2003; baxter - Supplements - Haematologica

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116T. VandenDriessche et al.• the vector should have the potential for longterm,preferably life-long FVIII gene expression;• the vector should be capable of efficient andpreferably selective gene transfer into theappropriate target cells, particularly hepatocytes,which normally express FVIII;• lack of de novo expression of viral genes in thetransduced target cells is required to avoidimmune rejection of transduced target cells;• the vector should not trigger any toxic oradverse side-effects;• inadvertent germline gene transfer should notoccur following somatic gene therapy;• vector preparations should be devoid of helpervirus or replication-competent virus;• the vector should not trigger inflammatoryimmune responses;• transduction in antigen-presenting cells(APCs) should be avoided to decrease the likelihoodof anti-FVIII antibodies;• vector administration should preferably benon-invasive;• the vector should not integrate into the targetcell genome to avoid insertional mutagenesisof cellular genes, particularly tumor suppressorgenes.There are currently no viral vectors that meetall these requirements. Nevertheless, there are anumber of promising vectors that can be usedfor hemophilia A gene therapy, each with theirown advantages and limitations, in particular:onco-retroviral and lentiviral vectors, highcapacityadenoviral vectors (HC-Ad) and adenoassociatedviral vectors (AAV). These viral vectorsare devoid of viral genes and consequentlycannot replicate in the transduced cells andpropagate in vivo, in contrast to the viruses theyhave been derived from. Furthermore, these replication-deficientvectors cannot express any viralantigens de novo in the transduced cells, herebysafeguarding the transduced cells from possibleimmune rejection by cytotoxic T-lymphocytes(CTL). Nevertheless, FVIII gene transfer mayresult in the presentation of endogenously synthesizedFVIII–derived peptides in the context ofMHC class I molecules and trigger CTL responsesthat could eliminate the FVIII-engineered targetcells. In addition, the viral vector particlesthemselves may induce a humoral immuneresponse that would lead to the formation ofneutralizing antibodies directed at the vector capsidor envelope structures. These antibodiescould potentially interfere with viral transduction,if the patient were to receive a subsequentviral vector challenge.Our work in particular, focuses on the use ofonco-retroviral, lentiviral and HC-Ad vectors forhemophilia A gene therapy which will be summarizedbelow.MoMLV-based retroviral vectorsMoloney murine leukemia virus (MoMLV)-based retroviral vectors can potentially give rise tostable gene expression by virtue of their stablechromosomal integration and lack of viral geneexpression. 4,5 Their integration allows passage ofthe transgene to all progeny cells. However, celldivision is required for MoMLV transduction andintegration, thereby limiting retroviral-mediatedgene therapy to actively dividing target cells. Genetherapy for hemophilia with MoMLV-based vectorsrequires either direct in vivo transduction ofcells that are naturally proliferating or induced toproliferate using growth factors, or ex vivo expansionand transduction of target cells followed bytheir readministration. Stable packaging cell linesare available that facilitate the large scale productionand characterization of recombinantvirus for potential use in clinical trials. No acutetoxicity or adverse effects have been reported inmore than 100 clinical trials with retroviral vectors.6 Furthermore, retroviral vector preparationsdevoid of replication-competent retroviruses(RCR) have not been shown to cause malignanttransformation in animals or in patients. 7,8 Rareclonal transformation events have only beenobserved in severely immunocompromized primatesreceiving a high dose of contaminatingreplication competent retroviruses (RCR). 9 Retroviralvector-mediated gene transfer is thereforerelatively safe for gene therapy of hemophilia.Retroviral vectors containing the FVIII geneshave been developed. However, first-generationFVIII-retroviral vectors were characterized by lowtiters and low FVIII expression levels which initiallyhampered their usefulness for gene therapy.The presence of an intact FVIII cDNA intoMoMLV retroviral vectors resulted in a 100 to1000-fold decrease in vector titer in comparisonwith the parental vector or vectors carrying othersimilarly sized cDNAs. 10-16 The inhibition ofFVIII expression and the low viral titer were dueto the presence of sequences within the FVIIIcDNA that inhibited RNA accumulation byinterfering with transcriptional initiation orelongation (see above). 12,14 Conservative mutagenesisof the entire 1.2 kb INS element failed toincrease FVIII expression or vector titer. 13 However,we and others showed that this impedimentcould be circumvented by including an intronupstream of the FVIII cDNA which led to a significantincrease in FVIII expression and restoredretroviral titer to normal levels (105 transducingunits/mL) (TU/mL). 13,17 Although otherpost-transcriptional mechanisms may also beinvolved 18, this design was based on the observationthat splicing can improve mRNA accumulationby increasing transcriptional efficiency,stabilizing RNA and/or increasing transport fromthe nucleus to the cytoplasm. 19,20 These intronbasedFVIII onco-retroviral vectors could be concentratedto even higher titers (109-1010TU/mL) following pseudotyping with the vesicularstomatitis virus G-protein (VSV-G). 21,22 Thisconstitutes a 107-fold improvement comparedto non-concentrated early generation FVIII retro-haematologica vol. 88(supplement n. 12):september <strong>2003</strong>
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 <strong>2003</strong>
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