Hemophilia: New Protein Therapeutics

Steven W. Pipe1

1Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Michigan, Ann Arbor, MI

Therapeutic advances for patients with hemophilia have resulted in reduced mortality, improved joint outcomes, safetyfrom blood-transmitted pathogens, improved quality of life, and a normalized life span in the developed world. Theproduction of recombinant coagulation factors has increased the worldwide capacity for replacement therapy andfacilitated aggressive prophylactic therapy. However, this has come at significant cost, and barriers remain to broadapplication of prophylaxis. Recombinant DNA technology remains a promising platform to develop novel hemophiliatherapeutics with improved functional properties to try to overcome some of these remaining barriers. Bioengineeringstrategies have produced novel therapeutics with increased production efficiency, increased potency and resistance toinactivation, prolonged plasma half-lives, and reduced immunogenicity. Alternative nonbiologic therapies may lead tonew treatment paradigms. The current pipeline of new technologies and products is promising and growing withseveral agents already advancing from preclinical to clinical trials.

The recurrent hemarthroses and resultant crippling arthropathy ofhemophilia have almost been completely eradicated through aggres-sive prophylactic therapy with clotting concentrates in the devel-oped world when initiated at a young age. Quality-of-life measuresin children now track similar to their unaffected peers, and patientsengage regularly in sporting activities unimagined in previousgenerations.1 Prophylaxis for severe hemophilia is now recognizedas the standard of care with optimal initiation very early in lifebefore the onset of repeated hemarthroses, typically between ages 1to 3 years.2 Nevertheless, barriers remain to realizing the benefits ofprophylaxis universally. The development of alloantibodies thatinhibit the activity of infused replacement products remains asignificant complication (up to 30% of patients with hemophilia A).The costs for prophylactic replacement therapy are much greaterthan $100,000 per patient per year. Repeated venous access(typically three times per week to every other day) is still a barrierfor many. Suboptimal adherence to a prophylactic regimen alsocompromises outcomes.3 There is also the frequent requirement forcentral venous access devices in the youngest boys to facilitateprophylactic infusion with associated risk for infection and thrombo-sis.4 Despite the increased capacity of factor concentrate production,worldwide demand for factor VIII is now over 5 billion units peryear, most of which is infused in North America and Europe, with80% of the world still without proper access to replacement therapy.Recombinant DNA technology is now serving as a platform forcontinued innovation to try to overcome some of the remaininghurdles for hemophilia care. This article summarizes the current andfuture trends in hemophilia therapeutics, with a particular focus ontargeted bioengineering strategies.

Bioengineered Factor VIIIPurified plasma-derived (pd) factor VIII (FVIII) concentrates havebeen available since the 1970s, but require rigorous plasma donorscreening and viral inactivation technologies to reduce the risk forblood-transmitted pathogens. Recombinant FVIII (rFVIII) concen-trates, available since 1992, are derived from expression withintransfected mammalian cell lines. rFVIII has proven to be aremarkable facsimile to pdFVIII with regard to biochemical andhemostatic properties, and has a similar pharmacokinetic profilewith a half-life of roughly 12 hours. After almost 20 years of

experience with rFVIII, there are clearly opportunities to extendrecombinant DNA technology to further enhance replacementtherapy. Bioengineering strategies have been directed at overcom-ing the inherent limitations of rFVIII biosynthesis and secretion,functional activity, half-life, and antigenicity/immunogenicity (Fig-ure 1). Some of these strategies have already reached commercializa-tion, several are in ongoing clinical trials, and a number of strategiesare in advanced preclinical development. Although many of thesestrategies may be promising for replacement therapy and prophy-laxis, others may find application in gene therapy studies.

Improved Production EfficiencyEarly on in the study of rFVIII expression, it was demonstrated thatthe B domain of FVIII, the equivalent of approximately 38% of theprimary cDNA sequence, could be removed without loss of FVIIIprocoagulant activity. This significantly improved the yield ofrFVIII due to markedly increased levels of mRNA and increasedtranslation. The reduced size of the B-domain–deleted (BDD)-FVIII cDNA also facilitated packaging within certain viral vectorsfacilitating its adoption for gene therapy strategies. BDD-rFVIIIremains the first and only modified human rFVIII molecule to cometo commercialization. Other strategies to improve the efficiency ofexpression have included introduction of a truncated factor IX (FIX)intron 1,5 point mutations that reduce interactions with endoplasmicreticulum (ER) chaperones6 or improve the efficiency of ER-Golgitransport (through inclusion of a short B-domain segment withinBDD-rFVIII7). Because these targeted modifications involve differ-ent steps within the secretion pathway, they can be combined in thesame molecule providing an additive effect in producing substantialincreases in the yield of FVIII in heterologous expression systems.7

