hemophilia b ( hb ) is an x - linked disorder occurring in 1:25,000 male births , caused by a deficiency in coagulating factor ix ( fix ) . in its severe form ( < 1% fix activity ) , it can result in frequent bleeding , reduced quality of life , and early death if left untreated . currently , treatment is based on intravenous ( iv ) administration of plasma - derived or recombinant fix concentrate . complications in the form of neutralizing antibodies ( inhibitors ) to the infused protein can occur in 1.5%3% of hb patients.2 , 3 although the frequency of inhibitor occurrence is low , life - threatening complications , such as allergic / anaphylactic reactions , are frequently associated with inhibitor development.4 , 5 current options for fix inhibitor management are limited , which places focus on alternative approaches for the prevention or treatment of inhibitors . lentiviral vectors ( lvs ) , derived from hiv-1 , are potent gene therapy vehicles for robust transduction and long - term stable expression into a wide variety of dividing and non - dividing cells , both in  vitro and in  vivo . as with other gene - therapy vectors , major hurdles faced by  lentiviruses in disease correction have been safety , specificity , and efficacy . in part due to setbacks caused by -retroviral vector integration into the target cell genome , third - generation lentiviral packaging systems have undergone rigorous construct optimization and development of self - inactivating long terminal repeats ( sin - ltrs ) and heterologous promoters , which minimize risks of replication competence , insertional mutagenesis , or aberrant expression of  adjacent cellular coding regions , thus addressing the need for safety  and efficacy.7 , 8 , 9 hematopoietic stem cell therapies that incorporate these new vectors have been tested in several human diseases ( wiskott - aldrich syndrome , x - linked severe combined immunodeficiency , -thalassaemia , adrenoleukodystrophy , metachromatic leukodystrophy , etc).8 , 10 most synthetic lvs are pseudotyped with an alternative envelope glycoprotein such as vsv - g ( vsv - lv ) for increased stability and broad cellular tropism . for most in  vivo applications , although pseudotyping by specific envelope glycoproteins may alter tropism toward particular cell types such as macrophages or neurons , there is a risk for off - target gene delivery that may result in aberrant expression or immune responses to the transgene or vector . strategies such as the use of tissue - specific promoter elements or microrna ( mirna)-based detargeting of irrelevant cell types can restrict gene expression to the cell type relevant for a particular therapeutic application , but are limited to a few targets.12 , 13 b cells are an important target for immunotherapy approaches.14 , 15 however , primary resting b cells , like other g0-arrested quiescent cells of the hematopoietic system , require cytokine- or cd40-mediated stimulation to the g1 phase to become efficiently transduced by conventional lentiviral or retroviral vectors , which is not desirable for in  vivo applications or for tolerogenic studies.16 , 17 , 18 measles virus ( mv ) hemagglutinin ( h ) and fusion ( f ) envelope protein pseudotyped lvs ( mv - lv ) have recently been shown to efficiently enter and integrate into non - activated b and t lymphocytes . importantly , mv - lv can also be used to generate targeted vectors that restrict gene delivery to cells of choice at the level of cell entry.20 , 21 this strategy involves mutating the receptor - binding sites in the h protein and fusion of a single chain variable fragment ( scfv ) specific for the target receptor of choice . among others , lvs delivering genes to lymphocytes with unprecedented selectivity have been generated . when targeted to cd19 and cd20 , selective gene delivery into resting human b lymphocytes was achieved.21 , 23 , 24 , 25 in this study , we expanded upon previous studies that used human cd20 ( hcd20 ) as a target antigen for scfv binding to b cells in an mv - lv vector system . we were able to confirm that mv - lv with receptor specificity for cd20 ( cd20-lv ) efficiently transduced resting primary human b lymphocytes or transgenic ( tg ) murine b cells expressing hcd20 . wild - type ( wt ) mouse b cells were not targeted by this vector , even upon lipopolysaccharide ( lps ) activation , demonstrating the remarkable specificity of this approach . cd20-lv was used to package an immunoglobulin g ( igg ) heavy chain conjugated human fix cassette ( cd20-lv ) , and autologous gene - modified hcd20-tg b cells were adoptively transferred to mediate immune tolerance to fix in a mouse model of hb . the use of resting b cells as tolerogenic antigen - presenting cells ( apcs ) , in combination with an igg fusion cassette to establish tolerance,26 , 27 resulted in significant prevention of inhibitory antibody development to fix . further , we were able to show that ex  vivo lentiviral transduction of human b cells did not result in a detectable elevation of inflammatory cytokines , suggesting minimal immune responses to this tolerance approach . previously , we had applied an established concept for tolerance induction to hb by ex  vivo retroviral transduction of lps - stimulated mouse b cells with a truncated human fix