(A) An example of transduction efficiency and logical gating strategy for UT vs 100 MOI transduced CD34 + cells differentiated into CD14 + cells. (A-B) FVIII expression in CD14 + monocytes differentiated from CD34 + cells after 14 days of monocyte-LTC (mo-LTC) assay. UM171 enriches CD14 + /CD31 + monocyte subset with high FVIII expression capacity. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34 + cells during transduction expanded a subpopulation of CD14 +/CD31 + monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector.

We discovered that FVIII production begins at the CD34 + progenitor stage after cytokine stimulation in culture. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs).

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