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

Abstract

Introduction 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). Aim 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. Methods Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results 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. Conclusion 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.

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy

“…Because of the packaging constraints of AAV vectors discussed above, only FIX fusion proteins would be amenable to incorporation into AAV vectors. However, recent work evaluating AAV gene therapy with transgenes of FIX fused with albumin or the Fc domain in HB mice was unsuccessful because of poor transgene expression compared with FIX-WT 162 . Intriguingly, several gene-editing approaches have also investigated inserting the FVIII or FIX gene into the albumin locus as an integrative gene therapy strategy to ensure long-term expression of WT FVIII or FIX despite hepatocyte cell division 163, 164, 165.…”

“…However, recent work evaluating AAV gene therapy with transgenes of FIX fused with albumin or the Fc domain in HB mice was unsuccessful because of poor transgene expression compared with FIX-WT. 162 Intriguingly, several gene-editing approaches have also investigated inserting the FVIII or FIX gene into the albumin locus as an integrative gene therapy strategy to ensure long-term expression of WT FVIII or FIX despite hepatocyte cell division. [163][164][165] Whether it is possible to modify these gene integrative approaches to express in vivo an FIX-albumin fusion protein similar to the current EHL product FIX-FP (Idelvion, Coagulation Factor IX [Recombinant], Albumin Fusion Protein) remains to be determined.…”

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

“…[9][10][11][12] Transgenes encoding longer-acting FIX-fusion proteins were also investigated but did not increase therapeutic efficacy in mice. 13 A particular substitution at position 338 (designated as R338L-Padua), associated with thrombophilia, 14 enhances FIX activity by fivefold to eightfold. Extensive preclinical and clinical experience with R338L-Padua gene therapy in hemophilia B revealed no increased thrombogenic or immunogenic risk.…”