Porcine BDD-rFVIII is in development for use in patients withinhibitors to human FVIII.8 Porcine BDD-rFVIII generates 10- to14-fold higher expression than human BDD-rFVIII when expressedin baby hamster kidney cell lines.9 Subsequently, HEK-293 celllines expressing porcine BDD-rFVIII demonstrated 36- to 225-foldhigher expression than human rFVIII constructs. Furthermore, thehigher protein production was not caused by significant increases insteady-state FVIII mRNA levels. This suggests a translational orposttranslational advantage for porcine rFVIII. Further study using

HEMOPHILIA

Hematology 2010 203

porcine/human hybrids has localized certain protein sequenceswithin porcine FVIII that confer this advantage, although additionalstudies will be necessary to characterize the mechanism.10 Bioengi-neering rFVIII for high-efficiency production could potentiallyreduce costs and increase the availability of concentrates fortherapy.

Improved Functional ActivityFollowing activation of FVIII by thrombin, activated FVIII (FVIIIa)exists in a heterotrimeric form with its activity limited by additionalproteolytic degradation and spontaneous decay through subunitdissociation. To address this limitation, an inactivation-resistantFVIII (IR8) was genetically engineered, which is not susceptible todissociation of the A2 domain subunit and proteolytic inactivationby activated protein C.11 This was achieved through modification ofthrombin cleavage sites and the introduction of point mutations. IR8exhibits an increased specific activity and prolonged cofactorfunction after thrombin activation in vitro and in vivo. Gale andcolleagues12 introduced a disulfide bond (DSB) engineered betweenthe A2 and A3 domains to stabilize FVIIIa by preventing A2 subunitdissociation following thrombin activation. A three-dimensionalhomology model of the FVIII A domains13 suggested that cysteinessubstituted at residues 664 and 1826 within FVIII would result in aDSB at the edge of the interface between the A1 and A3 domains,very near their solvent-exposed surfaces. This DSB-FVIII variant(C664-C1826) exhibited increased specific activity, prolongedcofactor activity following thrombin activation, and increasedpotency in whole-blood clotting assays.14 FVIII proteins withprolonged activity following thrombin activation have the potentialto increase the efficacy of FVIII in plasma potentially prolongingthe activity of FVIIIa, even when present in levels usually ineffec-tive for hemostasis.

Half-life ExtensionA major emphasis of current bioengineering efforts has been onhalf-life extension.15 Prolonging the half-life of FVIII could greatlyreduce the frequency and dose of infusions, thereby improving theefficacy of prophylaxis through better compliance, as well asimprove convenience and patient quality of life.16 The primarydeterminant of FVIII residence time in plasma is interaction withvon Willebrand factor (vWF), which protects it from proteolysis andcellular uptake. Clearance of FVIII has only recently begun to beelucidated. FVIII is too large in molecular weight to be cleared bythe kidneys. Cellular clearance (Figure 2), primarily in the liver,

occurs through interaction with a family of low-density lipoproteinreceptor-related proteins and heparan sulfate proteoglycan recep-tors, among others.17 Some of the half-life extension strategiesunder investigation include the following: sustained delivery throughassociation of rFVIII with polyethylene glycol (PEG)ylated lipo-somes, chemical modification (eg, PEGylation, polysialylation),bioengineering rFVIII through mutagenesis at putative binding sitesfor clearance receptors, or the generation of fusion proteins (eg, withan Fc antibody fragment).15 It may be surprising that PEGylatedrFVIII has not reached the clinic sooner. This has been used for anumber of biologics and successfully transitioned to the clinic (eg,PEG-asparaginase for acute lymphoblastic leukemia). However, theprimary advantage of PEGylation for a biologic is the incorporationof many water molecules within the hydrophilic PEG structures,functionally increasing the effective size of the conjugated proteinabove the filtration size of the kidney. This is of no particularadvantage for FVIII, because it is already too large for kidneyfiltration. Any advantage to PEGylation of FVIII is likely throughdisruption of interaction with cellular clearance receptors. However,such chemical modification could be a disadvantage to FVIII if itinterfered with key protein–protein interactions (eg, vWF, throm-bin, and activated FIX [FIXa]). These potential hazards of chemicalmodification of FVIII are particularly problematic without theability to target the sites where PEG polymers are conjugated to theprotein.

