2003; baxter - Supplements - Haematologica

124R.W. Herzogengineering (knock out) technology and containeda large deletion including the promoterand the first three exons of the F.IX gene. 19 Consequently,the animals did not produce endogenousF.IX transcript or protein. Anti-mF.IXresponses were weaker and more delayed as comparedto anti-hF.IX formation. 7,18 Inhibitor titersof 5-20 BU persisted for >12 months. HemophiliaB mice also formed inhibitors after repeatedIV infusion of recombinant mF.IX protein. 18These data are consistent with clinical experience,in which an increased risk of inhibitor formationhas been observed following proteintreatment in hemophilia B patients with substantialloss of F.IX coding information (e.g. genedeletions 20,21 ). This experimental system was usefulin evaluating strategies for avoiding inhibitorformation in a setting with high risk of anti-F.IXsynthesis. We had hypothesized that transientimmune modulation around the time of vectoradministration combined with sustained transgeneexpression from AAV-mediated gene transfermay be sufficient for prolonged systemic F.IXexpression without inhibitor formation. To testthis hypothesis, hemophilia B mice receivedAAV-mF.IX vector by IM injection at a single timepoint combined with IV or intraperitoneal (IP)injection of an immune suppressive drug thatwas given at the time of vector administrationand intermittently at several subsequent timepoints. 18 Only decreased inhibitor titers and partialsuccess was observed with agents that specificallyblock co-stimulatory pathways that areimportant for activation of B- and T-lymphocytes(signal 2 blockers such as anti-CD40L andCTLA4-Ig fusion protein). However, it is conceivablethat optimization of dosing and scheduleof drug administration could improve thesuccess rate. Inhibitor formation was initiallyefficiently blocked by infusing FK506 every otherday for one month, resulting in substantialshortening of the aPTT due to transgene expression.FK506 inhibits an intracellular signalingpathway required for T-cell activation. Nonetheless,correction of the coagulation deficiency waslost once administration of the drug was stopped,and low titer inhibitors were measured by 3-4months. The most promising results wereobtained with cyclophosphamide, a DNA alkylatingagent with a cytotoxic effect on dividingcells such as proliferating B- and T-cell clones.Cyclophosphamide is commonly used in treatmentof autoimmune diseases and in someimmune tolerance regimens in hemophiliapatients who developed inhibitors during proteintherapy. 22,23 This compound was given biweeklyup to week 6 after vector administration at dosesup to 50 mg/kg; such doses do not cause significantchanges in white blood cell counts inmice. Hemophilia B mice treated with this combinationof vector injection and transientimmune suppression showed correction of theaPTT without anti-mF.IX formation for >1 year(duration of the experiment).Scale-up to hemophilia B dogs and sustainedF.IX expression in the context of aF.IX missense mutationHemophilia B dogs represent an excellent largeanimal model for scale-up studies in a novel geneTable 1. Incidence of inhibitor formation in gene therapy for hemophilia B using hemophilia B dogs and mice and an AAV-2 vectorencoding a species-specific transgene. The table lists the animal model/mutation, route of administration (IM vs. hepatic),number of animals that developed inhibitors in the respective study, and the referenced paper.Hemophilia B animal model Route of administration Vector dose Incidence of References(vg/kg) inhibitor formationUNC-Chapel Hill dogs (F.IX missense mutation) IM ≤3x10 12 0/7 Herzog et al. 1999, Chao et al. 1999UNC Chapel Hill dogs IM 8.5x10 12 Transient (1/1) Herzog et al. 1999UNC Chapel Hill dogs Portal vein (for hepatic gene transfer) ≤4x10 12 0/7 Snyder et al. 1999, Wang et al. 2000,Mount et al. 2002Auburn dogs (F.IX null mutation) IM 1x10 12 2/2 Herzog et al. 2001IM + cyclophosphamide 1x10 12 0/1Auburn dogs Mesenteric vein (for hepatic gene transfer) ≤3x10 12 (1x10 12 )* 1/3 (0/2) 1 Mount et al. 2002Knock out mice with F.IX gene deletion IM° ≤4x10 12 10/10 Fields et al. 2001IM + cyclophosphamide 4x10 12 2/6 +IM: intramuscular. *Two dogs treated with 1x10 12 vg/kg did not develop anti-F.IX, while one dog treated with 3x10 12 vg/kg showed inhibitor formation by week 5 after genetransfer. Results from this animal are complicated by the fact that the dog had additionally inherited pyruvate kinase deficiency (associated with chronic hemolytic anemia)and anti-phospholipid. °Sustained expression of canine or human F.IX without inhibitor formation has been achieved by hepatic AAV-mediated gene transfer in hemophilia Bmice on a C57BL/6 genetic background (Snyder et al. 1999, Wang et al. 1999). The risk of inhibitor formation in other strain backgrounds remains to be evaluated. + Those 2mice with inhibitor formation received 25 mg cyclophosphamide/kg, while 0/3 animals treated with 50 mg/kg showed inhibitor formation. vg: vector genomes as titered byquantitative dot blot hybridization.haematologica vol. 88(supplement n. 12):september 2003