The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum–plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.

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.

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.

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We have previously reported that in utero transplantation (IUTx) of sheep fetuses (n=14) with human placental cells (PLC) transduced with a lentiviral vector encoding mcoET3, an expression/secretion-optimized, bioengineered fVIII transgene (PLC-mcoET3) increased plasma FVIII activity levels by 57%, 42%, and 35% at 1, 2, and 3 years post-IUTx, respectively, without the development of FVIII/ET3 inhibitors. We also demonstrated that immune tolerance to the cell/gene product was maintained after postnatal administration of PLC-mcoET3 (cells producing 20 IU/kg/24h were administered i.v. for 3 consecutive weeks). However, when IUTx-treated animals received weekly i.v. infusions of purified ET3 protein (20IU/kg) for 5 weeks, all recipients developed a robust ET3-specific IgG response that appeared at week 3 of infusion at titers ranging from 1:70 to 1:857 and inhibitory antibodies that ranged from 3-36 BU. Here, we investigated differences in the immune responses of animals that received IUTx with PLC-mcoET3 and were boosted postnatally with PLC-mcoET3 (IUTx-PLC-mcoET3) vs. ET3 protein (IUTx-ET3) to define the pathways by which the immune system differentially responds to protein vs. cell-secreted ET3. A sheep-specific multiplex gene expression analysis with 165 genes involved in immune cell signaling pathways (NanoString) was used to evaluate mRNA isolated from peripheral blood mononuclear cells collected at Weeks (W) 0, 1, and 5 of postnatal infusions. Significant fold-change expression in these mRNA targets was determined using NanoString nSolver 4.0 software. Animals in the IUTx-PLC-mcoET3 group (known to be devoid of inhibitors to ET3 post-boosting) showed that immunoregulation and immune tolerance gene clusters were among the top three clusters that increased expression from W0 to W5 (adj. p-value<0.01). Differential expression of genes in pathways involved in Th1, Th2, and Th17 responses was also found, at differing levels, in the IUTx-PLC group, suggesting a balance between immunity and tolerance was maintained. Surprisingly, the IUTx-ET3 group, which developed inhibitory antibodies after ET3 boosting, also showed significantly increased expression of immune tolerance genes, and downregulation of Th1 and Th17 cell signaling, when evaluated by direct global significance score. Nevertheless, 66% of these animals had a significant upregulation of Th2 cell signaling by W1 vs W0. To determine if the increase in expression of immune tolerance genes was due to the IUTx treatment, we also evaluated a group of aged-matched, non-transplanted sheep that received ET3 protein under the same dose and schedule. Results from Gene Set Analysis (GSA) demonstrated significant upregulation of genes involved in interferon signaling, class I MHC antigen processing, and Th17 signaling in these animals, suggesting the potential involvement of Th17 cells in the immune response in this group. In conclusion, IUTx with PLC-mcoET3 induces the upregulation of genes associated with immune tolerance, providing an explanation for the long-lasting elevation in plasma FVIII levels in these animals in the absence of inhibitors. Nevertheless, despite the continued expression of tolerogenic genes, administration of purified ET3 protein to these IUTx recipients induced upregulation of Th2 signaling, a pathway that was not observed in animals that only received ET3 protein, demonstrating that the mechanism by which tolerance is broken in IUTx recipients differs from that by which an immune response to ET3 occurs in animals with no prior exposure. Of note is that animals that develop inhibitors by the Th17 pathway had considerably higher inhibitor titers than the IUTx recipients that responded to ET3 infusion by the Th2 pathway. These studies underscore the need for a more complete understanding of the mechanisms by which immune tolerance to FVIII develops during ontogeny. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

