In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion (2021)

Abstract 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&amp;lt;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.