Prenatal transplantation of a clinical cell product engineered to secrete FVIII in a sheep with Hemophilia A corrects the severe bleeding phenotype and prevents hemarthroses

Abstract Prenatal treatment (PNT) of people with hemophilia A (HA) is a feasible, safe, yet unexplored clinical approach, consisting of an ultrasound-guided injection at 18-24 gestational wks (gw) that poses minimal risk to the fetus and mother. We manufactured an off-the-shelf GMP-compliant biological product consisting of human placental cells (PLC) transduced with a lentivector encoding a bioengineered high-expression fVIII transgene (mcoET3). PLC-mcoET3 secreted FVIII >10 IU/106cells/24h, and safety tests showed a VCN<5, the absence of adventitious agents and replication-competent lentivirus, and integration site analysis revealed no insertional mutagenesis risks. To test the in vivo safety and efficacy of this product, sheep HA carriers (n=5) were bred, and fetuses (n=8) received an intraperitoneal injection of 108/kg PLC-mcoET3 at 60-64 gestational days, corresponding to 18-24gw in humans. Of these 8 fetuses, 4 were female carriers, 3 unaffected sheep, and one was a hemophiliac male. As we previously reported, sheep with HA have a premature stop codon and frame-shift in exon 14, are cross-reactive material negative, and exhibit a severe bleeding phenotype starting at birth. All HA animals born from previous pregnancies had frequent spontaneous hemarthrosis, muscular hematomas, and episodes of hematuria and internal bleeding, leading to death if not treated. These HA sheep were housed individually and in padded environments and were bottled-fed, as kneeling to nurse caused hemarthrosis. Although these animals responded to hFVIII (»3600 IU FVIII in 3 months and >10 treatments), they bled from IV site/puncture and developed debilitating hemarthroses, leading to reduced locomotion and severe defects in posture and gait, with high-titer inhibitors detected in the majority of animals after hFVIII infusion. PNT with PLC-mcoET3 of animals that were born unaffected or were HA carriers resulted in increased levels of FVIII activity as we previously described with an equivalent non-GMP product (Nat.Commun. 2023,14(1):4206). Here we report, for the first time, that PNT of a HA fetus with PLC-mcoET3 resulted in the birth of a healthy lamb (5.75 kg). Despite the inadvertent trauma (dragging) to the umbilical cord caused by his twin sibling, this sheep was well and was soon seen kneeling and nursing. Cord tissue was collected for RFLP diagnostic test, as per protocol for all animals born from HA carriers. Around 8h post-birth, bleeding from the umbilical stump was detected. Because the RFLP diagnostic test was underway, it was not possible to distinguish between late bleeding due to umbilical trauma or HA. No other signs of bleeding were observed at this point such as hematomas, hemarthrosis, or mucosal bleeding; nevertheless, hFVIII (250U) was administered. Intradermal fluids were also administered, and the sheep was monitored for 2-3 hrs. No bleeding or hematoma was formed after injection of intradermal fluids, nor was any bleeding seen from the IV site. The bleeding from the umbilical cord stopped, and he was returned to his mother, whereupon he immediately began nursing. On the next day, RFLP results demonstrated that he was affected by HA. The decision was made to allow him to nurse naturally, instead of bottle-feeding, and as such, he was housed with the ewe and his twin. 2 and 4 weeks, later he exhibited lameness in the left hindlimb and right elbow, respectively. Although no measurable swelling of the joints was noted, since potential bleeds could not be excluded, the decision was made to treat with hFVIII 350IU (weight 6.45Kg) and 750 IU (weight 14Kg). Prior to administration of hFVIII, citrated plasma was collected and sent for FVIII measurement. Plasma was reported to have clots, so it was difficult to determine FVIII levels with certainty, but reported values varied from 1-4.1%. No anti-FVIII IgG developed after 3 infusions of hFVIII. The HA lamb is now 5 months, weighs 32kg, lives in his natural environment, and has not received any additional FVIII treatment. He never experienced hematuria or spontaneous mucosal bleeding, and he continued to kneel to nurse without developing hemarthroses. The few mild bruises/hematomas and episodes of slight lameness (that did not preclude mobility) were caused by traumas, and they spontaneously resolved in 24-48h. These data show the feasibility of treating HA prenatally and converting a severe phenotype to moderate, eliminating hemarthroses and allowing for an active sheep lifestyle.