently co - occurring tfbs . consequently , this in silico analysis is more comprehensive and allows 
 for the identification of tfbs elements that tend to cluster together 
 in crms of genes that are highly expressed in a given target tissue 
 ( in casu liver ) . it therefore takes into account the actual 
 context - dependent tfbs interactions from a broad genome - wide 
 perspective , instead of just relying on the over - representation of a single tfbs 
 element . our previous 
 studies had shown that the present computational approach was more reliable , 
 compared to other data - mining strategies that typically rely solely on the 
 over - representation of a given tfbs , regardless of its context . the current 
 in silico strategy that defines the hs - crms has been further 
 improved by taking into account cross - species evolutionary conservation of the 
 tfbs clusters . this approach is more comprehensive and increases 
 the likelihood that the superior performance of the hs - crms is 
 maintained in patients undergoing gene therapy , consistent with the robust fix 
 levels obtained in the non - human primate model . in addition , this study goes 
 beyond the initial description and validation of the algorithm and establishes a bona fide 
 causal relationship between the presence of these evolutionary conserved 
 tfbs clusters and high liver - specific expression . in contrast , the 
 previously published bioinformatics approach was applied in a different context 
 and merely established correlations between tfbs clusters and differentially 
 expressed genes following a given physiologic stimulus . however , that study did 
 not provide direct proof of the impact of such tfbs clusters on gene expression 
 levels . this study addresses this caveat by direct in vivo gene 
 transfer , hereby providing experimental in vivo validation of the 
 bioinformatics algorithm . though the majority of hs - crms resulted in a 
 significant increase in gene expression , a few elements had only limited impact . this suggests that other unknown factors may influence gene expression , such as 
 the spacing between a given hs - crm and the promoter / tata box 
 or epigenetic factors and chromatin remodeling . this study sets the stage to 
 further refine the in silico analysis to improve gene therapy vectors . moreover , the most potent hs - crm8 could be used in conjunction with 
 other liver - specific promoters , beyond those characterized in this study . this study has broad implications for the design of better gene therapy vectors 
 that target different diseases . the same principle could be applied to other 
 vectors or to other organs , 
 tissues or promoters , which we have validating experimentally in heart and 
 skeletal muscle ( rincon my , sarcar s , danso - abeam d , keyaerts m , matrai j , 
 samara - kuko e , et al . unpublished data ) . this may ultimately impact not 
 only on the clinical translation of gene therapy but also on fundamental 
 biological and transgenic studies that rely on the use of robust tissue - specific 
 gene expression . . the accession 
 numbers of the genes from which the hepatocyte - specific crms 
 ( i.e. , hs - crm1 to 14 ) are derived are listed in table 1 . identification of hepatocyte - specific crms . to identify the 
 hepatocyte - specific ( hs)-crms ( figure 
 1 and table 1 and 
 supplementary figure s1 and table s1 ) , we used a step - wise in 
 silico strategy : ( i ) we first identified liver - specific genes that are 
 highly expressed in the liver based on statistical analysis of micro - array 
 expression data of normal human tissues ; ( ii ) we then 
 extracted the corresponding promoter sequences up to 800  bp upstream of 
 the transcriptional start site ( tss ) from public databases ( ncbi36/hg18 
 genome assembly ) ; ( iii ) using the transfac database we subsequently mapped 
 tfbs elements to these promoters . we then identified the tissue - specific crm 
 using a multidimensional scaling ( mds)/ differential distance matrix ( ddm ) 
 approach , as described ; ( iv ) 
 finally , the cross - species conservation was taken into consideration to identify 
 highly conserved hs - crms . the different hs - crms 
 ( table 1 and supplementary table 
 s1 ) were synthesized by conventional oligonucleotide synthesis and 
 cloned upstream of the liver - specific minimal transthyretin ( ttr ) 
 ( figure 2a ) or paralemmin 
 ( palm ) promoters ( figure 2b ) , 
 driving the expression of the factor ix ( fix ) 
 mini - gene . alternatively , 
 the ttr promoter coupled to the hs - crm8 element was used to 
 drive wild - type fix ( figure 2c ) or 
 its codon - optimized counterpart ( figure 2d ) 
 in the context of a self - complementary ( sc ) aav backbone . these vectors also contained a minute 
 virus of mice ( mvm ) mini - intron . vector titers were determined by quantitative ( q ) 
 pcr ( see supplementary methods ) . wild - type or hemophilic mice were transfected hydrodynamically with 
 plasmids or injected intravenously with purified aav9 vectors . hfix antigen 
 levels were assayed in citrated mouse plasma using a hfix - specific enzyme - linked 
 immunosorbent assay ( elisa ) . the scaav9-hs - crm8-ttr - mvm - hfixco vector ( figure 2d ) 
 ( 9    10 vg / kg ) was intravenously injected 
 in cynomolgus macaques . animals were subjected to transient immune suppression 
 by cyclosporine a ( csa ) and rituximab treatment . immunological analysis , hfix antigen , anti - aav9 
 capsid igg , anti - hfix igg and bethesda assays are described in supplementary 
 methods . gene expression levels were evaluated by qrt - pcr and normalized 
 to mrna levels of the endogenous monkey gapdh gene . transduction 
 efficiency was determined by qpcr and transgene copy numbers were determined as 
 described previously . nucleotide sequence of the hs - crm elements ( hs - crm1 to hs - crm14 ) . hepatic hfix mrna levels and aav copy numbers following 
 intravenous injection of aav9-fix vectors in c57/bl6 mice ( n = 3 ) . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy 
 in non - human primates : analysis of coagulation parameters . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy 
 in non - human primates : analysis of hematological parameters . . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy 
 in non - human primates : blood chemistry . evolutionary conserved hs - crms enriched in 
 tfbs associated with high liver - specific expression . 

 methods . nucleotide sequence of the hs - crm elements ( hs - crm1 to hs - crm14 ) . hepatic hfix mrna levels and aav copy numbers following intravenous injection 
 of aav9-fix vectors in c57/bl6 mice ( n = 3 ) . click here for additional data file . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human 
 primates : analysis of coagulation parameters . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human 
 primates : analysis of hematological parameters . safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human 
 primates : blood chemistry . evolutionary conserved hs - crms enriched in tfbs associated 
 with high liver - specific expression .