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

Abstract

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.

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

“…While the low levels of mRNA for the fVIII transgene argue that ER stress was not likely the cause of low secreted FVIII activity, the upregulation of ER stress is obviously not desirable, as it will likely negatively impact the viability and functionality of the PLCs, precluding their use in cell therapy. The upregulation of TLR 3 and TLR 7 by the plasmids would also likely compromise one of the key attributes of PLCs for use as an off-the-shelf therapy, namely, their state of relative immune-inertness, a conclusion supported by our recent report showing activation of PLCs leads to production of γ-IFN ( 68 ). As such, another lucrative avenue for future studies would be the redesign of the plasmids to remove any bacterial sequences that might be serving as pathogen-associated molecular patterns (PAMPs) and be responsible for recognition by the TLRs within the PLCs.…”

“…This is especially true for the hLSECs, which exhibit very slow division kinetics and cannot be propagated for more than a couple of passages in vitro prior to senescing. The PLCs employed in the present study exhibit a phenotype and biological properties that closely resemble that of MSC from other tissues ( 3 , 22 , 68 ). It is noteworthy that prior studies have reported the downregulation of components of the DNA damage response (DDR) and HRR pathways in MSC with time in culture ( 94 – 96 ).…”

“…In an effort to make cell-based gene therapy a clinical reality for HA, we and others have performed studies over the past decades to identify the ideal cell type for delivering a fVIII transgene and the optimal vector to introduce the fVIII transgene into the desired cell population ( 3 , 22 , 31 , 34 , 37 – 48 ). Previously, we demonstrated the advantages of using lentivector-transduced PLCs as the cellular vehicle for delivering a fVIII transgene ( 22 , 68 ). The present study evaluated whether CRISPR/Cas9 could be used to deliver an EF1α-lcoET3 expression cassette into PLCs, with the hope of further enhancing safety by directing integration to the AAVS1 “safe harbor” genome locus, thereby eliminating the theoretical risk of insertional mutagenesis that is inherent to integrating vectors such as those based upon lentiviruses.…”

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

“…200 ng of RNA was converted into cDNA using the RT 2 First Strand Kit (Qiagen, Valencia, CA, United States). SYBR Green-based qPCR was performed using the qRT-PCR Primer Assay (Qiagen, Valencia, CA, United States) with the following human primers: (1) human fVIII (qRT-F8-FWD: 5′-ATGCACAGCATCAATGGCTAT-3′, qRT-F8-REV: 5′-GTGAGTGTGTCTTCATAGAC-3′) and (2) human vWF (qRT-vWF-FWD: 5′-GGATTCAGTGGATGCAGCAG-3′, qRT-vWF-REV: 5′-TAGGGAGGTCTTCGATTCGC-3′) ( Trevisan et al, 2021 ). Commercially available primers for Human GAPDH were used as an internal reference housekeeping gene (Qiagen, Valencia, CA, United States).…”