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).

Systemic delivery of factor IX messenger RNA for protein replacement therapy

“…Six early-stage clinical trials are currently underway, testing viral vector gene therapy in patients with hemophilia (7). However, AAV vectors can cause hepatitis and immune-mediated hepatocyte destruction in human patients (17,24). Viral vectors also carry a small, albeit definite risk of insertional mutagenesis (25).…”

“…Two catalytically enhanced FIX variants, R338A-hFIX and R338L-hFIX, have been shown to induce a 3-fold and 6-to 10-fold increase in clotting activity, respectively, compared with WT hFIX (12,17). We explored whether these hyperfunctional variants could further extend and improve the therapeutic efficiency of our LUNAR-mRNA formulation.…”

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice

“…These data are agreement with previous short-term gene therapy studies in mice. 46,47 The main determinant of death rates was the supraphysiologic levels of either FIX forms that occur in a time-dependent manner, ie, 8 to 10 months after vector injection, thus providing more stringent risk assessment than short-term 46,47 or long-term studies expressing FIX at relatively lower levels. 48 These data suggest that the safety of continuous expression of FIX-Padua does not differ from the FIX-WT expression and, thus FIX-Padua is not inherently more thrombogenic per se, but at extremely high levels behaves in a similar fashion as FIX-WT.…”

Novel approaches to hemophilia therapy: successes and challenges

“…63 The use of FIX-Padua in preclinical studies, including inhibitorprone HB canine model, showed excellent safety profile without increased immunogenicity. [64][65][66][67] Two clinical studies have used this variant. The study by Spark Therapeutics is using AAV-FIX-Padua at a single dose of 5 3 10 11 vg/kg (n 5 10) with FIX levels ;30%; all subjects have stopped prophylaxis for $12 weeks (ongoing).…”