protein lacking the pre - pro peptide ( rendering it non - functional ) , engineered in frame with an autologous igg1 heavy chain lacking the signal sequence for secretion . it has earlier been shown that endogenous processing of the protein by b cells could tolerize t  cells directly.28 , 29 the use of igg as a carrier , combined with the capability of b cells to function as tolerogenic apc , has been applied to various animal models of autoimmune and other diseases.26 , 27 , 30 , 31 , 32 , 33 we were able to demonstrate the prevention of inhibitor formation to fix and suppression of pre - existing inhibitors in fix - primed hb mice by this approach . however , because retroviral vectors can only transduce dividing cells , we sought to translate this strategy into lv using mice transgenic for hcd20 as a model . it has been shown that cd20-targeted , mv glycoprotein pseudotyped lv can effectively transduce resting primary human b cells with minimal activation , characterized by a small change in cell cycle progression from the resting g0 to the g1b phase . this makes these vectors a very attractive candidate in comparison to vsv - lv , especially for in  vivo cell targeting . we therefore engineered a truncated version of the human fix coding sequence ( retaining all known t  cell epitopes in mice ) at the n terminus of the igg heavy chain scaffold and cloned it into the psew lentiviral transfer vector ( figure  1a ) . although viral titers of cd20-lv were lower ( 2 to 3   10 transducing units / ml , titrated on raji cells ) than those obtained by conventional vsv - lv , targeted specificity allowed for 39%60% transduction of both primary human b cells and hcd20-tg resting b cells , as quantified by gfp expression of 7-aad - excluded live cells ( figure  2 ) . both primary human and hcd20-tg mouse cells were transduced at a similar frequency at various mois , confirming that transduction depended only on expression of the targeted receptor , in this case , cd20 ( figure  2 ) . similarly , previous studies confirmed high - target versus non - target cell discrimination upon transduction of mixed lymphocyte populations of human b and t  cells . notably , the high transduction frequencies observed in this study occurred in the absence of b cell stimulation by cytokines or other factors , with implications for tolerogenic studies and in  vivo gene therapy . in fact , lps stimulation had no effect on the frequency of transduced primary human or hcd20-tg mouse cells ( data not shown ) . in contrast , the transduction efficacy in primary b cells from wt balb / c mice was comparable to that of untransduced cells ( 0.7% gfp , figure  3a ) . transduction in wt mice was significantly lower as compared to hcd20-tg mouse b cells ( figure  3b ) . thus , we were able to ensure minimal off - target gene delivery by this approach . cd20-lv - based ex  vivo transduction was also found to elicit minimal inflammatory cytokine response by real - time rt - pcr quantification . mrna levels of interferon ( ifn)- , ifn- , tumor necrosis factor ( tnf)- , interleukin ( il)-6 , and il-10 remained unchanged in primary human b cells at 24  hr post transduction with cd20-lv particles ( data not shown ) . incubation of cells with the tlr9 agonist cpg ( 5  m odn 2066 ) as a positive control resulted in 31.56   2.7-fold upregulation in il-6 as compared to control untreated cells . we were unable to consistently quantify increased mrna expression of other inflammatory cytokines like ifn- , ifn- , and tnf- by the tlr9 agonist , which has also been documented by others . mouse b cells transduced with cd20-lv were adoptively transferred into balb / c - hb mice at 510   10 cells / mouse . the truncated fix protein was earlier shown to be just as effective in generating inhibitory antibodies as the full - length protein , while consistently yielding higher - titer virus . because the frequency of transduced cells was 39%60% , we did not flow sort for gfp cells before adoptive transfer . hb mice on the balb / c background do not respond as robustly to fix protein administration , resulting in a lower incidence of inhibitor formation compared to , for example , c3h / hej mice . in order to elicit a stronger immune response to fix administration , balb / c - hb mice were challenged 1  day following adoptive transfer of lentivirus - modified b cells with 1 iu fix in adjuvant delivered by the subcutaneous ( sc ) route . functional inhibitor and anti - fix igg1 titers were quantified 2 and 3  weeks later ( figure  4a ) . b cell gene transfer recipient mice developed significantly lower anti - hfix igg1 ( 4,428.01  ng / ml , figure  4b ) and bethesda titers ( 0.58 bu / ml , figure  4c ) as compared to control balb / c - hb mice that received nothing ( anti - hfix igg1 : 10,524.2  ng / ml , bu / ml : 2.33 ) at 2  weeks . this difference was sustained at 3  weeks ( 3,566.3  ng / ml versus 17,902.6 anti hfix igg1 ; 0.06 versus 2.79 bu / ml , figures 4b and 4c ) . next , prevention of inhibitor formation to hfix administered via the more clinically relevant iv route was tested . balb / c - hb mice received cd20-lv transduced hcd20-tg mouse b cells at 510   10 cells / mouse as before . the following day , mice were administered with 3 iu hfix , 1x / week for 8  weeks before quantification