The delivery of factor VIII (FVIII) through gene and/or cellular platforms has emerged as a promising hemophilia A treatment. Herein, we investigated the suitability of human placental cells (PLCs) as delivery vehicles for FVIII and determined an optimal FVIII transgene to produce/secrete therapeutic FVIII levels from these cells. Using three PLC cell banks we demonstrated that PLCs constitutively secreted low levels of FVIII, suggesting their suitability as a transgenic FVIII production platform. Furthermore, PLCs significantly increased FVIII secretion after transduction with a lentiviral vector (LV) encoding a myeloid codon-optimized bioengineered FVIII containing high-expression elements from porcine FVIII. Importantly, transduced PLCs did not upregulate cellular stress or innate immunity molecules, demonstrating that after transduction and FVIII production/secretion, PLCs retained low immunogenicity and cell stress. When LV encoding five different bioengineered FVIII transgenes were compared for transduction efficiency, FVIII production, and secretion, data showed that PLCs transduced with LV encoding hybrid human/porcine FVIII transgenes secreted substantially higher levels of FVIII than did LV encoding B domain-deleted human FVIII. In addition, data showed that in PLCs, myeloid codon optimization is needed to increase FVIII secretion to therapeutic levels. These studies have identified an optimal combination of FVIII transgene and cell source to achieve clinically meaningful levels of secreted FVIII.

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Microfluidic technology enables recapitulation of organ-level physiology to answer pertinent questions regarding biological systems that otherwise would remain unanswered. We have previously reported on the development of a novel product consisting of human placental cells (PLC) engineered to overexpress a therapeutic factor VIII (FVIII) transgene, mcoET3 (PLC-mcoET3), to treat Hemophilia A (HA). Here, microfluidic devices were manufactured to model the physiological shear stress in liver sinusoids, where infused PLC-mcoET3 are thought to lodge after administration, to help us predict the therapeutic outcome of this novel biological strategy. In addition to the therapeutic transgene, PLC-mcoET3 also constitutively produce endogenous FVIII and von Willebrand factor (vWF), which plays a critical role in FVIII function, immunogenicity, stability, and clearance. While vWF is known to respond to flow by changing conformation, whether and how shear stress affects the production and secretion of vWF and FVIII has not been explored. We demonstrated that exposure of PLC-mcoET3 to physiological levels of shear stress present within the liver sinusoids significantly reduced mRNA levels and secreted FVIII and vWF when compared to static conditions. In contrast, mRNA for the vector-encoded mcoET3 was unaltered by flow. To determine the mechanism responsible for the observed decrease in FVIII and vWF mRNA, PCR arrays were performed to evaluate expression of genes involved in shear mechanosensing pathways. We found that flow conditions led to a significant increase in KLF2, which induces miRNAs that negatively regulate expression of FVIII and vWF, providing a mechanistic explanation for the reduced expression of these proteins in PLC under conditions of flow. In conclusion, microfluidic technology allowed us to unmask novel pathways by which endogenous FVIII and vWF are affected by shear stress, while demonstrating that expression of the therapeutic mcoET3 gene will be maintained in the gene-modified PLCs upon transplantation, irrespective of whether they engraft within sites that expose them to conditions of shear stress.