Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A

Abstract

Liver gene therapy with adeno‐associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X‐linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti‐F8 antibodies which neutralise F8 activity. Taking advantage of split‐intein‐mediated protein trans‐splicing, we divided the coding sequence of the large and highly secreted F8‐N6 variant in two separate AAV‐intein vectors whose co‐administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti‐F8 antibodies unlike animals treated with the single oversized AAV‐F8 vector under clinical development. Therefore, liver gene therapy with AAV‐F8‐N6 intein should be considered as a potential therapeutic strategy for HemA.

Optimization strategies and advances in the research and development of AAV‐based gene therapy to deliver large transgenes

“…Conversely, full-length protein expression was evident in 5/9 and in 0/5 eyes injected with ABCA4 and CEP290 dual AAV vectors, respectively. 114 Another example of FVIII gene delivery using the inteins and AAV vector was attempted by Esposito et al 115 The authors used Npu dnaE split-inteins and FVIII-N6 variant with a size of 5 kB for packaging in dual AAV8. The dual AAV8 at the dose of 5 × 10 11 genome copies was retro-orbitally injected in C57Bl/6 mice.…”

“…The dual AAV8 at the dose of 5 × 10 11 genome copies was retro‐orbitally injected in C57Bl/6 mice. Dual AAVs effectively expressed the full version of F8‐N6 in the liver cells of mice, thereby achieving the therapeutic level of the FVIII protein 112–115 …”

“…Dual AAVs effectively expressed the full version of F8-N6 in the liver cells of mice, thereby achieving the therapeutic level of the FVIII protein. [112][113][114][115] A hybrid approach using recombination and TS has also been applied for 'big genes' transfer with AAV (Figure 1). In this approach, the expression cassette-coding transgene is broken up and packaged into two independent AAV vectors.…”

“…It has been found that cells transduced by an AAV vector loaded with only one part of a transgene with appropriate genetic regulatory elements can initiate gene expression. 115 Moreover, the therapeutic efficiency of dual AAV vectors carrying a transgene could correlate with virus load. As was shown by Yan et al the level of expression of Epo protein delivered by dual AAV2 and TS was higher (9.4-fold) in the high dosage (4 × 10 11 vg/muscle) mouse group than in the low (6 × 10 10 vg/muscle) group at 110 days post treatment.…”

“…Another example of FVIII gene delivery using the inteins and AAV vector was attempted by Esposito et al 115 . The authors used Npu dnaE split‐inteins and FVIII‐N6 variant with a size of 5 kB for packaging in dual AAV8.…”