of functional inhibitor and anti - fix igg1 titers ( figure  5a ) . three of four mice receiving b cell gene transfer did not develop inhibitors against fix ( 0 bu ) , whereas one - fourth of the animals ( 25% ) formed an inhibitor ( figure  5c ) . this is in contrast to the 81% inhibitor response rate seen in control animals that received protein therapy only ( figure  5c ) . a similar difference was seen for anti - hfix igg1-binding antibodies , with a 4.6-fold reduction in the average titer ( figure  5b ) . because of the one mouse that failed to respond to the tolerogenic therapy , the differences in average antibody formation ( 856 versus 3,931  ng igg1/ml and 2 versus 3.4 bu / ml ) failed to reach statistical significance . statistical analysis defined this animal as an outlier . on outlier removal , the treated group and control group were found to be significantly different for anti - fix igg1 titers ( figure  5b , p  = 0.0014 ) . tolerance induction using gene - modified primary b cells is highly promising in various mouse models of genetic disease , such as hemophilia and autoimmune diseases.26 , 27 , 31 , 33 , 36 however , inability to efficiently transduce human b cells has kept this approach from moving forward . here , we addressed this problem by developing a lentiviral delivery system that specifically targets hcd20 . not only were we able to demonstrate remarkable cell - specific targeting by this method , but the availability of a hcd20 transgenic mouse strain also allowed us to test for the effectiveness of this approach for tolerance induction in  vivo . taken together , we report a highly specific and effective protocol for tolerance induction , which indicates the feasibility of this approach for translational studies . prevention and management of inhibitors in hb is not addressed as  commonly as that of hemophilia a because inhibitor development is less frequent . however , 9%23% of severely affected patients develop inhibitors , of which approximately 80% are high responding ( 5 bu / ml).2 , 37 in addition , the frequent and simultaneous occurrence of allergic / anaphylactic reactions with the development of inhibitors is unique to hb.4 , 5 tolerance approaches to prevent or reverse fix inhibitors are , therefore , highly desirable . we were able to show in an earlier study that b cell - based therapy for hb effectively prevented inhibitor formation to fix and desensitized mice with a pre - existing response . in addition , it is known that lvs transduce b cells poorly and can not enter truly quiescent b cells , making gene transfer studies difficult for b cells . our approach to retarget the vector to cd20-expressing b cells allowed for transduction of resting b cells without the risks of an immune response caused by activation signals provided to the b cells during gene transfer . we demonstrated receptor - specific targeting and improved tolerance to fix by gene - modified hcd20-tg mouse b cells , resulting in a significant prevention in the development of inhibitory antibodies to fix in a mouse model of hb . importantly , incidence in inhibitor formation was lower in mice receiving gene - modified b cells , suggesting that this protocol may be advantageous in preventing humoral responses to coagulation factor therapy . in our previous studies in c3h hb mice , using a murine retroviral vector , we found that transplant of b cells expressing the hfix - igg fusion was also able to reverse pre - existing inhibitors and eliminate anaphylactic reactions . because the lv expresses the same hfix - igg fusion , it is likely that this vector will also be effective in inhibitor reversal . to prove this point , it will be necessary to backcross hcd20-tg mice onto the c3h background to subsequently test for inhibitor reversal in c3h / hej fix mice ( without intervention , these animals have a more robust and lasting inhibitor response to hfix , which is needed to distinguish active tolerance induction from spontaneous decline in titers ) . we had previously shown that nucleofection of plasmid dna , although achieving high gene transfer efficacy , yielded strong immune responses to fix , which was due to tlr9-dependent activation of innate immune mediators and inflammatory cytokine expression . ex  vivo lentiviral transduction did not result in a detectable immune response , as confirmed by a lack of inflammatory cytokine elevation . consistent with results initially described by kneissl et  al . , we also found that transduction by cd20-lv caused minimal activation in resting b cells , eliminating the need for potential activators of innate immunity . toward clinical translation of this approach , upscaling and production of cd20-lv in a gmp - like process large - scale production of vector may be difficult to reach with the engineered mv glycoproteins used in this study . high transduction efficiencies on b cells allowing active hfix secretion have recently been described for lentiviral vectors pseudotyped with the baboon envelope glycoprotein . although this could be very efficient for ex  vivo b cell transduction , this vector lacks selectivity for b cells , making direct in  vivo application difficult . a recent paper demonstrated that the nipah virus ( niv ) glycoproteins can be engineered for receptor targeting