Bioengineered Factor IX Molecules with Increased Catalytic Activity Improve the Therapeutic Index of Gene Therapy Vectors for Hemophilia B

Abstract

Although the desire to develop gene therapy for hemophilia B is high, safety remains a concern. Therefore, improving the therapeutic index of gene therapy vectors is an important goal. Thus, we evaluated the use of three bioengineered factor IX (FIX) variants with improved catalytic activity in the context of the helper-dependent adenoviral vector. The first vector expressed R338A-FIX, an FIX variant with the arginine at position 338 changed to an alanine, which resulted in a 2.9-fold higher specific activity (IU=mg) compared with the wild-type FIX. The second vector expressed FIX VIIEGF1 , a variant with the EGF-1 domain replaced with the EGF-1 domain from FVII, which resulted in a 3.4-fold increase in specific activity. The third expressed R338A + FIX VIIEGF1 , a novel variant containing both aforementioned modifications, which resulted in a 12.6-fold increase in specific activity. Highlevel, long-term, and stable expression of these three variants was observed in hemophilia B mice with no evidence of increased thrombogenicity compared with wild-type FIX. Thus, these bioengineered FIX variants can increase the therapeutic index of gene therapy vectors by permitting administration of lower doses to achieve the same therapeutic outcome. Furthermore, these variants may also be valuable for recombinant FIX protein replacement therapy.

The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

“…In an early study, we demonstrated that replacement of arginine 338 by alanine (R338A) was associated with an ϳ 3-fold increase in the protein specific activity in murine models of HB receiving AAV-FIX-R338A 9 as later confirmed in other models. 10,11 Recently, we described a naturally occurring gain-of-function mutation in humans characterized by leucine at position 338 (R338L), which exhibits normal antigen levels, but an ϳ 8-fold higher specific activity. 12 Notably, the arginine at position 338 in FIX is conserved among mammals, and this region of the enzyme appears to be part of the substrate exosite for factor X.…”

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

“…Another group generated a similar increase in specific clotting activity (12.6 fold higher than wt-FIX) through replacement of the FIX-EGF-1 domain by the corresponding sequence of FVII supplementary to the R338A substitution [41]. Moreover, expression of FIX-R338A variants following liver-directed gene transfer led to an improvement in specific clotting activity and hemostasis [42] in hemophilia B mice following delivery of both adeno-associated viral (AAV) vectors [40,42] as well as helper-dependent adenoviral (HDAd) vectors [41]. Lastly, even one more efficacious variant with a single substitution at position 338 from arginine to leucine was identified in a patient with juvenile thrombophilia in the Padua University Hospital [43].…”

“…The observed increase in catalytic efficiency was rather the outcome of the greater binding affinity of FIXa-R338A to FVIIIa [40]. Since those reports several independent studies were implemented in the bioengineering of new combined variants with the substitution R338A in order to J Genet Syndr Gene Ther Gene Therapy for Hemophilia ISSN:2157-7412 JGSGT an open access journal obtain an additive effect in catalytic activity [40,41] and thereby to succeed more efficacious gene therapy approaches by means of novel FIX variants with enhanced procoagulant potential for treatment of hemophilia B [40,42]. A further increase in FIX activity could be achieved by combining R338A with two other mutations (V86A and E277A) resulting in a FIX triple variant which exhibited 13-fold higher specific coagulation activity and 10-fold higher affinity for human FVIIIa [40].…”

“…A further increase in FIX activity could be achieved by combining R338A with two other mutations (V86A and E277A) resulting in a FIX triple variant which exhibited 13-fold higher specific coagulation activity and 10-fold higher affinity for human FVIIIa [40]. Another group generated a similar increase in specific clotting activity (12.6 fold higher than wt-FIX) through replacement of the FIX-EGF-1 domain by the corresponding sequence of FVII supplementary to the R338A substitution [41]. Moreover, expression of FIX-R338A variants following liver-directed gene transfer led to an improvement in specific clotting activity and hemostasis [42] in hemophilia B mice following delivery of both adeno-associated viral (AAV) vectors [40,42] as well as helper-dependent adenoviral (HDAd) vectors [41].…”

Adenoviral Vectors for Hemophilia Gene Therapy

“…One such strategy makes use of more potent expression cassettes. For example, in the case of hemophilia B, three bioengineered FIX variants with improved catalytic activity have been tested in the context of HDAd [38]. The first vector expressed R338A-FIX, a FIX variant with the arginine at position 338 changed to an alanine [39,40], which resulted in a 2.9-fold higher specific activity (IU/mg) compared with the wild-type FIX.…”