O2150: Transamniotic Delivery of Hematopoietic Stem Cells Genetically Modified to Carry a Human Hemoglobin Subunit Beta Gene (HBB) in a Healthy Rodent Model

Background: Genetic mutations in the hemoglobin subunit beta gene (HBB) can lead to a variety of congenital disorders including sickle cell disease and β-thalassemia, both of which may have severe manifestations already in the perinatal period. Hematopoietic stem cell (HSC)-based gene therapy for these diseases has long proven viable experimentally and have reached clinical trials postnatally. Transamniotic stem cell therapy (TRASCET) with HSCs has emerged experimentally as an effective, minimally invasive route of prenatal administration of autologous HSCs. From the amniotic fluid, donor HSCs have been shown to reach the fetal circulation and bone marrow via hematogenous routing through the placenta. In this study, we sought to determine whether TRASCET could be a practical alternative for the fetal administration of genetically modified HSCs carrying a human HBB gene (hHBB) in a healthy syngeneic rat model.

Methods: After IACUC approval, time-dated pregnant Lewis dams underwent volume-matched intra-amniotic injections in all their fetuses (n=61) of a suspension of donor HSCs (2x106 cells/mL) genetically modified using a custom lentiviral vector designed to permanently insert either both a hHBB gene and a firefly luciferase reporter gene (hHBB group, n=42; figure A) or the firefly luciferase reporter gene only to control for HBB-derived protein interspecies homology (control group, n=19) on gestational day 17 (E17; term=E21). Donor HSCs consisted of syngeneic cells isolated from Lewis rat amniotic fluid documented to be at least 80% positive for CD34, CD117, SSEA3, SSEA4, Sox2, Oct4, and alkaline phosphatase on flow cytometry. Successful transduction was confirmed by ELISA prior to HSC administration in vivo. At term, fetal samples from five anatomical sites relevant to hematopoiesis were screened for the presence of human hemoglobin subunit beta by ELISA. In addition, multiple random samples from the hHBB group were also screened for the presence of the hHBB gene by digital droplet PCR (ddPCR). Tissue from control fetuses were used to confirm primer specificity. Statistical analysis was by Wilcoxon rank sum test for ELISA and Fisher’s exact test for survival analyses (p < 0.05).

Results: There was no maternal mortality and no premature deliveries. Overall fetal survival was 61% (37/61), with no significant difference between the groups (p=0.784). When controlled by HSCs without hHBB injections, human hemoglobin subunit beta production was documented at term in the fetal bone marrow and spleen (p < 0.001 and p=0.028 respectively; figure B). Positive hHBB expression by ddPCR was detected in the spleen (54%), bone marrow (46%), blood (46%), liver (23%), and thymus (15%).

Conclusion: Genetically modified hematopoietic stem cells carrying a human hemoglobin subunit beta gene can reach fetal hematopoietic sites after simple intra-amniotic injection in a healthy syngeneic rat model. Transamniotic hematopoietic stem cell-based gene therapy could become a novel strategy for the perinatal management of select hemoglobinopathies.