2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia 117viral vectors. 10,11,13In vivo gene therapy for hemophilia A withMoMLV-based vectors requires direct transductionof cells that are either naturally proliferatingin vivo or that are induced to proliferate. High,stable levels of functional human FVIII could beachieved in newborn, FVIII-deficient mice injectedintravenously with high-titer VSV-G pseudotypedonco-retroviral vectors containing the B-domain deleted FVIII cDNA. 21 High-levels (>200mU FVIII/mL) of functional human FVIII productioncould be detected in about 50% of theanimals, some of which expressed physiologic orhigher levels (up to 12500 mU/mL). Most highexpresserssurvived an otherwise lethal tail-clipping,unequivocally demonstrating phenotypiccorrection of the bleeding disorder. 21 To ourknowledge, this is the first demonstration thathemophilia A can be cured by gene therapy in aclinically relevant animal model. Efficient genetransfer occurred into liver, spleen and lungs butnot in other organs including testes, with predominantFVIII mRNA expression in the liver. 21Long-term correction of hemophilia has beenachieved in about 50% of the FVIII-deficient animals,whereas the other half was not corrected. 21This differential response is due to a specificimmune mechanism since the latter mice developedinhibitory antibodies to human FVIII (rangingbetween 7 and 350 Bethesda units/mL),whereas none of the corrected mice did. 21 Furthermore,in the transient FVIII expressing mice,induction of antibodies was causally related to thedecrease in FVIII expression. 21 Finally, in theabsence of a specific immune response in FVIII-KO/SCID mice, human FVIII expression could bedetected in all recipient animals. 21 The differentialresponse is reminiscent of the lack of therapeuticefficacy in 10-20% of the hemophilia A patientsthat develop inhibitory antibodies to FVIII followingprotein replacement therapy. The higher proportionof non-expressers in FVIII-deficient micecould be due to the inherently higher immunogenicityof xenogenic human FVIII in non-humanspecies. Since we observed a lower level of FVIIIgene transfer in the transient and non-expressorrecipient mice compared to the long-term expressors,a mechanism whereby transduced cells wereeliminated by a cellular immune response cannotbe ruled out. It is therefore likely that the FVIIItransducedhepatocytes are recognized and eliminatedby FVIII-specific CTLs.The cause of the heterogeneous antibodyresponse and FVIII levels among the differentrecipients is not clear. Since the FVIII-deficientmouse is not an inbred strain, genetic differencesmay have contributed to this variability (Figure1). Alternatively, high levels of FVIII may havebeen required to induce immune tolerance toFVIII in the FVIII-deficient mice and preventinduction of inhibitory antibodies (Figure 2).This would be reminiscent of tolerance-inductionby repeatedly injecting FVIII proteins at highconcentrations in hemophilia A patients. 23 However,expression of a xenoprotein in neonatalmice is quite different from giving a species-specificprotein to a subject with a mature immunesystem. In particular, neonatal mice can becometolerant to soluble xenoantigens because neonatalsplenic B-cells are tolerance susceptible forseveral days after birth during which B-cell clonesmay be expanding. The proportion of tolerancesusceptiblesplenic B-cells decreases to less than10% after the first week after birth. It is indeedpossible that exposure of neonatal FVIII-deficientanimals to sufficiently high levels of human FVIIIprotein following FVIII gene transfer resulted inthe induction of tolerance to human FVIII, atleast in some of the recipients (Figure 2). Thishypothesis would be consistent with the observedcorrelation between high FVIII gene transfer andexpression and lack of inhibitory antibodies inthese mice and with the induction of tolerance ofFVIII in neonatal mice receiving high doses ofhuman FVIII. 24 Conversely, when gene transferefficiency was low, FVIII expression may havebeen below a critical threshold during the neonatalphase to induce tolerance, resulting in theinduction of inhibitory antibodies and/or CTLs(Figure 2). The potential immunogenicity ofhuman FVIII in some of the recipient FVIII-deficientmice is consistent with previous reportsindicating that repeated administration ofhuman FVIII protein in adult FVIII-deficient miceled to the induction of inhibitory antibodies. 25The efficient hepatic gene transfer in neonatalmice could be due primarily to the higher hepatocyteturn-over rate in newborn versus adultanimals. In adult mice most hepatocytes arerefractory to gene transfer using onco-retroviralvectors since disassembly of the nuclear membraneduring cell division is required to allow theonco-retroviral preintegration complex to enterthe nucleus. 26 Hence, gene delivery strategiesthat could overcome the requirement for hepaticcell division and be used in adults, would bedesirable.Lentiviral vectorsIn order to overcome the need for active celldivision, retroviral vectors derived from lentiviruseshave been developed, with HIV as the prototype.27 The lentiviral life cycle differs from thatof onco-retroviruses in that the preintegrationcomplex is readily transported into the nucleus,thus enabling efficient transduction of nondividingcells, including neurons. Gene transferwith lentiviral vectors in dividing cells is moreefficient than with MoMLV-based retroviral vectors.28 While present efforts are aimed at the generationof stable packaging cell lines 29 and safe 30and clinically acceptable vectors, the attributesof the lentiviral vector system may be well suitedfor the treatment of hemophilia A. The latestgeneration lentiviral vector packaging systemrelies on a self-inactivating HIV-derived vectorbackbone which does not contain any HIV-1genes. Instead, only the cis-acting elements nec-haematologica vol. 88(supplement n. 12):september 2003