Page 291 - Haematologica April 2020

mRNA-based therapy for hemophilia A
mRNA than in mice treated with rFVIII. Of note, the incu- bation of FVIII-encoding mRNA, TransIT® or TransIT®-for- mulated mRNA with immature human monocyte-derived dendritic cells failed to induce the production of tumor necrosis factor-α and interleukin-6 (Figure 5E, F), indicat- ing that mRNA and TransIT® do not activate innate immune cells and are not responsible for triggering the anti-FVIII immune response. In additional experiments, we investigated the site of production of mRNA-encoded proteins following formulation in TransIT®. To this end, Balb/c mice were injected with luciferase-encoding mRNA formulated in TransIT®. The luminescence meas- ured 24 h later was at least 2-fold greater in the spleen than in the liver (Online Supplementary Figure S1), suggest- ing that the liver and hepatocytes are not the main target for TransIT®-formulated mRNA. Attempts to detect FVIII in FVIII-encoding mRNA-treated FVIII-deficient mice led to occasional FVIII signals in the marginal zone of the spleen and constant absence of FVIII detection in the liver (data not shown).
Discussion
The present work documents the sustained endogenous production of pro-coagulant FVIII following the injection of FVIII-encoding mRNA into FVIII-deficient mice, and is of potential relevance for the improvement of treatment for patients with hemophilia A. FVIII levels achieved 24 h after the injection of mRNA were about 40% of the levels in normal plasma. The levels of FVIII expression are noto- riously low because of poor transcriptional and translation efficacies27-29 as well as retention of the protein in the endo- plasmic reticulum.30,31 Attempts to improve the levels of expression of FVIII include partial removal of the B domain with conservation of essential N-glycosylation
sites,24,31 mutation of an immunoglobulin binding-protein (BiP) binding site32 to ensure improved transfer from the endoplasmic reticulum to the Golgi apparatus,33 and codon optimization. Here, mRNA was generated using wild- type23 or codon-optimized cDNA encoding human BDD- FVIII. While codon optimization improved FVIII produc- tion following transfection of eukaryotic cells with plas- mid DNA in vitro, it did not increase the levels of FVIII:C or FVIII:Ag following in vitro mRNA transfection of cells or in vivo transfection of FVIII-deficient mice. Codon opti- mization aims at improving translation rates by using codons for which the cognate tRNA levels are not limit- ing. Thus, codon optimization of FVIII-encoding cDNA cloned in lentiviral vectors or adenoviral-associated vec- tors led to more than 10-fold increased FVIII levels after in vitro transfection of cell lines and after injection into wild- type or FVIII-deficient mice.24,25 The lack of improvement in protein production associated with the administration of codon-optimized mRNA encoding FVIII suggests that codon optimization of FVIII preferentially targets tran- scriptional rather than translational events, as previously shown.34,35 Alternatively, levels of mRNA introduced into each cell upon in vivo transfection with TransIT® may be much lower than that transcribed endogenously following transfection using DNA, and insufficient to exhaust non- abundant tRNA. Increasing the amount of mRNA injected in vivo did not, however, have drastic effects on the FVIII levels reached in the circulation.
Frequent spontaneous joint and muscle bleeds in patients with severe hemophilia A eventually lead to the development of arthropathy and functional joint impair- ment. An association between joint bleeds and baseline FVIII activity levels was demonstrated,36 with FVIII levels above 5% of the normal values drastically reducing the occurrence of joint bleeds. Because the half-life of human FVIII in patients with hemophilia A is between 12 and 18
AB
Figure 3. Time-dependent production of endogenous factor VIII after a single injection of factor VIII-encoding mRNA. mRNA encoding codon-optimized B domain- deleted (BDD) factor VIII (CoFVIIIHSQ) was formulated in TransIT®. (A) FVIII-deficient mice were then injected intravenously with 1, 3 or 5 μg FVIII-encoding mRNA. FVIII:C was measured in plasma after 24 and 72 h. Individual symbols on the graphs represent individual mice; horizontal bars represent means ± standard error of mean. The dotted line indicates the critical FVIII level (i.e., 5%) required to drastically reduce joint bleeds. Statistical differences were assessed using a two-tailed t test (ns: non-significant, *P<0.05). (B) Mice were injected with 3 μg CoFVIIIHSQ-encoding mRNA formulated in TransIT®, or with 100 μL of 10 nM recombinant BDD-FVIII (3 IU rFVIII). FVIII:C in plasma was measured after 30, 60, 90, 120, 180, 240, 360, 390 and 480 min in the case of rFVIII, in the case of mRNA. The dotted line depicts the non-linear fit (two-phase exponential decay: y=0.62*e-0.15x+0.2211*e-0.80x) of the experimental data obtained with rFVIII. The full circles and full line curve represent means ± standard error of mean of six mice treated with mRNA (representative of two independent experiments; fitted with a one-phase exponential decay for the values obtained at 24, 48 and 72 h: y=0.99*e-0.03874x). Areas under the curves were calculated using Prism GraphPad (version 6).
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