Page 288 - Haematologica April 2020

J. Russick et al.
gle-stranded RNA were found to trigger innate immunity upon ligation of TLR3, 7 and 8, and RIG-1.9-12 The replace- ment of uridines by 1-methylpseudouridines and the removal of double-stranded RNA by high performance liq- uid chromatography was demonstrated to abrogate the activation of innate immune cells,13,14 and allowed the in vivo production of different proteins, including erythropoietin, factor IX and anti-human immunodeficiency virus antibod- ies without the induction of overt neutralizing immune responses.15-19 Conversely, the administration of synthetic mRNA was also used in vaccination strategies either by direct injection20 or upon adoptive transfer of ex vivo-trans- fected dendritic cells.21,22
Because of its monogenic nature and the requirements for low amounts of FVIII to correct the bleeding pheno- type of affected patients, hemophilia A is a particularly suitable disease for treatment with mRNA. Furthermore, transfection with mRNA is by essence not integrative, and thus avoids risks of uncontrolled insertional mutage- nesis that may occur with DNA-based gene therapy approaches. In addition, mRNA is translated only tran- siently and is degraded by physiological pathways, thus ensuring its safety and facilitating the control of the bioavailability of the encoded protein. Here, we investi- gated whether the intravenous administration of FVIII- encoding mRNA enables the production of therapeutic levels of pro-coagulant FVIII in FVIII-deficient mice.
FVIIIHSQ or CoFVIIIHSQ cloned in the ReNeo vector (0.1 μg) using lipofectamine (Invitrogen, Carlsbad, CA, USA). For in vitro trans- fection using mRNA, mRNA (0.4 μg) was mixed with TransIT®- mRNA reagent (0.45 μL, Mirus Bio, Madison, WI, USA) and Boost reagent (0.29 μL) in a final volume of 50 μL of Dulbecco modified Eagle medium (DMEM) for 2 min at room temperature. HEK293 cells (50,000 cells/130 μL) were incubated with the formulated mRNA overnight in DMEM-F12 (Thermo Fisher). FVIII was meas- ured in the supernatant after 24 h. Supernatant was kept frozen at -80°C until use.
Treatment of mice
Mice were 8- to 12-week old F8 exon 16 knockout C57BL/6 mice (a kind gift from Prof H.H. Kazazian, Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA). Mice were injected intravenously with recombinant BDD-FVIII (rFVIII, Refacto®, Pfizer, 150 IU/kg), or with mRNA (1 to 5 μg) formulated in TransIT® (100 to 350 μL final volume). Blood was collected from the retro-orbital sinus 6, 24, 48, 72 or 120 h following the injection of mRNA. Plasma was kept frozen at -80°C until use. Animals were handled in agreement with local ethical authorities (approval by Charles Darwin ethics committee, authorization #3335 2015121718044892). FVIII:Ag, FVIII:C, anti-FVIII IgG and FVIII inhibitors were measured as described in the Online Supplementary Methods.
Results
Codon-optimization of cDNA encoding factor VIIIHSQ improves in vitro factor VIII production
We first investigated whether codon-optimization of FVIIIHSQ improves the production of FVIII by BHK trans- fected cells. To this end, CoFVIIIHSQ was synthesized and inserted in the Reneo vector. BHK cells were transiently transfected with 0.1 μg FVIIIHSQ or CoFVIIIHSQ-encoding cDNA (Figure 1A). FVIII:Ag and FVIII:C were measured in the supernatant 24 h later by enzyme-linked immunosor- bent assay (ELISA) and chromogenic assay. Transfection with CoFVIIIHSQ-encoding cDNA produced 4.2-fold more FVIII:Ag (mean ± standard error of mean: 0.11±0.01 IU/mL vs. 0.03±0.00 IU/mL, respectively; P<0.01) and 4.8- fold more FVIII:C (0.14±0.02 IU/mL vs. 0.03±0.01 IU/mL, respectively; P<0.01), than transfection with the non-opti- mized FVIIIHSQ-encoding cDNA. Our data confirm previ- ous findings obtained upon gene therapy in preclinical models of hemophilia A,24,25 on the capacity of codon opti- mization to increase the yields of FVIII production.
Transfection with factor VIII-encoding mRNA leads to factor VIII production in vitro
We then validated the capacity of mRNA transcribed in vitro, using the FVIIIHSQ and CoFVIIIHSQ-encoding cDNA as templates, to promote FVIII production. As the codon- optimization is for Homo sapiens, mRNA encoding FVIIIHSQ or CoFVIIIHSQ formulated in TransIT® was used to trans- fect human HEK293 cells (Figure 1B). As a negative con- trol, HEK293 cells were transfected with luciferase- encoding mRNA. Transfection with the two FVIII-encod- ing mRNA led to the in vitro production of similar amounts of FVIII (FVIII:Ag: 0.28±0.03 IU/mL vs. 0.36±0.04 IU/mL for FVIIIHSQ and CoFVIIIHSQ, respectively) and similar activities (FVIII:C: 0.20±0.01 IU/mL vs. 0.24±0.02 IU/mL, respectively). Accordingly, the specific activity of FVIII produced using both mRNA did not dif-
Methods
Cloning of factor VIII
The cDNA encoding human B domain-deleted (BDD) FVIII (FVIIIHSQ), containing the 14-amino acid segment SFSQNPPVLKRHQR in place of the B domain, cloned in the ReNeo mammalian expression plasmid with geneticin resist- ance, has been described previously.23 Codon optimization of the DNA sequence encoding human BDD-FVIII was adapted to the bias of Homo sapiens using in-house proprietary software (GeneOptimizer) from GeneArt (Thermo Fisher, Darmstadt, Germany). The GeneOptimizer software also calculates removal of cis-acting sequence motifs, including internal TATA-boxes, chi-sites and ribosomal entry sites, AT- or GC-rich sequence stretches, AU-rich elements, inhibitory and cis-acting repressor sequence elements, repeat sequences, RNA secondary struc- tures, and all cryptic splice sites. The codon-optimized BDD- FVIII-encoding cDNA was also cloned in the ReNeo vector.
In vitro transcription of mRNA
mRNA were transcribed as previously described15 using the
linearized plasmids encoding BDD-FVIII (FVIIIHSQ), the codon- optimized BDD-FVIII (CoFVIIIHSQ) and firefly luciferase (Luciferase). The Megascript T7 RNA polymerase kit (Thermo Fisher) was used for transcription, and UTP was replaced with 1- methylpseudouridine triphosphate (m1ΨTP; TriLink, San Diego, CA, USA) to generate m1Ψ-containing mRNA. All mRNA were transcribed to contain 100-nucleotide long poly(A) tails. To obtain cap1, RNA was incubated with guanylyltransferase and 2´-O-methyltransferase (Vaccinia capping system; New England Biolabs, Frankfurt, Germany). All mRNA were purified and stored at -20°C.
In vitro transfection
For transient in vitro production of FVIII, baby hamster kidney
(BHK) cells (0.5x106 cells in 48-well plates) were transfected with
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