PEGylationRostin et al18 published their experience with PEGylation ofBDD-rFVIII. Random coupling of PEG at amino groups of lysinesled a significant reduction in specific activity, as well as a significantproportion of molecules that could not bind vWF. Thus, recentstrategies have focused on novel chemical conjugations that allowtargeted PEGylation. Mei et al17 recently described their success by

Figure 1. Examples of bioengineering strategies to improve thefunctional properties of rFVIII.

Figure 2. Life cycle of FVIII. Following intravenous infusion, FVIII isstabilized in plasma through noncovalent association with vWF,protecting it from proteolysis and cellular uptake. With a hemostaticchallenge, thrombin activation releases FVIII from vWF so that it canexert its procoagulant function. The majority of infused FVIII is cleared inthe liver through interaction with the low-density lipoprotein receptor-related protein (LRP) family of cell surface receptors. A PEGylated formof FVIII would have reduced cellular uptake and a resultant prolongationof plasma half-life. The elimination of PEG-FVIII that is internalized in thehepatocyte has not been fully characterized, but may follow urinary andfecal excretion routes limiting intracellular accumulation.

204 American Society of Hematology

screening targeted PEGylated rFVIII mutants achieved throughlinkage to free surface-exposed cysteine residues introduced throughmutagenesis. They identified variants that retained full procoagulantfunction and vWF binding in vitro, exhibited improved pharmacoki-netics in hemophilic mice and rabbits, and prolonged efficacy inbleeding models in mice consistent with their enhanced half-life invivo. Their results confirm that the site of PEGylation on FVIII iscritical to PEGylation efficiency, preservation of procoagulantactivity, and pharmacokinetic impact.

PolysialylationPolysialic acid (PSA) modification of therapeutic proteins is analternative strategy to PEGylation for increasing the size of aprotein. PSAs are linear, hydrophilic polymers of N-acetylneura-minic acid that occur abundantly on the surface of many cells andproteins. They can be conjugated to therapeutic proteins andpotentially alter their pharmacokinetics, including prolonging thehalf-life.19 PSA also produces a “watery cloud” around the therapeu-tic molecule protecting it from immune-mediating cells, proteolyticenzymes, and clearance receptors. The use of PSA has already beensuccessfully applied to several therapeutic proteins.20 Some of theadvantages that have been demonstrated with PSA technologyinclude reduced immunogenicity and antigenicity, and preservationof function with increased stability. Furthermore, PSA, unlike PEG,are naturally occurring and biodegradable, which offers a potentialadvantage when administering large doses of a biologic therapyover a prolonged period of time. PSA modification is now beingexplored through direct conjugation of rFVIII, FIX, FVIIa, andvWF.

PEGylated LiposomesFormulation with PEGylated liposomes is an established processthat has been used to extend the half-life of a broad range oftherapeutic proteins, including FVIII.21 For this approach, thetherapeutic protein is reconstituted with PEGylated liposomes as acarrier. Because liposomes are typically cleared very quickly fromthe circulation, the addition of PEG can extend the circulatoryhalf-life of the liposomes considerably. This approach can effec-tively modify pharmacokinetic and pharmacodynamic properties ofproteins and has been utilized to develop a potentially longer actingFVIII. With this strategy, rFVIII molecules remain unmodified, sothere is no loss of normal protein–protein interactions and func-tional activities. In preclinical trials within a hemophilia A mousemodel, prophylactic infusion of rFVIII reconstituted with PEGy-lated liposomes (rFVIII-PEG-Lip) prolonged some pharmacoki-netic parameters, compared with standard rFVIII and correlatedwith an enhanced hemostatic efficacy.22 In addition, rFVIII-PEG-Lip has been examined in patients with severe hemophilia A in ablinded, controlled, crossover, multicenter trial.16 A single prophy-lactic infusion of rFVIII-PEG-Lip resulted in a longer bleed-freeinterval, compared with standard rFVIII. The rFVIII-PEG-Lipformulation was well tolerated, and no significant adverse eventswere reported during the trial. These findings generated greatinterest, and a subsequent, double-blind, randomized, crossoverphase I trial was conducted to compare the pharmacokinetics of asingle infusion of rFVIII-PEG-Lip with that of rFVIII in 26 menwith severe hemophilia A.23 However, rFVIII-PEG-Lip and stan-dard rFVIII demonstrated similar pharmacokinetic parameters.Additional preclinical studies in a hemophilia A mouse modelsuggested that rFVIII-PEG-Lip results in prolonged apparent FVIIIactivity beyond what would be expected by the plasma pharmacoki-netics when whole blood clotting was assayed by rotational

thromboelastography.24 Furthermore, rFVIII-PEG-Lip increased P-selectin surface expression on platelets in response to collagen, andthe enhanced procoagulant activity was retained until ultracentrifu-gation of the plasma, suggesting it may be acting through sensitiza-tion of platelets and the generation of procoagulant microparticles.25

Nevertheless, a phase II clinical trial was stopped midstage when aninterim analysis indicated that the trial would not meet its efficacyendpoint.

