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).

FIG. 1.

Expression in plasma of wild-type factor IX and FIXR338L from otherwise identical liver-directed expression cassettes in FIX^–/– mice. Codon optimization of the factor IX cDNA used in each vector was identical. The Leu-338 variant results from a single nucleotide substitution in the codon-optimized FIX cDNA. The number of C57BL/6 FIX^–/– mice that received intraportal venous vector was 13 in the scAAV8.WTFIX treatment group (WTFIX) and 10 in the scAAV8.FIXR338L group. (A) Total human factor IX protein expressed. (B) Specific activity of circulating factor IX. The normal specific activity of recombinant factor IX is ∼200 U/mg. (C) Circulating factor IX activity as assayed in a one-stage clotting assay (factor IX-specific activated partial thromboplastin time) demonstrated sustained FIXR338L expression at supraphysiological levels for most of the 40 weeks of observation. (D) At the end of the follow-up, plasma factor IX activity was measured and then transection of the tail tip was performed at a uniform circumference of 1.5 mm. After this bleeding challenge, hemophilia B mice (FIX^–/–) experienced severe bleeding, which resulted in diminished activity or distress requiring euthanasia of 8 of 14 mice. All mice that had received either factor IX gene therapy vector as a single injection 40 weeks earlier survived the tail transection and remained active, similar to the hemostatically normal mice (WT) that underwent the same bleeding challenge.

FIG. 2.

Dose response after scAAV8.FIXR338L vector was delivered by peripheral (tail) vein. FIX^–/– mice (six to eight per group) received the indicated doses of scAAV8.FIXR338L as a single tail vein injection to model the peripheral venous route of dosing planned for human clinical application. So as to allow comparison of tail vein (TV) and portal vein (PV) administration using the same vector dosing examined in Fig. 1, separate groups of age-, weight-, and sex-matched FIX^–/– mice received 4.0×10¹¹ VG/kg by PV, with factor IX expression displayed by the green triangles. (A) Circulating factor IX protein. (B) Circulating factor IX activity. At any given dose, the specific activity of plasma factor IX (factor IX activity/factor IX antigen, expressed in units/mg protein) was five to eight times normal, with factor IX expression persisting over months. Plasma was examined at each time point for the development of antibodies directed against factor IX. No factor IX inhibitors (as measured by Bethesda assay) or anti-FIX IgG (as measured by ELISA) was detected in any animal throughout the observation. (C) Dose-dependent protection from hemorrhage in the tail transection bleeding challenge. At 32 weeks after vector delivery, all mice from each dose group had plasma factor IX activity determined (as recorded on the x axis below each column) and then underwent a tail transection bleeding challenge and the amount of blood loss in 10 min after wounding was collected and quantitated by measuring the optical density (OD) of the shed hemoglobin.

FIG. 3.

Dose-dependent protection from the development of hemarthrosis and bleeding-induced joint pathology is improved by the substitution of FIXR338L. FIX^–/– mice were treated via the tail vein with scAAV8.FIXR338L (squares) or the corresponding vector expressing WTFIX (×). Four weeks after vector delivery, the mice received a unilateral right hindlimb knee joint needle puncture to induce hemarthrosis. Two weeks after injury, knee joints were collected and scored for synovitis, using the Valentino mouse synovitis score. (A) At both the lower dose of 2×10¹⁰ VG/kg (left) and the higher dose of 2×10¹¹ VG/kg (right), the expression from scAAV8.FIXR338L (squares) results in higher plasma levels of factor IX activity when compared with the WTFIX vector (×). The average synovitis score of 4.4 was previously reported for a large cohort of untreated FIX^–/– mice that underwent this bleeding challenge, and this value is represented by the dashed line. A large group of strain-matched hemostatically normal C57BL/6 mice exposed to this bleeding challenge developed a mean synovitis score of 0.14, with a range of 0–2.0 (demarcated by the shaded rectangle). (B) Representative histopathology of knee joints 2 weeks after single knee joint puncture injury. The area bounded by the black rectangle in each top panel (original magnification, ×40) is shown at an original magnification of ×200 in the immediately underlying bottom panel. Normal joint anatomy was preserved in hemostatically normal C57BL/6 wild-type control mice (left) by an open joint space (*), thin flat synovial epithelial cells (arrowhead) supported by an unremarkable fatty and fibrovascular stroma, and subsynovial fat pad (**) composed predominantly of scant small blood vessels (arrows) (bottom left). In contrast, FIX^–/– mice given scAAV8.FIXR338L (2×10¹⁰ VG/kg) that expressed FIXR338L (middle) demonstrated minimal to mild changes of synovitis (synovitis score, ∼2/10); an increase in the size and number of blood vessels (neovascularization) (arrows) in the subsynovial fat pad (**) is present; however, there is little to no increase in synovial epithelial cell thickness (arrowhead). Mice given WTFIX vector (2×10¹⁰ VG/kg) had severe synovitis (synovitis score, >4/10) (right) with a marked increase in synovial thickness in concert with effacement and replacement of the subsynovial fat (**) pad by proliferation of synovial epithelial cells and granulation tissue, admixed with inflammatory cells. As observed in clinically severe hemarthrosis, we see pannus formation (P) when these proliferating cells grow over the articular cartilage, resulting in erosion (*) in combination with marked neovascularization (arrows), and foci of fibrinonecrotic debris (D). Hematoxylin–eosin. Scale bars, 200 μm.

FIG. 4.

Manufacture of GLP vector produced from scalable suspension cell culture eliminates most contaminating empty AAV capsids. Left: Transmission electron micrograph of the clinical vector used in the dose–response and toxicity/biodistribution studies, demonstrating a favorable ratio of therapeutic particles to defective interfering particles (empty capsids make up less than 10% of vector stock). Black circles are superimposed to show examples of the relatively hollow appearance that is typical of empty vector capsids. Right: Silver stain of clinical vector demonstrates only three proteins, corresponding to the AAV structural proteins Vp1, Vp2, and Vp3, present at the expected ratio of ∼1:1:9. Lanes 2 and 4 contain identical vector.