2003; baxter - Supplements - Haematologica

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[Gene Therapy]review paperImmunologic sequelae andpotential for inhibitor developmentin adeno-associated viralgene therapy for hemophilia Bhaematologica <strong>2003</strong>; 88(suppl. n. 12):122-126http://www.haematologica.org/free/immunotolerance2001.pdfROLAND W. HERZOGDept. Pediatrics, The Children’s Hospital of Philadelphia andUniversity of Pennsylvania Medical Center, Philadelphia, PA,USAFormation of inhibitory antibodies against a coagulationfactor antigen in a gene therapy setting is currentlybeing characterized for adeno-associated viral(AAV) gene transfer in animal models of hemophilia.Hemophilia B dogs of two different colonies witha missense or null mutation in the factor IX (F.IX)gene as well as hemophilia B mice with a large F.IXgene deletion were subjected to AAV-mediatedtransfer of species-specific F.IX transgenes to skeletalmuscle or liver. Treated animals were analyzed fortransgene expression and F.IX-specific B- and T-lymphocyteresponses. Particular emphasis in thisanalysis was given to the role of the underlying mutation,the route of vector administration, and immunemodulation. All of these were found to have a significantimpact on the risk of anti-F.IX formation,which represents a T helper cell-dependent process.These studies provide the groundwork for a betterunderstanding of F.IX-specific CD4 + T-cell activationin AAV-mediated gene transfer that may lead to neutralizinganti-F.IX IgG formation, and thus help definegene transfer protocols with minimal risk for suchimmune responses.Correspondence: Roland W. Herzog, The Children’s Hospital ofPhiladelphia, Abramson Research Center 310, 34th St. andCivic Center Blvd., Philadelphia, PA 19104, USA. Phone: international+1.215.590-4907. Fax: international +1.215.590-3660. E-mail: rwherzog@mail.med.upenn.eduConventional treatment of the inheritedbleeding disorder hemophilia is based onintravenous (IV) infusion of functionalcoagulation factor VIII (F.III, hemophilia A) orfactor IX (F.IX, hemophilia B). Formation ofinhibitory anti-F.VIII or anti-F.IX currently representsthe most serious complication of this proteinreplacement therapy. 1,2 More recently, genetherapy strategies have been introduced into theclinic in the form of phase I clinical trials. 3-6While inhibitor formation is well documented inprotein therapy with a prevalence of 3-4% inhemophilia B and 20-30% in hemophilia A, therisk of such an immune response in gene-basedtreatment is only beginning to be defined. A historyof inhibitor formation has been an exclusioncriterion for enrollment of patients in genetherapy trials, and inhibitors have indeed notbeen observed in these trials. However, it is notclear yet whether the risks and characteristics ofinhibitor formation are similar or different ingene therapy as compared with protein therapy.Moreover, a particular combination of genetransfer vector, DNA construct, and target tissue/routeof administration is likely to produceits own unique set of signals to the immune system.7 Therefore, the risk and characteristics ofimmune responses caused by gene transfer haveto be defined for each particular protocol. Here,animal experiments designed to define anti-F.IXresponses in adeno-associated virus (AAV)-mediatedgene transfer are summarized.AAV vectors have been shown to transfer a F.IXtransgene efficiently to skeletal muscle fibers orhepatocytes following intramuscular (IM) injectionof the vector or infusion into the portal circulation,resulting in sustained expression andpartial to complete correction of the coagulationdeficiency in hemophilia B mice and dogs. 8-12AAV vectors are based on a single-stranded DNAvirus with a small (4.7-kb) genome flanked byinverted terminal repeats (ITRs). The wild-typevirus is a non-pathogenic, replication defectivemember of the parvovirus family. 13 The vectordoes not contain viral coding sequences (whichhave been replaced by a F.IX expression cassette)and can be produced in the absence of a helpervirus. 14 A phase I trial of IM administration ofthe vector in patients with severe disease (