Reduced Antigenicity/ImmunogenicityAnother line of investigation involves developing FVIII withreduced antigenicity/immunogenicity. The most sophisticated con-structions are human/porcine hybrid BDD-rFVIII molecules thathave reduced antigenicity within human inhibitor plasmas,26 andhave guided the design of bioengineered variants with reducedimmunogenicity in a hemophilia A mouse model without anyapparent loss of function.27 In another strategy, BDD-rFVIII hasbeen produced in the human embryonic kidney cell line, HEK293Fcells.28 This was done to produce a human pattern of posttransla-tional modifications of FVIII, such as glycosylation, to reduce theimmunogenic profile, compared with existing rFVIII produced inhamster cells. Further clinical studies will need to determine if thereis a significant advantage of these types of FVIII variants inpreviously untreated patients with hemophilia.

Bioengineered FIX (Figure 3)Purified pdFIX has been available since 1992, followed by recombi-nant FIX (rFIX) in 1998, produced in mammalian cell lines similarto rFVIII. Although, rFIX also shares nearly identical hemostaticproperties with pdFIX, there are differences in posttranslationmodifications of rFIX that result in altered pharmacokinetics.Specifically, rFIX exhibits an � 30% reduced recovery in plasma atequivalent dosing to pdFIX, requiring dose modification for treat-ment and prophylactic regimens. However, the plasma half-life forboth pdFIX and rFIX is similar at roughly 16 hours.

Alternative Expression SystemsAn innovative expression technology has been used to enhance theexpression of rFIX in stable cell lines.29 This strategy involvesflanking rFIX cDNA with the transcriptional control regions fromChinese hamster elongation factor 1�, a highly expressed genewithin Chinese hamster ovary (CHO) cells. Within this system, theexpression levels of rFIX are increased 10-fold over traditionalstably transfected CHO cells. Such technology could be exploited

Figure 3. Examples of bioengineering strategies to improve thefunctional properties of rFIX.

Hematology 2010 205

for efficient production of rFIX, or any other recombinant protein,and hopefully facilitate reduced costs.

Transgenic animals have shown promise as an efficient source ofproducing abundant recombinant proteins.30 The yields from suchsystems could improve the availability of recombinant clottingfactors worldwide or serve as an efficient source material forchemical modification to alter pharmacokinetic or biochemicalproperties, or to pursue alternative delivery systems (eg, oraladministration). Recombinant coagulation protein production re-quires that the cell culture bioreactor systems can replicate thecomplex biochemistry of their plasma-derived counterparts and isinfluenced by the cell density within the bioreactor system. Themammary gland of livestock has both the cellular machinery andhigh cell density to produce recombinant proteins at two orders ofmagnitude or greater than traditional cell culture bioreactor systems.The first approved transgenic recombinant product was recombinantantithrombin produced in the milk of transgenic goats.31 However,in contrast to ruminants, the pig mammary gland has demonstratedthe capacity to carry out the posttranslational modifications (eg,glycosylation, sulfation) necessary to enable high efficiency produc-tion of recombinant proteins with the biologic activity and pharma-cokinetic properties needed to be considered as a therapeutic forhemophilia. Recombinant human FIX has been produced in the pigmammary gland at very high yields (100 IU/mL), compared withplasma source material (1 IU/mL) or CHO cell bioreactors (2IU/mL), such that it has been estimated that the milk from 60 pigs(� 12,000 L/year) could supply the entire amount of rFIX neededfor prophylaxis in the United States.32

Increased PotencySeveral bioengineering strategies have generated rFIX variants withincreased potency. These include point mutations, such as variantFIX-R338A, which exhibits 3- to 7-fold higher specific activity invitro33 and in vivo.34 Recently, the importance of this particularresidue was highlighted by the identification of a family with apotent X-linked thrombophilia who were identified as carrying aR338L missense mutation of FIX.35 The plasma FIX activity of theproband was 800% of normal, and an rFIX-R338L exhibited a 5- to10-fold higher specific activity in vitro. Hopfner et al36 used insightsfrom the structural and functional homology between FIXa andactivated factor X (FXa) to bioengineer a recombinant FIX-FXhybrid that exhibited a catalytic efficiency 130-fold that of thewild-type rFIX. Such variants could considerably reduce theinfusion requirements for hemostatic efficacy.

