Molecular Profiling of Liver Sinusoidal Endothelial Cells in Comparison to Hepatocytes: Reflection on Which Cell Type Should Be the Target for Gene Therapy

Human factor VIII (FVIII), which deficiency leads to hemophilia A, is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the characteristics of these cells that secrete FVIII are not well known. We have previously reported that based on genome-wide expression and CpG methylation profiling, LSECs have a distinct molecular profile that distinguishes them from other endothelial cells. Hepatocytes are targeted by gene therapy protocols to treat hemophilia A. However, the hepatocyte is not the natural site for FVIII synthesis and current gene therapy protocols are eliciting immune responses that require immune suppression with corticosteroid therapy in a fairly high proportion of patients over a significant period of time. Cellular stress because of ectopic FVIII expression and codon optimization are discussed as potential underlying mechanisms. Here, we highlight the molecular differences between LSECs and hepatocytes.

Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.

Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.

Extended Half-Life of the Double-Chain Recombinant Factor VIII-Fc Fusion Protein (FRSW107) in Hemophilia a Patients

Objective:To determine pharmacokinetics (PK), safety and tolerability of a double-chain recombinant human factor VIII-Fc fusion protein (FRSW107) in previously treated patients (PTPs) in clinical trial which was registered at www.clinicaltrials.gov as NCT03747653 and scheduled to finish at end of July, 2019. Methods:At least 12 PTPs (12 to 60 years old) with FVIII:C<2% and without history of FVIII inhibitors received a single dose of rFVIII (Advate) at 25 or 65 IU/kg followed by an equal dose of FRSW107. Results: Up-on the data from 7 subjects received 25 IU/kg , the geometric mean t1/2 of FRSW107 was 26.23 hours. This represents a 1.98-fold improvement over that of rFVIII (13.34 hours) at equivalent doses. All adverse events were unrelated to study drug. None of the study subjects developed FVIII inhibitors. Conclusion:According to the preliminary data from this phase 1 clinical trial, FRSW107 shows a significantly extended half- life compared to control FVIII while no safety issue identified. Disclosures No relevant conflicts of interest to declare.

Successful Perioperative Combination of High-Dose FVIII Therapy Followed by Emicizumab in a Patient with Hemophilia A with Inhibitors

We managed perioperative hemostasis for a 72-year-old man with hemophilia A and low inhibitor titers (3 BU/mL), who underwent osteosynthesis for supracondylar fracture of the left humerus. He was treated perioperatively using the combination of high doses of factor VIII (FVIII) with recombinant human Factor VIII Fc fusion protein (rFVIIIFc), followed by emicizumab. On the day of surgery (day 0), he was administered bolus infusion of 150 IU/kg rFVIIIFc, followed by continuous infusion at a dose of 4 IU/kg/h. Emicizumab, 3 mg/kg, was injected subcutaneously once a week, on days 5, 12, 19, and 26. Inhibitors were detected on day 6 at a titer of 4 BU/mL and FVIII:C decreased to below assay sensitivity limits on day 10. The rate of increase in inhibitor titers was high, with inhibitors increasing to 343.4 BU/mL on day 14. The transition of thrombin production by thrombin generation assay (TGA) showed temporary decrease in thrombin production on day 7, although it was restored by day 10, i.e., five days after commencement of emicizumab therapy. Rotational thromboelastometry displayed consistent results with TGA, showing that clotting time was prolonged and the alpha angle decreased to less than measurable levels on day 6, although they were improved by day 10. There were no bleeding-related events or other adverse events throughout the perioperative period. In conclusion, emicizumab was effective for the management of perioperative hemostasis after development of an anamnestic response in a patient with hemophilia A with inhibitors. Combination therapy with high doses of FVIII followed by emicizumab could be a workable alternative for patients with hemophilia A with inhibitors.