Preclinical evaluation of a next-generation, subcutaneously administered, coagulation factor IX variant, dalcinonacog alfa.

INTRODUCTION: The rapid clearance of factor IX necessitates frequent intravenous administrations to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration has historically been limited by low bioavailability and potency. Dalcinonacog alfa was developed using a rational design approach to be a subcutaneously administered, next-generation coagulation prophylactic factor IX therapy. AIM: This study aimed to investigate the pharmacokinetic, pharmacodynamic, and safety profile of dalcinonacog alfa administered subcutaneously in hemophilia B dogs. METHODS: Two hemophilia B dogs received single-dose daily subcutaneous dalcinonacog alfa injections for six days. Factor IX antigen and activity, whole blood clotting time, and activated partial thromboplastin time were measured at various time points. Additionally, safety assessments for clinical adverse events and evaluations of laboratory test results were conducted. RESULTS: There was an increase in plasma factor IX antigen with daily subcutaneous dalcinonacog alfa. Bioavailability of subcutaneous dalcinonacog alfa was 10.3% in hemophilia B dogs. Daily subcutaneous dosing of dalcinonacog alfa demonstrated the effects of bioavailability, time to maximal concentration, and half-life by reaching a steady-state activity sufficient to correct severe hemophilia to normal, after four days. CONCLUSION: The increased potency of dalcinonacog alfa facilitated the initiation and completion of the Phase 1/2 subcutaneous dosing study in individuals with hemophilia B.

Systemic delivery of factor IX messenger RNA for protein replacement therapy.

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.

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.

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).

PEGylated therapeutic proteins for haemophilia treatment: a review for haemophilia caregivers.

PEGylation is the technology involving the covalent attachment of polyethylene glycol (PEG) to a protein-, peptide- or small-molecule drug to improve their pharmacokinetic, pharmacodynamic and immunological profiles, and thus, enhance the therapeutic effect. Today, PEGylation of proteins is a well-established technology and is being used in the treatment of a variety of clinical disorders. Several PEGylated coagulation proteins for haemophilia A and B are under development with the goal of prolonging the circulation half-life of factor VIII (FVIII) or factor IX. The prolongation of half-life, resulting in less frequent injections can provide significant benefits in improving the quality of life of subjects with haemophilia and improvement in adherence to treatment. A review of published literature on PEGylated therapeutic products currently approved for human use and a discussion of a PEGylated recombinant FVIII molecule (BAY 94-9027, Bayer HealthCare, Berkeley, CA, USA) currently being investigated in the pivotal clinical trial prior to registration is provided. Available safety information of PEGylated proteins containing high molecular weight PEG does not indicate any safety concerns to date, following long-term (chronic) use in animal models or patients. Chronic use of currently available PEGylated products has been shown to be safe, paving the way for chronic use of PEGylated coagulation products in persons with haemophilia.

Prolonged activity of factor IX as a monomeric Fc fusion protein.

Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.

Evaluation of recombinant human factor IX: pharmacokinetic studies in the rat and the dog.

The pharmacokinetics of intravenously administered recombinant human factor IX (rhFIX) were studied in Sprague-Dawley rats and Beagle dogs. Rats received rhFIX (50 IU/kg once daily) for 28 days, and the plasma half-life was 5 h. Anti-Human Factor IX serum antibody levels were found in only 1 of 12 rats. The pharmacokinetic profiles of rhFIX or Mononine, a purified human plasma-derived factor IX, after single 100 IU/kg i.v. doses in dogs, were similar. Peak plasma concentrations of rhFIX and Mononine were 4-5 micrograms/ml. The mean plasma half-lives were 13.2 +/- 1.6 h for rhFIX and 13.3 +/- 1.6 h for Mononine. Dogs also received rhFIX (40 IU/kg i.v., daily) for 28 days or Mononine (40 IU/kg i.v. daily) for 14 days. Anti-human Factor IX serum antibody levels were determined for each compound. Pharmacokinetic half-lives decreased in these treated dogs which developed antihuman Factor IX antibodies. The antibody responses in 28 day rhFIX (40 IU/kg) dogs were similar to 14 day Mononine (40 IU/kg) dogs.

Subcutaneous injection of factor IX for the treatment of haemophilia B.

Haemophilia B patients are normally treated, either prophylactically or in response to bleeding episodes, by frequent intravenous injections of factor IX purified from blood donors. Here we show in model animal experiments that purified human factor IX, when injected subcutaneously, is rapidly (in 3-11 h) and reasonably efficiently (30-40% of an equivalent intravenous dose) transported at least partly by the lymphatic drainage of the skin into the bloodstream, mostly in a biologically active form. This suggests that patients could be treated prophylactically by subcutaneous rather than intravenous injection, where the short delay in raising plasma factor IX to haemostatic levels would be clinically acceptable. More generally, our studies emphasize that the subcutaneous route of injection should be useful for other therapeutic proteins, including other clotting factors, which have to be delivered to the bloodstream, as long as their half-life is at least a few hours allowing time for transport into the general circulation.