Half-life ExtensionStrategies to extend the half-life of FIX include direct, targetedmodification with PEGylation and fusion protein technology, whichlinks FIX to another protein with a much longer plasma half-life.rFIX was fused to the constant region (Fc) of immunoglobulin G(rFIXFc).37 The presence of the Fc portion protects the fusionprotein from catabolism through interaction with the neonatal Fcreceptor (FcRn). The Fc domain binds FcRn at acidic pH (� 6.5),but not at physiologic pH (7.4). Fc-containing proteins that areinternalized by endothelial cells bind to FcRn present in theacidified endosome in a pH-dependent manner and are then recycledback to the cell surface where they are released back into plasma atphysiologic pH, rather than targeted for degradation in the lyso-some. Preclinical data with rFIXFc demonstrated a 3- to 4-foldlonger terminal half-life in mice, rats, and cynomolgus monkeys.The functional impact was demonstrated as the whole-blood

clotting time was corrected through 144 hours for rFIXFc, com-pared with 72 hours for unconjugated rFIX in the hemophilia Bmouse model. Similar results were obtained in two hemophilia Bdogs. In a second strategy, rFIX was fused to albumin (rFIX-FP) viaa cleavable peptide linker.38 This allows for release of rFIX fromalbumin on activation. rFIX-FP demonstrated significantly pro-longed pharmacokinetics in rats, rabbits, and hemophilia B micewith effective hemostasis. Both of these fusion protein strategies arepresently being evaluated in clinical trials.

Bioengineered Activated Factor VIIRecombinant activated FVII (rFVIIa) has proved to be a safe andeffective therapeutic for the management of bleeding in hemophiliapatients with inhibitors. It has both tissue factor-dependent andindependent mechanisms of action. However, its short plasmahalf-life requires a short interval for follow-up dosing and limits itsapplication in prophylaxis. In addition, clinical studies have sug-gested that higher initial doses may result in a more rapid onset ofhemostasis,39,40 and the time from initiation of bleeding to initiationof treatment may have a significant impact on efficacy.41 Accord-ingly, several bioengineering strategies have been implemented inan attempt to improve rFVIIa functionality. Some of these includeglycoPEGylation,42 targeted PEGylation to specific residues ofFVII, formulation with PEG-Lip,43 and fusion of rFVIIa withalbumin,44 all directed at half-life extension. Other strategies haveattempted to increase the potency and rate of onset of action ofrFVIIa through directed molecular evolution and rational de-sign.45,46 Several of these approaches have already moved to theclinical testing phase.

Alternative Delivery StrategiesNonintravenous (ie, oral, intratracheal, subcutaneous, and intramus-cular) delivery of hemostatic agents for hemophilia would liberatepatients from the challenges of regular direct venous access and thecomplications of central venous catheters. However, the very lowbioavailability observed with such nonintravenous delivery ofalready expensive clotting factor concentrates remains a barrier. Inaddition, some routes of administration could actually increaseclotting factor immunogenicity. Alternative delivery strategiescould be realized if the production costs of concentrates could bedramatically reduced. Recombinant clotting factor expression withintransgenic animals and plants may be able to reach such productionscales. Additional bioengineering strategies could also improvebioavailability via the enteric route. Transgenic plants expressingrFIX fused to a transmucosal carrier protein have been prepared.47

The rFIX remains encapsulated within the plant cells, protecting itfrom enteric degradation. Interestingly, hemophilia B mice thatwere fed this bioencapsulated transgenic rFIX plant material werealso protected from inhibitor formation and anaphylaxis to intrave-nous challenge with rFIX, suggesting such oral delivery strategiescould be a strategy to avoid another major complication of proteinreplacement therapy.

Alternative Hemostatic AgentsResearch toward novel therapeutics for hemophilia is not limited tobioengineering recombinant coagulation proteins. Alternative pro-tein and nonprotein agents are showing promise in preclinicaldevelopment. Kopecky et al48 synthetically designed peptide se-quences that were screened and tested for their ability to bind toinhibitory antibodies effectively neutralizing anti-FVIII antibodiesin vitro. The promising peptides typically have sequences that aresimilar to FVIII. However, owing to their short half-life, strategiessuch as PEGylation may be required to extend their circulation time.