of lv , resulting in substantially improved vector production yields , while retaining the high selectivity of the mv - based system . in particular , when the same cd20-specific scfv that was used here on the mv glycoproteins was used on the niv glycoproteins , vector production yields increased by more than two orders of magnitude , making a scale - up for the production of clinical vector lots conceivable . briefly , the cd20-scfv was linked to the ectodomain of the pcg - hmut plasmid via the factor xa cleavage site . this composed an 18 amino acid ( aa ) cytoplasmic tail truncation and mutations in the cd46 ( y481a , s548l , and f549s ) and signaling lymphocyte activation molecule ( slam , r533a ) interaction regions of the h protein of the mv edmonston strain . cytoplasmic tail truncations of mv h and f proteins facilitated efficient incorporation of these proteins into the lentiviral membrane . cd20-lv particles were produced by polyethyleneimine ( pei)-based co - transfection of hek293 t cells with the hiv-1 packaging plasmid pcmvr8.9 , the gfp transfer plasmid psew - gfp , and the two plasmids encoding the modified h and f proteins , respectively ( h : f ratio of 1:7 ) , as described . vector particles were concentrated by ultracentrifugation over a 20% ( weight / vol ) sucrose cushion ( 100,000   g for 3  hr at 4c ) and titrated by transduction on raji cells . to generate the lv transfer vector plasmid cd20-lv encoding the hfix - igg fusion protein , a 0.9-kb truncated version of the human fix coding sequence ( lacking the n terminus ) was pcr amplified from the aav2-ef1-fix vector . this was subcloned into  bssk - igg , a murine igg1  h chain cassette plasmid containing noti and xhoi cloning sites at the n terminus of the igg1  h chain ( a gift of dr . the signal sequence of the igg h chain was previously deleted from the bssk - igg vector by site - directed mutagenesis . the hfix - igg region was then inserted into the transfer plasmid psew - gfp , thereby replacing the gfp reading frame , and lv particles were produced as described . wt mouse b cells and hcd20-tg b cells were purified using the mouse b cell isolation kit ( miltenyi biotec ) , which isolates untouched resting b cells . primary human cd19 b cells ( mostly cd20 ) , negatively selected from peripheral blood mononuclear cells ( pbmcs ) , were purchased from precision for medicine . resting human and mouse b cells were transduced with cd20-lv or cd20-lv , respectively , by spinoculation at 1,200   g for 90  min on retronectin ( 20  g / ml , clontech ) coated six - well plates . the percentage of gfp cells was determined by flow cytometric analysis after 72  hr . data were collected on an lsr - ii flow cytometer ( bd biosciences ) and analyzed with fcs express 5 ( de novo software ) . animals were housed under specific pathogen - free conditions under institutional animal and care and use committee approved protocols . all animals were male and 68  weeks old at the onset of the experiments . hb mice with a targeted deletion ( promoter and first three exons ) of murine f9 ( null mutation ) were bred on a balb / c background for > 10 generations . mice were generated by injecting a bacterial artificial chromosome carrying the gene for hcd20 into the pronuclei of c57bl/6 x sjl / j embryos . balb / c - hb mice were adoptively transferred with 510   10 primary murine hcd20-tg b cells transduced with cd20-lv . this was followed by either sc immunization with 1 iu hfix ( benefix , pfizer ) in adjuvant ( sigma adjuvant system ) or 3 iu hfix replacement therapy by the iv route , once a week , for 8  weeks . plasma samples were collected by tail bleed into 0.38% sodium citrate , and inhibitory antibodies to fix were measured by bethesda assay , as described . statistical significance was determined using two - way anova or  student s t test with graphpad prism 5 software . c.j.b . is listed as inventor on patent applications for receptor - targeted lentiviral vectors that have been out - licensed .
gene - modified b cells expressing immunoglobulin g ( igg ) fusion proteins have been shown to induce tolerance in several autoimmune and other disease models . 
 however , lack of a vector suitable for gene transfer to human b cells has been an obstacle for translation of this approach . to overcome this hurdle 
 , we developed an igg - human factor ix ( hfix ) lentiviral fusion construct that was targeted to specifically transduce cells expressing human cd20 ( hcd20 ) . 
 receptor - specific retargeting by mutating envelope glycoproteins of measles virus ( mv)-lentiviral vector ( lv ) and addition of a single - chain variable fragment specific for hcd20 resulted in gene delivery into primary human and transgenic hcd20 mouse b cells with high specificity . 
 notably , this protocol neither required nor induced activation of the b cells , as confirmed by minimal activation of inflammatory cytokines . using this strategy 
 , we were able to demonstrate induction of humoral tolerance , resulting in suppression of antibody formation against hfix in a mouse model of hemophilia b ( hb ) . in conclusion , 
 transduction of receptor - specific retargeted lv into resting b cells is a promising method to develop b cell therapies for antigen - specific tolerance induction in human disease .