Gene therapy for hemophilia B using CB 2679d-GT: a novel factor IX variant with higher potency than factor IX Padua.

Sustained expression of therapeutic factor IX (FIX) levels has been achieved after adeno-associated viral (AAV) vector-based gene therapy in patients with hemophilia B. Nevertheless, patients are still at risk of vector dose-limiting toxicity, particularly liver inflammation, justifying the need for more efficient vectors and a lower dosing regimen. A novel increased potency FIX (designated as CB 2679d-GT), containing 3 amino acid substitutions (R318Y, R338E, T343R), significantly outperformed the R338L-Padua variant after gene therapy. CB 2679d-GT demonstrated a statistically significant approximately threefold improvement in clotting activity when compared with R338L-Padua after AAV-based gene therapy in hemophilic mice. Moreover, CB 2679d-GT gene therapy showed significantly reduced bleeding time (approximately fivefold to eightfold) and total blood loss volume (approximately fourfold) compared with mice treated with the R338L-Padua, thus achieving more rapid and robust hemostatic correction. FIX expression was sustained for at least 20 weeks with both CB 2679d-GT and R338L-Padua whereas immunogenicity was not significantly increased. This is a novel gene therapy study demonstrating the superiority of CB 2679d-GT, highlighting its potential to obtain higher FIX activity levels and superior hemostatic efficacy following AAV-directed gene therapy in hemophilia B patients than what is currently achievable with the R338L-Padua variant.

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.

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites.

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.

Development of long-acting recombinant FVIII and FIX Fc fusion proteins for the management of hemophilia.

INTRODUCTION: Prophylactic treatment with replacement clotting factor is the recommended regimen for patients with severe hemophilia to prevent bleeding episodes. However, currently available replacement clotting factors are limited by their relatively short half-lives and require intravenous injections up to three times weekly to maintain protective levels, which can impact compliance and, thus, patient outcomes. AREAS COVERED: The potential advantages of long-acting coagulation factors, including reduced injection frequency, increased treatment adherence, and improved clinical outcomes, are discussed. Fragment crystallizable (Fc) fusion technology is introduced and the development of long-acting recombinant factor VIII Fc (rFVIIIFc) and recombinant factor IX Fc (rFIXFc) fusion proteins for the treatment of hemophilia A and B, respectively, are described. Preclinical and clinical studies of rFVIIIFc and rFIXFc showing improved pharmacokinetics over currently available products are reviewed. EXPERT OPINION: Long-acting coagulation factors, including rFVIIIFc and rFIXFc, have the potential to change current paradigms of care for hemophilia A and B, respectively. Less frequent infusions may provide prolonged protection from bleeding and bleed resolution with fewer injections. In addition, long-acting coagulation factors provide an opportunity for improved individualized treatment for hemophilia.

Cloning, sequencing, and analysis of the full-length CDNA of rhesus monkey factor IX.

OBJECTIVE: Coagulation factor IX (FIX) is a vitamin K-dependent serine protease, which plays a key role in the coagulation cascade. The rhesus monkey may be an indispensable substitute for humans in research of pig-to-human xenotransplantation, due to its close relationship. But the coagulation function concordance between rhesus monkey and human is unknown. In this study, we cloned the full-length cDNA of rhesus monkey FIX (rFIX) to investigate the genomic backgrounds of the coagulation systems. METHOD: We cloned the full-length cDNA from the cDNA library of rhesus monkey liver tissue. Polymerase chain reaction was used to screen the positive clones. Based on a partial sequence obtained by cDNA library screening and a homologous sequence from the database, we designed a second pair of primers to obtain the full sequence. For further analysis of rFIX, we used several online ExPASy Proteomic tools. RESULT: We obtained the full-length cDNA of rFIX, which has 2668 nucleotides, predicting an open reading frame of 1383 nucleotides corresponding to 461 amino acids. The deduced protein sequence indicated functional domains of signal peptide, Gla, two epidermal growth factor, and trypsin-like serine protease, which were consisted with those of human FIX (hFIX). Sequence alignments showed that rFIX is highly homologous to hFIX with nucleotide identity of 96% and amino acid identity of 97%. CONCLUSION: We have report herein the full-length cDNA of rFIX. The high homology between rhesus monkey and human coagulation factor ensure the reliability and feasibility of rhesus monkey as a recipient in studies on coagulation disorders in xenotransplantation.

Protein engineering of the hydrophobic domain of human factor IX.

Vitamin K-dependent plasma proteins contain a highly conserved hydrophobic domain located between the gamma-carboxyglutamic acid (Gla) domain and the first epidermal growth factor (EGF)-like domain. Here we have used protein engineering of the hydrophobic domain in human factor IX to investigate its function in intact factor IX. Mutant proteins were generated by site-directed mutagenesis and in vitro expression in Madin-Darby canine kidney (MDCK) cells. All of our mutants, including one with a deletion of the entire hydrophobic domain, were activated by factor XIa, showing that this domain is not required for factor IX activation. The results with the mutant Phe41-->Val suggest that the hydrophobic domain interacts with the adjacent EGF-like domain. Our data for the Phe41-->Asp mutant is consistent with, but cannot prove, a role for this residue in the maintenance of a phospholipid-binding structure required for factor IX function.