Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice.

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Le Quellec S ，Dane AP ，Barbon E ，Bordet JC ，Mingozzi F ，Dargaud Y ，Marais T ，Biferi MG ，Négrier C ，Nathawani AC ，Enjolras N ... -  《-》

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins.

The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV-based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half-life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Adeno-associated viral vectors (AAV) mediating expression of hFIX-Alb and hFIX-Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half-life translates to higher plasma levels of FIX. Single-stranded cross-packaged AAV2/8 vectors expressing hFIX-Alb, hFIX-Fc and hFIX were evaluated in vitro, and in mice. Both hFIX-Alb and hFIX-Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV-mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX-fusion proteins were comparable to wild-type hFIX. However, their expression levels were threefold lower than wild-type hFIX in vivo most likely due to inefficient secretion. This, the first, evaluation of hFIX-fusion proteins in the context of AAV gene transfer suggests that the hFIX-fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

Le Quellec S ，Dane A ，Enjolras N ，McIntosh J ，Rosales C ，Negrier C ，Nathwani A ... -  《-》

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer.

Cohn EF ，Zhuo J ，Kelly ME ，Chao HJ ... -  《-》

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.

Monahan PE ，Sun J ，Gui T ，Hu G ，Hannah WB ，Wichlan DG ，Wu Z ，Grieger JC ，Li C ，Suwanmanee T ，Stafford DW ，Booth CJ ，Samulski JJ ，Kafri T ，McPhee SW ，Samulski RJ ... -  《-》

Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo.

Kao CY ，Yang SJ ，Tao MH ，Jeng YM ，Yu IS ，Lin SW ... -  《-》