Gene therapy utilizing split-intein-mediated protein trans-splicing was able to produce therapeutic levels of clotting factor VIII (FVIII) without circulating antibodies in a hemophilia A mouse model, as published in EMBO Molecular Medicine.
The researchers split the highly secreted and active FVIII-N6 variant to fit into separate adeno-associated viral (AAV) intein (intervening protein) vectors. The separate N- and C-polypeptides were then codelivered by independent viruses and reconstituted through protein trans-splicing to excise the inteins and restore the full-length FVIII-N6.
“Here, we show that dual AAV vectors armed with Npu DnaE split-inteins efficiently and precisely reconstitute the large and highly secreted [FVIII]-N6 (N6) variant in the mouse liver resulting in stable therapeutic levels of [FVIII],” the authors said.
A codon optimization method was also applied to the split FVIII-N6 (CodopN6) variants which increased the production of FVIII by cells from ~70 IU/dl for the regular FVIII-N6 variant to ~200 IU/dl.
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Systemic administration of a single dose of FVIII-N6 inteins, CodopN6 inteins, or a single AAV-codon-optimized FVIII-V3 variant (CodopV3) resulted in wild-type levels of FVIII in a hemophilia A mouse model. FVIII expression levels tended to decline over time, however. Both the FVIII-N6 and CodopN6-treated mice showed significantly decreased coagulation times compared to baseline.
The mice injected with CodopV3 in the study also developed FVIII-antibodies over time. The antibody levels were inversely correlated with FVIII activity with higher antibodies being present in animals with no detectable FVIII activity. The mice that received FVIII-N6 or CodopN6 inteins did not develop antibodies.
The use of inteins may allow for the therapeutic administration of genes that exceed the roughly 4.7 kb cargo capacity for AAV vectors such as the 5 kb FVIII-N6 variant in this case. By splitting the FVIII-N6 variant and adding intein regions, sizes of 3.7 kb for the 5’ half and 3 kb for the 3’ half were able to be achieved.
All AAV-based hemophilia gene therapies currently under clinical investigation utilize B-domain-deleted versions of the FVIII transgene of roughly 4.4 kb in size. This large size leaves little room for regulatory elements and restricts promotor options, the authors said.
Esposito F, Lyubenova H, Tornabene P, et al. Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A. EMBO Mol Med. Published online May 2, 2022. doi:10.15252/emmm.202115199