The Severe Spontaneous Bleeding Phenotype in a Novel Hemophilia a Rat Model with an Inversion Mutation Is Rescued By Platelet-Targeted FVIII Expression

Previous studies by our group and others have demonstrated that platelet targeted FVIII expression can rescue the bleeding phenotype in hemophilia A (HA) mice in both the inhibitor and non-inhibitor models. The clinical efficacy has been further proven in a dog HA non-inhibitor model. While these are informative, neither the canine nor mouse model entirely mimic human disease because 1) neither HA mice nor dogs exhibit the frequent spontaneous bleeding in severe HA patients, and 2) canine platelets do not contain FVIII carrier protein VWF and therefore store and release FVIII differently, thus precluding examining the efficacy of platelet gene therapy in the presence of inhibitors.

The efficacy of platelet-FVIII gene therapy, therefore, needs to be examined in an animal model more representative of human disease. Since the inversion of FVIII gene is the most common mutation that causes severe HA in patients, using a CRISPR/Case9 strategy we generated a novel HA rat model in a Dahl/Salt Sensitive (Dahl) inbred congenic background (Dahl-F8KO) with the nearly entire murine FVIII gene inverted causing a translational stop 6 amino acids after the signal sequence of FVIII. There is no detectable FVIII in plasma in Dahl-F8KO rats (LOD is 1 U/dL). Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 56.5% of homozygous affected animals (n=46) by the age of 8-weeks and up to 82.6% by the age of 32-weeks. This was significantly higher (P<0.0001) than the occurrence of spontaneous bleeding in another colony in Sprague Dawley (SD) background developed by Nielsen et al. using zinc finger nuclear strategy (J Thromb Haemost 2014) with a 13-bp deletion in exon 16 of FVIII. In SD-F8KO rat model, spontaneous bleeds occur in 23.3% and 42.1% of homozygous affected animals (n=85) that were housed in our facility by the age of 8- and 32-weeks, respectively. Likewise, the percentages of homozygous affected animals with 2, 3, or 4 bleeding episodes in the Dahl-F8KO colony were significantly higher than those in the SD-F8KO colony. In contrast, none of the heterozygous (F8^+/-) animals ever have a spontaneous bleed. Similar to SD-F8KO rats, Dahl-F8KO rats can develop anti-FVIII inhibitory antibodies after 2 doses of recombinant human FVIII (rhF8) treatment. Thus, our novel Dahl-F8KO rat model is a unique animal model with the severest spontaneous bleeding phenotype yet reported.

To investigate if platelet-derived FVIII can rescue the spontaneous bleeding phenotype in HA rats, we generated transgenic rats (2bF8^Tg) in both SD- and Dahl-background in which FVIII expression is under control of the platelet-specific αIIb promoter (2bF8) using lentivirus-mediated oocyte transduction transgenesis. Bone marrow (BM) mononuclear cells were isolated from femurs of 2bF8^Tg rats and transplanted into F8^-/- rats preconditioned with 950 R TBI. Animals were closely monitored after BM transplantation (BMT), and blood samples were collected for FVIII assays and whole blood thrombin generation assay (wbTGA). After BMT from SD-2bF8^Tg rats, no spontaneous bleeding was observed in SD-F8KO recipients within the entire study course (1 year at the time of this report) with a sustained platelet-FVIII expression of 20.9±8.1 mU/10⁸ platelets (n=3). Similarly, we performed BMT from Dahl-2bF8^Tg rats into Dahs-F8KO and found that 1 of 3 recipients had a bruise at the early stage of BM reconstitution. However, no any other spontaneous bleeding has yet been observed (5 months). To confirm that the bleeding diathesis in F8^-/- rats was ameliorated after 2bF8 gene expression, wbTGA was performed. All parameters, including Lag Time, Peak Time, Peak Thrombin, Endogenous Thrombin Potential in 2bF8^Tg-BMT recipients were significantly different compared to F8^-/- control rats, but were similar to those obtained in WT control animals. Of note, neither detectable levels of plasma FVIII nor anti-F8 antibodies were detected in F8^-/- recipients after receiving BMT from 2bF8^Tg rats.

In summary, we have developed a novel HA rat model with both the pathophysiology and clinical phenotype found in severe HA patients, which could be an ideal model for evaluating the clinical efficacy of new therapeutics. Our studies demonstrated that transplantation of BM cells that are genetically modified to express FVIII only in platelets can efficiently prevent the severe spontaneous bleeding in HA rats with no anti-F8 antibody development.