2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia 125therapy protocol and for safety studies such asassessment of the risk of inhibitor formation.There are two characterized colonies with no circulatingF.IX antigen. The dogs at UNC-ChapelHill have a missense mutation in the F.IX gene.Modeling studies predict that the mutant F.IXmolecule does not fold correctly and therefore islikely destroyed intracellularly. 24,25 IM administrationof an AAV vector (expressing canine F.IX)at multiple sites at a single time point directedsustained systemic expression of vector dosedependentlevels of biologically active canine F.IX(>4 years) without vector-related toxicity. 8,12 Furthermore,these experiments illustrated the simplicityand safety of this non-invasive method ofgene transfer. Expression was demonstrated byELISA and by partial correction of the whole bloodclotting time (WBCT) and, at higher vector doses,of the aPTT. Dose escalation showed that~5×10 12 -1×10 13 vector genomes [vg]/kg wererequired for therapeutic levels of expression (>1%of normal). At the reported vector doses (up to8.5×10 12 vg/kg), inhibitors were either absent(4/5 dogs) or transient (spontaneous remissionwithin 2 months, 1/5 animals). 8 Inhibitor formationmay be observed at higher vector doses, inparticular at high doses per site of injection (Herzoget al., Hum Gen Ther, in press). Thus, althoughthe risk of inhibitor formation in the context of amissense mutation is greatly reduced, it may benecessary to include transient immune suppressionat high vector doses in muscle-directed genetransfer.Similar success in the UNC hemophilia B dogshas been reported for AAV-mediated hepatic genetransfer following portal vein infusion of the vector,albeit at lower vector doses. 9,11,26 No anticF.IXwas reported in these studies, and expressionwas sustained for at least several years. Similarto IM injections, the AAV vector does notcause heptotoxicity, and vector administration isgenerally well tolerated by the animals. Recentprogress in vector delivery techniques and the useof alternate AAV serotypes (all data discussed herewere obtained with AAV-2 vector) with greatertransduction efficiency of skeletal muscle are likelyto reduce if not eliminate the dose advantageof the liver route over the muscle route (refs. no.27,28 and Arruda et al., unpublished results). However,the risk of inhibitor formation in theseapproaches remains to be defined. In particular,one has to consider that levels of locally producedF.IX may be much higher following gene transferthat is the case for IM injection of the AAV-2 vector,which may significantly influence immuneresponses.Sustained F.IX expression in hemophilia Bdogs with a F.IX null mutation followinghepatic gene transferAnalogous to results in the hemophilia B mice,dogs with a F.IX null mutation (an early stopcodon and unstable mRNA, hemophilia B dogcolony at Auburn University), IM administrationof an AAV vector expressing cF.IX induced persistenthigh titer inhibitors. 29,30 This immuneresponse was observed at vector doses that did notcause inhibitor formation but rather resulted insustained cF.IX expression in the model characterizedby a missense mutation. Inhibitor formationwas not observed in a null mutation dog thatadditionally received cyclophosphamide asdescribed above for treatment of murine hemophiliaB. Inhibitor formation was also observed ina null mutation dog that received IV infusion ofpurified plasma-derived cF.IX. 29 Surprisingly, 2animals of this colony that were treated by hepaticgene transfer did not form anti-cF.IX and continueto express cF.IX at levels of 5-12% of normalwith complete or substantial correction ofthe WBCT and of the aPTT. 26 This was achieved bymesenteric vein infusion of an AAV vector containinga strong hepatocyte-specific enhancer/promotercombination. Similar results hadbeen obtained previously in hemophilia B miceusing hepatic gene transfer. However, these micehave been bred on a C57BL/6 genetic background,which is more promiscuous for transgeneexpression without immune responses followingthis route of administration than otherstrains of inbred mice, thus making definite statementsabout the risk of inhibitor formation moredifficult. 31,32 Nonetheless, other data showed thatliver-directed AAV-mediated gene transfer may becharacterized by a reduced risk of anti-F.IX formationeven in strains other than C57BL/6. 33Our results from the F.IX null mutation dogsillustrate the lower likelihood of inhibitor formationby AAV-mediated liver-directed gene therapyin a large animal model of hemophilia B as comparedto other treatment modalities. 26 Theimmunologic mechanism responsible for theseobservations is currently under active investigationin our laboratory. Table 1 summarizes theincidence of inhibitor formation as reported inseveral studies using muscle- or liver-directedAAV-mediated gene transfer of a species-specificF.IX gene in animal models of hemophilia B.In summary, AAV-mediated gene transfer ischaracterized by a reduced potential for cellularimmune responses and inflammation but mayresult in T helper cell-dependent antibody formationto the F.IX transgene product. The datastrongly suggest a crucial role of the gene transferrecipient’s underlying F.IX mutation on therisk of inhibitor formation. In muscle-directedgene transfer, inhibitor formation is observed inanimals with substantial loss of F.IX codinginformation such as a large gene deletion or anearly stop codon, whereas sustained systemicexpression without or with only transientinhibitor formation was achieved in the contextof a crm- missense mutation. These differencesin the incidence of antibody formation may beexplained by lack of expression of potentiallyimmunodominant T-cell epitopes during devel-haematologica vol. 88(supplement n. 12):september 2003