Prenatal Transplantation of Placental Cells Engineered to Express FVIII Leads to Corrective Plasma Levels of FVIII after Birth

Prenatal transplantation (PNTx) is a clinically viable procedure that has been performed in ~50 human patients for 14 different genetic disorders. When performed using a minimally invasive, ultrasound-guided approach, there have been no reported adverse events, proving that this procedure poses minimal risk to both the fetus and the mother. Hemophilia A (HA) is an ideal disease to treat by PNTx, since 75% of HA patients have a family history of HA, and prenatal diagnosis is feasible, available, and encouraged in most Western and developing countries. Moreover, digital PCR now allows analysis of free fetal DNA in maternal plasma, and can diagnose HA in utero non-invasively as early as 7 weeks. Successful PNTx for HA could promise the birth of a healthy infant who required no further treatments, removing heavy burdens on patients, their families, and the healthcare system. Even if not curative, a modest increase in FVIII levels, provided by the transplanted FVIII-expressing cells, would convert a severe, life-threatening bleeding disorder to a mild phenotype, devoid of spontaneous bleeding events, significantly reducing the need for postnatal treatments. In an effort to develop an off-the-shelf therapy that could be provided to HA patients prior to birth, we first performed studies to determine the optimal cellular vehicle for delivering FVIII for prenatal therapies. Analysis in vitro of 3 human cell sources - amniotic fluid, bone marrow stromal cells, and placental cells - demonstrated that placental cells (PLCs) were the most robust at FVIII production and secretion following lentiviral vector (LV) transduction. Based on these promising results, human PLCs were isolated based on c-Kit positivity under GMP conditions, and a master cell bank established for a definitive preclinical study and for future clinical trials. Characterization prior to transduction demonstrated that PLCs (Passage 3-15) expressed vWF, and constitutively produced and secreted functional FVIII at 0.4-0.5 IU/10⁶ cells/24h, demonstrating that these cells possess all of the requisite machinery to efficiently produce and process this complex protein. Upon transduction at passage 3 with LV encoding a bioengineered and codon optimized FVIII transgene, designated mcoET3, under the control of the EF1a promoter, PLCs stably secreted 27.9 IU FVIII/10⁶ cells/24h. To assess the relative safety of these gene-modified cells, qPCR was performed with vector-specific primers, and amplification compared to a known single-copy gene. These analyses yielded a vector copy number of 0.35±0.05 per diploid genome equivalent. To test the therapeutic potential of this PNTx-based treatment for HA, EF1α-mcoET3-LV transduced and expanded PLCs were transplanted into sheep fetuses (n=4) at a dose of 10⁷ cells/kg fetal weight, at 65 gestation days (gd; term 145 gd), the equivalent of 16-17 gestation weeks in humans, using a clinically-employed ultrasound-guided injection procedure. Evaluation at 1 month after birth (4 months post-transplant), of the 1 male and 1 female animal that have been born to-date, showed that they had a 42% and 82% increase, respectively, in plasma FVIII activity levels, as measured by aPTT, when compared to control non-transplanted animals. Of particular note is that plasma analysis at 2 months of age (5 months post-transplant) demonstrated that plasma FVIII levels were maintained at 30% and 71%, in the male and female, respectively, despite the more than tripling in plasma volume (540 ml to 1822 ml) in the male and the 2.6-fold increase in plasma volume (540 ml to 1395 ml) in the female, that occurred during this neonatal and infancy period of rapid growth. ELISA analyses performed on the plasma samples, using an antibody specific to ET3-FVIII, confirmed the results obtained by aPTT. In conclusion, we have shown that human PLC represent an ideal off-the-shelf cellular vehicle for delivering a FVIII transgene, and that PNTx with PLC engineered to express an expression/secretion-optimized variant of FVIII (mcoET3) results in curative plasma levels of FVIII following birth, in a large preclinical animal model.