206 American Society of Hematology

Fucoidans, known also as nonanticoagulant-sulfated polysaccha-rides (NASP), are heparin-like molecules that at certain concentra-tions exhibit hemostatic properties rather than anticoagulant activ-ity. They are believed to act through blockade of tissue factorpathway inhibitor, effectively removing the brakes from coagula-tion. NASP can accelerate the clotting times of plasma fromhemophilia patients, and improve hemostasis when administeredsubcutaneously to hemophilic mice49 and dogs.50 Most notably,NASP improved hemostasis when administered orally to severehemophilia A dogs.50

Aptamers are single-stranded nucleic acids that can directly inhibitfunction by folding into a specific three-dimensional structure witha high affinity for the target. They can theoretically be generated tobind to any protein of interest. This technology has been explored inclinical trials of anticancer and antiviral therapeutics.51 Moreover,opportunities also exist for therapeutic aptamers as antithromboticand hemostatic agents. Aptamers that target tissue factor pathwayinhibitor have shown similar hemostatic effects in vitro as NASP.52

Finally, missense mutations leading to premature stop codons inFVIII or FIX occur in approximately 10% to 15% of persons withhemophilia A or B, respectively. Small molecules have beendeveloped that can facilitate translational readthrough of prematurestop codons. A lead candidate has been tested in transgenic miceengineered with a nonsense FIX mutation (R338X) and resulted indetectable plasma levels (3-5 ng/mL) in up to 20% of the mice.53 Aphase II clinical trial is underway in hemophilia patients known tocarry premature stop codons.

ConclusionsAlthough a cure for hemophilia has not yet been realized, there is atremendous pipeline of novel therapeutic agents. Promising newbioengineering strategies are being driven by detailed structure andfunction analysis of coagulation proteins complimented by high-resolution crystal structures. A broad range of species now serves aspreclinical models to guide lead candidate selection. In addition,alternative therapeutics with novel mechanisms of action maydramatically alter therapeutic paradigms. The best ideas from thebench have moved from preclinical studies to clinical trials (Figure4). We can look forward to many of these strategies successfullyovercoming some of the remaining challenges that remain for thetreatment of hemophilia.

DisclosuresConflict-of-interest disclosure: The author has received honorariafrom Baxter BioScience, Inspiration Biopharmaceuticals, CSLBehring, and Novo Nordisk, and research funding from InspirationBiopharmaceuticals. He has also been a consultant for BaxterBioScience. He has a membership on an entity’s Board of Directorsor advisory committees for Baxter BioScience and Novo Nordisk.

Off-label drug use: None disclosed.

CorrespondenceSteven W. Pipe, MD, Department of Pediatrics and CommunicableDiseases, Hemophilia and Coagulation Disorders Program, Univer-sity of Michigan, MPB D4202, 1500 E. Medical Center Dr., AnnArbor, MI 48109; Phone: (734) 647-2893; Fax: (734) 615 (0464);e-mail: ummdswp@med.umich.edu

References1. Ross C, Goldenberg NA, Hund D, Manco-Johnson MJ. Athletic

participation in severe hemophilia: bleeding and joint outcomesin children on prophylaxis. Pediatrics. 2009;124:1267-1272.

2. Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophy-laxis versus episodic treatment to prevent joint disease in boyswith severe hemophilia. N Engl J Med. 2007;357:535-544.

3. Berntorp E. Joint outcomes in patients with haemophilia: theimportance of adherence to preventive regimens. Haemophilia.2009;15:1219-1227.

4. Blanchette VS, Manco-Johnson M, Santagostino E, Ljung R.Optimizing factor prophylaxis for the haemophilia population:where do we stand? Haemophilia. 2004;10(Suppl 4):97-104.

5. Plantier JL, Rodriguez MH, Enjolras N, Attali O, Negrier C. Afactor VIII minigene comprising the truncated intron I of factorIX highly improves the in vitro production of factor VIII.Thromb Haemost. 2001;86:596-603.

6. Swaroop M, Moussalli M, Pipe SW, Kaufman RJ. Mutagenesisof a potential immunoglobulin-binding protein-binding siteenhances secretion of coagulation factor VIII. J Biol Chem.1997;272:24121-24124.

7. Miao HZ, Sirachainan N, Palmer L, et al. Bioengineering ofcoagulation factor VIII for improved secretion. Blood. 2004;103:3412-3419.

8. Barrow RT, Lollar P. Neutralization of antifactor VIII inhibi-tors by recombinant porcine factor VIII. J Thromb Haemost.2006;4:2223-2229.

9. Doering CB, Healey JF, Parker ET, Barrow RT, Lollar P. Highlevel expression of recombinant porcine coagulation factorVIII. J Biol Chem. 2002;277:38345-38349.

10. Doering CB, Healey JF, Parker ET, Barrow RT, Lollar P.Identification of porcine coagulation factor VIII domainsresponsible for high level expression via enhanced secretion.J Biol Chem. 2004;279:6546-6552.

11. Pipe SW, Kaufman RJ. Characterization of a geneticallyengineered inactivation-resistant coagulation factor VIIIa. ProcNatl Acad Sci U S A. 1997;94:11851-11856.

