In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion

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.

Bisphenol A, Bisphenol F, and Bisphenol S: The Bad and the Ugly. Where Is the Good?

Background: Bisphenol A (BPA), a reprotoxic and endocrine-disrupting chemical, has been substituted by alternative bisphenols such as bisphenol F (BPF) and bisphenol S (BPS) in the plastic industry. Despite their detection in placenta and amniotic fluids, the effects of bisphenols on human placental cells have not been characterized. Our objective was to explore in vitro and to compare the toxicity of BPA to its substitutes BPF and BPS to highlight their potential risks for placenta and then pregnancy. Methods: Human placenta cells (JEG-Tox cells) were incubated with BPA, BPF, and BPS for 72 h. Cell viability, cell death, and degenerative P2X7 receptor and caspases activation, and chromatin condensation were assessed using microplate cytometry and fluorescence microscopy. Results: Incubation with BPA, BPF, or BPS was associated with P2X7 receptor activation and chromatin condensation. BPA and BPF induced more caspase-1, caspase-9, and caspase-3 activation than BPS. Only BPF enhanced caspase-8 activity. Conclusions: BPA, BPF, and BPS are all toxic to human placental cells, with the P2X7 receptor being a common key element. BPA substitution by BPF and BPS does not appear to be a safe alternative for human health, particularly for pregnant women and their fetuses.

Delivery of Fviii-mcoET3 to Previously Untreated Sheep Using Human Placental Cells Enables Durable Elevation of Plasma FVIII Levels and Avoids Inhibitor Formation

Hemophilia A (HA) is characterized by a decrease in the functional clotting protein factor VIII (FVIII). Current hemophilia treatments consist of either prophylactic or on-demand administration of FVIII protein. Despite the vast improvements and availability of these treatments, which have increased the life expectancy and quality of life of HA patients, up to 30% of patients who receive FVIII infusions develop inhibitors to FVIII, rendering subsequent treatments ineffective and placing the patient at risk of a life-threatening bleeding event. The current method used to eradicate inhibitors is immune tolerance induction (ITI), in which the patient is repeatedly infused with high doses of FVIII protein to induce tolerance. While effective in some patients, ITI is extremely expensive, the mechanism whereby tolerance is induced is not well understood, and as many as 35% of patients fail to become tolerized. In this study, we created a Master Cell Bank of human placental cells (PLC) transduced with a lentiviral vector (0.5 vector copies/diploid genome equivalent) to produce high levels (4.9IU/10^6 cells/24 hr) of mcoET3, an expression/secretion-optimized FVIII protein (PLC-mco). We hypothesized that the immunomodulatory properties of PLC could be exploited to deliver FVIII while preventing inhibitor formation, and to determine whether the route of administration impacted the efficacy and safety of this cell-based treatment. To accomplish these objectives, we administered one of the following to normal healthy juvenile sheep recipients: a) 3 weekly IV infusions of 4x10^6 PLC-mco/kg (calculated to provide ~20IU/kg mcoET3 each 24 hr); or b) a single intraperitoneal (IP) infusion of 10^7 PLC-mco/kg or c) to enable comparison of the relative immunogenicity of mcoET3 when delivered as a bolus protein injection versus when constitutively secreted from transplanted PLC-mco, we also included a control/reference group in which we administered 5 weekly IV injections of 20 IU/kg recombinant mcoET3 protein. Prior to the first injection, and once per week for 5 weeks after the first injection, we collected blood from each animal and isolated plasma and peripheral blood mononuclear cells (PBMC). The plasma was used to determine FVIII activity by aPTT and to assess the presence of mcoET3-specific IgM and IgG by ELISA. PBMC were used to perform ELISpot assays to determine whether mcoET3-specific Th1 and Th2 cells developed following each infusion paradigm. After IV PLC injection, the sheep occasionally exhibited transient labored breathing; this was never observed in animals that received IV protein infusions nor those that received PLC via the IP route. One week after the IP injection of PLC-mcoET3, plasma FVIII levels had increased by 6.3% over baseline and then further increased to remain in a range between 63-108% for weeks 2-5. Following IV PLC infusions, sheep showed a 12.9% increase in plasma FVIII levels at 1-week post-infusion which persisted in weeks 2-4 and rose to a maximum of 30% five weeks after the first injection. As expected, given FVIII's short half-life, the sheep that received mcoET3 protein exhibited baseline plasma FVIII levels when evaluated 1 week after each IV protein infusion. To evaluate humoral immunity in these animals, ELISA analysis demonstrated that all sheep in this study were devoid of mcoET3-specific IgM antibodies at all time points. Similarly, no mcoET3-specific IgG antibodies were ever detected in sheep transplanted IV or IP with PLC-mco. In contrast, the IV infusion of recombinant mcoET3 protein resulted in the generation of a specific IgG response by 2 weeks after the first infusion. With respect to cell-mediated anti-mcoET3 immunity, ELISpot assays performed on PBMC demonstrated that the only cohort that developed Th1 or Th2 cells specific to mcoET3 was the one that received mcoET3 protein. In this group, Th2 cells became detectable by 3 weeks after the first protein infusion, while Th1 cells only became evident by week 4. Taken together, our results show that human PLC can be transduced to produce high levels of FVIII protein, and that these cells can then be transplanted into previously untreated animals to yield a prolonged increase in plasma FVIII levels. Our findings also support our hypothesis that delivering a fVIII transgene via PLC enables avoidance of an immune response to a FVIII protein (mcoET3) that is immunogenic to sheep when delivered as an IV bolus. Disclosures Doering: Expression Therapeutics, LLC: Current equity holder in private company, Patents & Royalties, Research Funding; Kilpatrick, Townsend & Stockton: Consultancy.