title: Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.
abstract: Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).
authors.affiliation: 1 Gene Therapy Center, University of North Carolina , Chapel Hill, NC 27599.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Monahan
authors.forename: Junjiang
authors.initials: J
authors.lastname: Sun
authors.forename: Tong
authors.initials: T
authors.lastname: Gui
authors.forename: Genlin
authors.initials: G
authors.lastname: Hu
authors.forename: William B
authors.initials: WB
authors.lastname: Hannah
authors.forename: David G
authors.initials: DG
authors.lastname: Wichlan
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Wu
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Grieger
authors.forename: Chengwen
authors.initials: C
authors.lastname: Li
authors.forename: Thipparat
authors.initials: T
authors.lastname: Suwanmanee
authors.forename: Darrel W
authors.initials: DW
authors.lastname: Stafford
authors.forename: Carmen J
authors.initials: CJ
authors.lastname: Booth
authors.forename: Jade J
authors.initials: JJ
authors.lastname: Samulski
authors.forename: Tal
authors.initials: T
authors.lastname: Kafri
authors.forename: Scott W J
authors.initials: SW
authors.lastname: McPhee
authors.forename: R Jude
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000032:analysis; D002213:Capsid / Q000737:chemistry / Q000276:immunology; D002986:Clinical Trials as Topic; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism / Q000493:pharmacokinetics; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D002836:Hemophilia B / Q000097:blood / Q000235:genetics / Q000503:physiopathology / Q000628:therapy*; D006470:Hemorrhage / Q000097:blood / Q000235:genetics / Q000503:physiopathology / Q000517:prevention & control*; D006801:Humans; D008099:Liver / Q000276:immunology / Q000821:virology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D013623:Tail; D014018:Tissue Distribution; D014771:Virion / Q000235:genetics
chemical_list: D057134:Antibodies, Neutralizing; D011994:Recombinant Proteins; D005164:Factor IX