Administration of Cells Engineered to Secrete Fviii-mcoET3 in Prenatal Sheep Recipients Results in Sustained Curative Fviii Plasma Levels for 3 Years after Birth, without Immune or Toxicity-Related Adverse Events

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 (T_h1) and IL-4 (T_h2) were performed at 3 different time points. No mcoET3-specific T_h1 or T_h2 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.