The treatment of hemophilia A (HA) patients by prenatal transplantation (PNT) is a feasible, yet underestimated and unexplored clinical approach. The procedure, similar to that of an amniocentesis, poses minimal risk to both the fetus and the mother. Herein, we report on the PNT of sheep fetuses (n=23) with human placental cells (PLC) transduced with a lentiviral vector encoding a bioengineered high-expression fVIII transgene (mcoET3), at a dose of 107-108/kg at 60-64 gestational days, which corresponds to 16-18 gestational weeks in humans. The cells secreted 5.1-9.7IU-fVIII/106 cells/24h, with a vector copy number of 0.35-0.99 per diploid genome equivalent. 9 animals were lost to natural causes unrelated to the treatment or the procedure, or were euthanized for tissue histopathology. Fourteen animals were available for long-term evaluation. Animals followed up over a 1-year (n=14), 2-year (n=9), and 3-year (n=6) period had increased mean plasma fVIII activity levels of 62.1%, 45.6%, and 50.98% respectively, demonstrating that despite the rapid growth of the animals from approximately 0.1kg at the time of PNT to an average of 80kg by year 3, the fVIII produced by the transplanted cells was sufficient to maintain steady levels throughout the duration of the study. We also examined whether PNT-treated animals developed liver inflammation and/or mounted an immune response to the transplanted cells or the fVIII produced by the mcoET3 transgene. At all-time points post-PNT, hematological parameters and liver enzymes (AST, ALT, and alkaline phosphatase) were normal, demonstrating the absence of any liver toxicity. To determine if PNT-treated animals developed a humoral response to mcoET3, an ELISA was performed at 3-6 and 10-16 months postnatally and demonstrated that PNT-treated animals were devoid of anti-mcoET3 IgG. To establish whether these animals had developed memory T cell responses to mcoET3, ELISpot assays for IFN-g (Th1) and IL-4 (Th2) were performed at 3 different time points. No mcoET3-specific Th1 or Th2 cells were ever detected in any of the PNT recipients. To determine if the PNT recipients developed an immune response to the transplanted PLC, we performed one-way mixed lymphocyte reactions (MLR) against the transplanted PLC, and used a screening panel-reactive antibody test (PRA) to identify the development of anti-HLA antibodies specific for the HLA phenotype of the transplanted PLC. MLR demonstrated robust reactivity towards third-party human lymphocytes but not to the transplanted PLC. In addition, no PLC specific anti-HLA antibodies were found, however 2 of the treated animals had detectable levels of xenogeneic antibodies, towards other HLA phenotypes. RTqPCR analysis demonstrated engraftment of transplanted PLC in all major organs and histopathologic examination showed no evidence of any lentiviral-related or procedural toxicity in any tissue examined. In conclusion, PNT of fetal sheep recipients resulted in sustained high fVIII plasma levels for more than 3 years after birth, with no evidence of therapy-related toxicity, nor the development of fVIII inhibitors. Thus, these studies attest to the feasibility, immunologic advantage, and safety of treating HA prior to birth. Disclosures Doering: Kilpatrick, Townsend & Stockton: Consultancy; Expression Therapeutics, LLC: Current equity holder in private company, Patents & Royalties, Research Funding.

Membrane contact sites (MCSs) are regions in cells where membranes of two organelles are in close apposition. MCSs are proposed to facilitate non‐vesicular trafficking of lipids. Lipid transfer proteins (LTPs) are one of the most commonly‐found components in the MCS system and are speculated to mediate lipid transfer between membranes. However, both the biological functions of MCSs and the functional role of LTP in MCSs remain largely elusive. Herein, we examine in detail a previously‐proposed MCSs model, the ER‐Golgi/endosome MCS facilitating the phosphatidylserine (PtdSer) decarboxylation 2 (Psd2) pathway, which converts PtdSer into phosphatidylethanolamine (PtdEtn) in Saccharomyces cerevisiae. Particularly, we investigate the biological role of a yeast LTP, one of the yeast Phosphatidylinositol (PtdIns)‐Transfer Proteins (PITPs), Sfh4, and a PtdIns 4‐OH kinase Stt4, in this Psd2‐MCS system.We found that Psd2, Sfh4, and Stt4 are the only essential components facilitating Psd2‐dependent PtdEtn synthesis while other previously proposed elements constituting the Psd2‐MCS (Scs2, Scs22, and Pbi1) are dispensable. Surprisingly, neither the PtdIns‐transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via an Sfh4‐Psd2 physical interaction involving the F175 residue on the surface of Sfh4. Stt4 also displays physical interaction with Psd2 in an Sfh4‐independent manner. Instead of directly activating Psd2, Stt4 regulates the substrate pool accessible to Psd2, thereby indirectly controlling the Psd2‐mediated synthesis of PtdEtn. These results demonstrate that the ER‐endosomal MCS model is an inaccurate description of the Psd2 system in yeast, and provide an outstanding example where PITP biological function is uncoupled from its ‘canonical’ activity as a PtdIns transfer protein. Additionally, our findings reveal a novel mechanism in which a PITP and a PtdIns 4‐OH kinase regulate cellular phospholipid homeostasis.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (GM44530) and the Robert A. Welch Foundation (BE‐0117) to VAB. The authors declare no financial conflicts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.