IV International Workshop on Immune Tolerance in Hemophilia 123Biology of lymphocyte responses in muscledirectedgene transfer – murine models ofgene transferIn early preclinical experiments, we observedthat IM injection of an AAV vector or a first generationadenoviral vector, both expressinghuman F.IX (hF.IX) from the strong viral CMVenhancer/promoter, resulted in rapid inductionof neutralizing anti-hF.IX IgG in immunocompetentmice. 7,15 While these results may not besurprising given the use of a non-species-specifictransgene, this experimental system was usefulin delineating lymphocyte subsets involved inanti-F.IX formation following muscle-directedgene transfer (as summarized in Figure 1). AntihF.IXIgG formation occurred by day 14 after vectoradministration resulting in neutralization ofsystemic hF.IX expression in the context of eithervector system. However, a series of B- and T-cellassays revealed substantial differences in the typeof immune responses for the two vectors. Adenoviralgene transfer resulted in activation of cytotoxicT-lymphocytes specific for hF.IX expressingcells as well as for adenoviral antigens. Musclefibers expressing the transgene were targeted byinflammatory infiltrates including CD8 + (CTL)lymphocytes, thereby causing destruction oftransduced fibers and elimination of transgeneexpression. These results highlight an importantdifference between protein and gene therapy,namely that cells expressing the coagulation factorafter gene transfer are capable of presentingantigen-derived peptides by their MHC class Imolecules. If this occurs in the target cells suchas muscle fibers, these cells become potential targetsfor CTL responses. If MHC class I presentationoccurs in professional antigen-presentingcells (APCs), such as dendritic cells, this maycause activation of CTLs. In the case of adenoviralgene transfer, MHC class II-resticted T helpercells (CD4 + T-cells) of Th1 and Th2 subsets wereactivated resulting in formation of primarilyIgG2a (the murine equivalent of human IgG1)anti-hF.IX. In contrast, AAV-transduced muscletissue continued to express the transgene even inthe presence of antibody formation without evidenceof inflammation or activation of hF.IXspecificCTL. Data from Jooss et al. suggest thatAAV vectors transduce APCs (dendritic cells) relativelyinefficiently, and that this accounts forthe lack of CTL activation against the transgeneproduct. 16,17 Activation of T helper cells by AAVvector encoded hF.IX showed a stronger biastoward the Th2 subset resulting in predominantsynthesis of IgG1 (the murine equivalent ofhuman IgG4). 7,18 These characteristics suggestthat inhibitor formation in AAV-mediated genetherapy is mechanistically similar to antibodyformation in the context of protein infusion, andis probably not similar to the immune responsetypically seen in the context of a viral infection.Immune modulation may prevent inhibitor formationin the context of a F.IX gene deletionIn order to address the risk of inhibitor formationin AAV-mediated gene transfer, we constructedvectors that express species-specifictransgenes. Hemophilia B mice that received anAAV vector expressing murine F.IX (mF.IX)developed inhibitory anti-mF.IX within 4-8weeks after IM injection of the vector. 18 Thesemice had been generated by means of geneticFigure 1. Model for lymphocyte activation resultingin anti-F.IX formation after AAV-mediated gene transferto skeletal muscle. Note that T-cell activation andantibody (inhibitor) formation is more likely to occurin the context of a F.IX null mutation (e.g. gene deletion,early stop codon, etc.) than in the context of amissense mutation. This diagram summarizes resultsobtained in murine models of gene transfer. AAVmediatedgene transfer to skeletal muscle results insecretion of F.IX from transduced myofibers. Uponuptake of the F.IX antigen by professional APCs suchas dendritic cells, peptide fragments are displayed byMHC class II molecules, which may lead to activationof CD4 + T helper cells. In the context of AAV-mediatedgene transfer to skeletal muscle, these are primarilyof the Th2 subset, thus promoting IgG1 secretionby B cells, while limited activation of Th1 cellsmay result in some synthesis of IgG2a or IgG2b. Activationof F.IX-specific, MHC class I restricted CD8 +cytotoxic T lymphocytes was not observed.haematologica vol. 88(supplement n. 12):september <strong>2003</strong>
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