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.

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII.

Introduction: Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods: We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results: PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion: Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

Investigating Optimal Autologous Cellular Platforms for Prenatal or Perinatal Factor VIII Delivery to Treat Hemophilia A.

Patients with the severe form of hemophilia A (HA) present with a severe phenotype, and can suffer from life-threatening, spontaneous hemorrhaging. While prophylactic FVIII infusions have revolutionized the clinical management of HA, this treatment is short-lived, expensive, and it is not available to many A patients worldwide. In the present study, we evaluated a panel of readily available cell types for their suitability as cellular vehicles to deliver long-lasting FVIII replacement following transduction with a retroviral vector encoding a B domain-deleted human F8 transgene. Given the immune hurdles that currently plague factor replacement therapy, we focused our investigation on cell types that we deemed to be most relevant to either prenatal or very early postnatal treatment and that could, ideally, be autologously derived. Our findings identify several promising candidates for use as cell-based FVIII delivery vehicles and lay the groundwork for future mechanistic studies to delineate bottlenecks to efficient production and secretion of FVIII following genetic-modification.

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.

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.