12. Gale AJ, Pellequer JL, Griffin JH. An engineered interdomaindisulfide bond stabilizes human blood coagulation factor VIIIa.J Thromb Haemost. 2003;1(9):1966–1971.

13. Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EG,Kemball-Cook G. A molecular model for the triplicated Adomains of human factor VIII based on the crystal structure ofhuman ceruloplasmin. Blood. 1997;89:2413-2421.

14. Radtke KP, Griffin JH, Riceberg J, Gale AJ. Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and

Figure 4. The pipeline status of bioengineering strategies for noveltherapeutics in hemophilia.

Hematology 2010 207

improved potency in whole blood clotting assays. J ThrombHaemost. 2007;5:102-108.

15. Lillicrap D. Extending half-life in coagulation factors: where dowe stand? Thromb Res. 2008;122(Suppl 4):S2-S8.

16. Spira J, Plyushch OP, Andreeva TA, Andreev Y. Prolongedbleeding-free period following prophylactic infusion of recom-binant factor VIII reconstituted with pegylated liposomes.Blood. 2006;108:3668-3673.

17. Mei B, Pan C, Jiang H, et al. Rational design of a fully active,long-acting PEGylated factor VIII for hemophilia A treatment.Blood. 2010;116(2):270–279.

18. Rostin J, Smeds AL, Akerblom E. B-domain deleted recombi-nant coagulation factor VIII modified with monomethoxypolyethylene glycol. Bioconjug Chem. 2000;11:387-396.

19. Rottensteiner H, Turecek PL, Pendu R, et al. PEGylation orpolysialylation reduces FVIII binding to LRP resulting inprolonged half-life in murine models [abstract 3150]. Blood.2007;110.

20. Gregoriadis G, Jain S, Papaioannou I, Laing P. Improving thetherapeutic efficacy of peptides and proteins: a role for polysi-alic acids. Int J Pharm. 2005;300:125-130.

21. Powell JS. Liposomal approach towards the development of alonger-acting factor VIII. Haemophilia. 2007;13(Suppl 2):23-28.

22. Baru M, Carmel-Goren L, Barenholz Y, et al. Factor VIIIefficient and specific non-covalent binding to PEGylated lipo-somes enables prolongation of its circulation time and haemo-static efficacy. Thromb Haemost. 2005;93:1061-1068.

23. Powell JS, Nugent DJ, Harrison JA, et al. Safety and pharmaco-kinetics of a recombinant factor VIII with pegylated liposomesin severe hemophilia A. J Thromb Haemost. 2008;6:277-283.

24. Pan J, Liu T, Kim JY, et al. Enhanced efficacy of recombinantFVIII in noncovalent complex with PEGylated liposome inhemophilia A mice. Blood. 2009;114:2802-2811.

25. Polgar J, Matuskova J, Wagner DD. The P-selectin, tissuefactor, coagulation triad. J Thromb Haemost. 2005;3:1590-1596.

26. Barrow RT, Healey JF, Gailani D, Scandella D, Lollar P.Reduction of the antigenicity of factor VIII toward complexinhibitory antibody plasmas using multiply-substituted hybridhuman/porcine factor VIII molecules. Blood. 2000;95:564-568.

27. Parker ET, Healey JF, Barrow RT, Craddock HN, Lollar P.Reduction of the inhibitory antibody response to human factorVIII in hemophilia A mice by mutagenesis of the A2 domainB-cell epitope. Blood. 2004;104:704-710.

28. Sandberg H, Agerkvist I, Kannicht C, Ramstrom M, StenlundP, Dadaian M. Characteristics of the the first human cell linerecombinant human factor VIII (human-cl rhFVIII). J ThrombHaemost. 2007;5:PP-MO-574.

29. Running Deer J, Allison DS. High-level expression of proteinsin mammalian cells using transcription regulatory sequencesfrom the Chinese hamster EF-1alpha gene. Biotechnol Prog.2004;20:880-889.

30. Van Cott KE, Monahan PE, Nichols TC, Velander WH.Haemophilic factors produced by transgenic livestock: abun-dance that can enable alternative therapies worldwide. Haemo-philia. 2004;10(Suppl 4):70-76.

31. Niemann H, Kues WA. Transgenic farm animals: an update.Reprod Fertil Dev. 2007;19:762-770.

32. Gil GC, Velander WH, Van Cott KE. Analysis of the N-glycansof recombinant human factor IX purified from transgenic pigmilk. Glycobiology. 2008;18:526-539.

33. Chang J, Jin J, Lollar P, et al. Changing residue 338 in human

factor IX from arginine to alanine causes an increase incatalytic activity. J Biol Chem. 1998;273:12089-12094.

