Gene Therapy For Hemophilia B Using CB 2679d-GT: A Novel Factor IX Variant With Higher Potency Than Factor IX Padua

Abstract

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 three amino acid substitutions (R318Y, R338E, T343R), significantly outperformed the R338L-Padua variant after gene therapy. CB 2679d-GT demonstrated a statistically significant ~3-fold improvement in clotting activity when compared to R338L-Padua after AAV-based gene therapy in hemophilic mice. Moreover, CB 2679d-GT gene therapy showed a significantly reduced bleeding time (~5 to 8-fold) and total blood loss volume (~4-fold) compared with mice treated with the R338L-Padua, thus achieving a more rapid and robust hemostatic correction. FIX expression was sustained for at least 20 weeks with both CB 2679d-GT and R338L-Padua while 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.

A Machine Learning Framework Predicts the Clinical Severity of Hemophilia B Caused by Point-Mutations

“…Along these lines, we also verified that the Padua (Simioni et al, 2009) and the CB 2679d-GT mutants (Nair et al, 2021), known to considerably increase the activity of FIXa, have a high surface exposure and centrality values tending towards the least connected residues of the FIXa RIN. Interestingly, we observed that while most FIXa surface residues interacting with FVIIIa display low connectivity and high surface exposure, some residues eluded this trend by taking part in multiple molecular interactions with other residues (i.e., Lys347, Arg379, Leu383), and caused a major reduction of FXa generation if mutated to alanine (Kolkman et al, 1999;Bajaj et al, 2001).…”

“…In this sense, as attested by the fact that some FIX mutants—either natural ( Simioni et al, 2009 ) or engineered ( Nair et al, 2021 )—dramatically increase the catalytic activity and the half-life of FIX, it is clear that a deep understanding of the FIX structure and function is crucial to accelerate the development of more potent and less immunogenic FIX constructs.…”

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

“…A triple variant of fIX containing R318Y, R338E, and T343Y outperformed the Padua mutant as a novel gene therapeutic in treating hemophilia B with no risk of thrombophilia. 111 Residues R338 and T343 are located at the putative fVIIIa/fIXa interface and may form stabilizing contacts with the fVIIIa A2 domain. Together, these data suggest that binding between the fVIIIa A2 domain and fIXa helix-338 is a strong determinant in the formation and stabilization of the Xase complex and highlight several fIX residues to mutate in the design of next-generation hemophilia B gene therapeutics.…”

Gene Therapy in Hemophilia: Recent Advances

“…Nair et al [ 81 , 82 ] tested a new modification of the Padua variant of FIX. In clinical trials with FIX-R338L, they found that some patients showed an increase in liver transaminase levels, which was correlated with the loss of expression of FIX, even in immunosuppressed subjects.…”