title: Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy.
abstract: Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.
authors.affiliation: Department of Pediatrics and Pathology, University of Pennsylvania Medical Center and Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: Roland W
authors.initials: RW
authors.lastname: Herzog
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fields
authors.forename: Valder R
authors.initials: VR
authors.lastname: Arruda
authors.forename: Jeff O
authors.initials: JO
authors.lastname: Brubaker
authors.forename: Elina
authors.initials: E
authors.lastname: Armstrong
authors.forename: Darryl
authors.initials: D
authors.lastname: McClintock
authors.forename: Dwight A
authors.initials: DA
authors.lastname: Bellinger
authors.forename: Linda B
authors.initials: LB
authors.lastname: Couto
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Nichols
authors.forename: Katherine A
authors.initials: KA
authors.lastname: High
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D004285:Dogs; D005164:Factor IX / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D002836:Hemophilia B / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D007273:Injections, Intramuscular; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology*; D008934:Mitogens / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes
chemical_list: D000906:Antibodies; D016207:Cytokines; D007074:Immunoglobulin G; D008934:Mitogens; D005164:Factor IX