34. Schuettrumpf J, Herzog RW, Schlachterman A, Kaufhold A,Stafford DW, Arruda VR. Factor IX variants improve genetherapy efficacy for hemophilia B. Blood. 2005;105:2316-2323.

35. Simioni P, Tormene D, Tognin G, et al. X-linked thrombophiliawith a mutant factor IX (factor IX Padua). N Engl J Med.2009;361:1671-1675.

36. Hopfner KP, Brandstetter H, Karcher A, et al. Converting bloodcoagulation factor IXa into factor Xa: dramatic increase inamidolytic activity identifies important active site determinants.EMBO J. 1997;16:6626-6635.

37. Peters RT, Low SC, Kamphaus GD, et al. Prolonged activity offactor IX as a monomeric Fc fusion protein. Blood. 2010;115:2057-2064.

38. Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S.Genetic fusion to albumin improves the pharmacokineticproperties of factor IX. Thromb Haemost. 2009;102:634-644.

39. Kenet G, Lubetsky A, Luboshitz J, Martinowitz U. A newapproach to treatment of bleeding episodes in young hemo-philia patients: a single bolus megadose of recombinant acti-vated factor VII (NovoSeven). J Thromb Haemost. 2003;1:450-455.

40. Parameswaran R, Shapiro AD, Gill JC, Kessler CM. Doseeffect and efficacy of rFVIIa in the treatment of haemophiliapatients with inhibitors: analysis from the Hemophilia andThrombosis Research Society Registry. Haemophilia. 2005;11:100-106.

41. Lusher JM. Early treatment with recombinant factor VIIaresults in greater efficacy with less product. Eur J Haematol.1998;63(Suppl):7-10.

42. Ghosh S, Sen P, Pendurthi UR, Rao LV. Activity and regulationof glycoPEGylated factor VIIa analogs. J Thromb Haemost.2008;6:1525-1533.

43. Yatuv R, Dayan I, Carmel-Goren L, et al. Enhancement offactor VIIa haemostatic efficacy by formulation with PEGy-lated liposomes. Haemophilia. 2008;14:476-483.

44. Weimer T, Wormsbacher W, Kronthaler U, Lang W, LiebingU, Schulte S. Prolonged in-vivo half-life of factor VIIa byfusion to albumin. Thromb Haemost. 2008;99:659-667.

45. Bornaes C, Jensen RB, Ropke M, et al. Improved procoagulantand pharmacokinetic properties of two novel recombinanthuman factor VIIa variants prepared by directed molecularevolution and rational design. J Thromb Haemost. 2007;5:O-W-038.

46. Allen GA, Persson E, Campbell RA, Ezban M, Hedner U,Wolberg AS. A variant of recombinant factor VIIa withenhanced procoagulant and antifibrinolytic activities in an invitro model of hemophilia. Arterioscler Thromb Vasc Biol.2007;27:683-689.

47. Herzog RW, Verma D, Moghimi B, Singh HD, LoDuca PA,Daniell H. Oral delivery of bioencapsulated factor IX protectsfrom inhibitor formation and anaphylaxis in protein replace-ment therapy for hemophilia B [abstract 222]. Blood. 2009;114.

48. Kopecky EM, Greinstetter S, Pabinger I, Buchacher A, Ro-misch J, Jungbauer A. Combinatorial peptides directed toinhibitory antibodies against human blood clotting factor VIII.Thromb Haemost. 2005;94:933-941.

49. Liu T, Scallan CD, Broze GJ Jr, Patarroyo-White S, Pierce GF,Johnson KW. Improved coagulation in bleeding disorders bynon-anticoagulant sulfated polysaccharides (NASP). ThrombHaemost. 2006;95:68-76.

50. Prasad S, Lillicrap D, Labelle A, et al. Efficacy and safety of a

208 American Society of Hematology

new-class hemostatic drug candidate, AV513, in dogs withhemophilia A. Blood. 2008;111:672-679.

51. White RR, Sullenger BA, Rusconi CP. Developing aptamersinto therapeutics. J Clin Invest. 2000;106:929-934.

52. Waters EK, Kurz JC, Genga R, Nelson JA, McGinness KE,

Schaub RG. An aptamer antagonist of tissue factor pathwayinhibitor improves coagulation in hemophilia A and FVIIIantibody-treated plasma [abstract 544]. Blood. 2009;114.

53. Liu Y-L, Margaritis P, Khazi F, et al. Nonsense suppressionapproaches in treating hemophilia [abstract 512]. Blood. 2008;112.

Hematology 2010 209