Transplanting FVIII/ET3-secreting cells in fetal sheep increases FVIII levels long-term without inducing immunity or toxicity.

Hemophilia A is the most common X-linked bleeding disorder affecting more than half-a-million individuals worldwide. Persons with severe hemophilia A have coagulation FVIII levels <1% and experience spontaneous debilitating and life-threatening bleeds. Advances in hemophilia A therapeutics have significantly improved health outcomes, but development of FVIII inhibitory antibodies and breakthrough bleeds during therapy significantly increase patient morbidity and mortality. Here we use sheep fetuses at the human equivalent of 16-18 gestational weeks, and we show that prenatal transplantation of human placental cells (107-108/kg) bioengineered to produce an optimized FVIII protein, results in considerable elevation in plasma FVIII levels that persists for >3 years post-treatment. Cells engraft in major organs, and none of the recipients mount immune responses to either the cells or the FVIII they produce. Thus, these studies attest to the feasibility, immunologic advantage, and safety of treating hemophilia A prior to birth.

Autologous bone marrow-derived MSCs engineered to express oFVIII-FLAG engraft in adult sheep and produce an effective increase in plasma FVIII levels.

Introduction: Hemophilia A (HA) is the most common X-linked bleeding disorder, occurring in 1 in 5,000 live male births and affecting >1 million individuals worldwide. Although advances in protein-based HA therapeutics have improved health outcomes, current standard-of-care requires infusion 2-3 times per week for life, and 30% of patients develop inhibitors, significantly increasing morbidity and mortality. There are thus unmet medical needs requiring novel approaches to treat HA. Methods: We tested, in a highly translational large animal (sheep) model, whether the unique immunological and biological properties of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) could enable them to serve as cellular delivery vehicles to provide long-term expression of FVIII, avoiding the need for frequent infusions. Results: We show that autologous BM-MSCs can be isolated, transduced with a lentivector to produce high levels of ovine (o)FVIII, extensively expanded, and transplanted into adult animals safely. The transplanted cells engraft in multiple organs, and they stably produce and secrete sufficient quantities of FVIII to yield elevated plasma FVIII levels for at least 15 weeks. Discussion: These studies thus highlight the promise of cellular-based gene delivery approaches for treating HA.

Effects of Shear Stress on Production of FVIII and vWF in a Cell-Based Therapeutic for Hemophilia A.

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.

Microfluidic devices for studying coagulation biology.

The ability to study the behavior of cells, proteins, and cell-cell or cell-protein interactions under dynamic forces such as shear stress under fluid flow, provides a more accurate understanding of the physiopathology of hemostasis. This review touches upon the traditional methods for studying blood coagulation and platelet aggregation and provides an overview on cellular and protein response to shear stress. We also elaborate on the biological aspects of how cells recognize mechanical forces and convert them into biochemical signals that can drive various signaling pathways. We give a detailed description of the various types of microfluidic devices that are employed to study the complex processes of platelet aggregation and blood coagulation under flow conditions as well as to investigate endothelial shear-response. We also highlight works mimicking artificial vessels as platforms to study the mechanisms of coagulation, and finish our review by describing anticipated clinical uses of microfluidics devices and their standardization.

Defining the Optimal FVIII Transgene for Placental Cell-Based Gene Therapy to Treat Hemophilia A.

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.