Transamniotic Fetal Administration of Genetically Modified Hematopoietic Stem Cells Carrying a Human Transgene in a Syngeneic Rat Model

Background: Hematopoietic stem cell (HSC)-based gene therapy has long been proven viable experimentally for the treatment of certain hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders, having already reached clinical reality in a few applications. Fetal administration of genetically modified HSCs has only been feasible to date via invasive and therefore considerably morbid methods. However, it has been recently shown that native donor HSCs can actually reach the fetal circulation and bone marrow after simple delivery into the amniotic fluid, at least in a syngeneic healthy model. We sought to determine whether the transamniotic route could also be a practical alternative for the fetal administration of genetically-modified HSCs in a comparable model.

Methods: After IACUC approval, pregnant Lewis rat dams underwent volume-matched (50μL) intra-amniotic injections in all their fetuses (n=47) on gestational day 17 (E17; term=E21-22) of donor HSCs (2x10^6 cells/mL) genetically modified using a custom lentiviral vector designed to constitutively express both a firefly luciferase reporter gene and a human adenosine deaminase (ADA) transgene (figure). The gene for ADA was chosen because of the single gene mutation that causes congenital ADA deficiency, a well-known disorder that affects fetal development, leads to severe immunodeficiency, and yet has shown to be curable by gene therapy. Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid, which were at least 80% positive for CD34, CD117, SSEA3, SSEA4, Sox2, Oct4, and alkaline phosphatase on flow cytometry. Transduction was confirmed by both RT-qPCR and ELISA prior to administration in vivo. Fetuses were euthanized at term, when 7 select sites relevant to HSC-based therapies were screened for either luciferase activity by luminometry or for presence of human ADA mRNA by digital droplet PCR (ddPCR). Preference was given to the more sensitive and specific ddPCR. Positive luminescence was defined as relative light unit values exceeding the mean plus 2 standard deviations of blank wells. For ddPCR, values above any background noise from the no-template primer wells were taken as positive; fetal tissue from non-manipulated Lewis rats were used to confirm primer specificity.

Results: Among survivors (30/47; 64%), positive luminescence and positive human ADA expression were detected in the bone marrow (respectively 33% and 76%), liver (respectively 11% and 81%), spleen (respectively 11% and 67%), thymus (respectively 33% and 67%), lungs (respectively 44% and 86%), and brain (respectively 22% and 90%). Nucleated peripheral blood cells were analyzed only by ddPCR, showing positive human ADA expression at 54%.

Conclusion: Genetically modified hematopoietic stem cells can reach the fetal circulation and fetal bone marrow after simple intra-amniotic administration in a syngeneic rat model. Gene therapy via transamniotic hematopoietic stem cell delivery may become a practicable, minimally invasive strategy for the prenatal treatment of select hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders.