251. Intrauterine Haemopoietic Stem Cell Therapy Followed By Postnatal Cell Re-Infusion To Enhance Engraftment in a Murine Thalassaemia Model
Authors: Jerry Chan, Lay-Geok Tan, Niraja Dighe, Kang-Wei Tan, Dedy Sandikin, Mahesh Choolani, Citra Mattar
Journal: Molecular Therapy (2015)

Intrauterine haemopoietic stem cell therapy (IUHSCT) can be considered for treating haemoglobinopathies and certain metabolic diseases in early pregnancy, for the ability to easily escalate cell dose for higher engraftment and to target the pre-immune fetus. The potential therapeutic benefits are currently unrealised due to sub-therapeutic cell engraftment from host cell competition for haemopoietic niches or neutralising immune response. We investigated the strategy of IUHSCT followed by post-natal donor cell re-infusion to achieve clinically-relevant long-term engraftment in an HbbTh3-/+ mouse model of thalassaemia. Congenic IUHSCT was performed with murine fetal liver-derived mononuclear cells (MNC, 20% Lin-), followed by selective postnatal donor cell re-infusion to microchimeric pups. Adult bone marrow MNC were used when fetal liver was unavailable. Both injected pups and donor cells were derived from C57BL6 strains, with the former expressing surface antigen CD45.2. Donor cells expressed CD45.1 or GFP and were CD26-inhibited with Diprotin A prior to transplantation. Each pup received 2 or 5E+6 GFP or CD45.1 cells in utero at E14-17. Surviving pups were examined serially for chimerism after weaning. Chimeric mice (>1% engraftment) were treated with IV busulphan and received serial postnatal transplantation of the same strain of donor cells. Increasing cell doses ranging from 5E+6, and 1E+7 to 4E+7 cells per mouse were administered at six to 20 weeks postnatal. Initial donor cell engraftment exhibited a dose-response relationship following IUHSCT. Median chimerism in pups receiving 2E+6 cells in utero was 2% (range 1-6%) and 3% (3-5%) at three and eight postnatal weeks respectively. In comparison, recipients of 5E+6 cells showed 6% chimerism at three weeks (2-7%, p=0.1). Postnatal reinfusions maintained chimerism only transiently. The first dose of 5E+6 cells/pup maintained chimerism at 2-5% for the following eight weeks, and this steadily declined to 1% over the next four weeks. The second postnatal dose (1E+7 cells) had no effect as chimerism decreased to 0-2% by 20 weeks. Donor cell levels increased to 2-6% following the third dose (3E+7 cells) before becoming undetectable levels by 32 weeks. Following IUHSCT with 5E+6 cells, postnatal reinfusion (4E+7 cells) achieved stable chimerism of 4% (range 3-5%) for the following 12 weeks, eventually declining to 2% by 16 weeks. Unchallenged chimeric pups showed a steady decline in engraftment to <1% by 16 weeks (p=0.1). High cell doses are required to overcome host competition to reach engraftment levels of >2%. Sub-therapeutic chimerism predicts engraftment failure despite postnatal boost, possibly reflecting a non-tolerant environment despite fetal exposure to donor cells. However the main problem remains an inability to sustain a relevant chimerism long-term. Immune-modulation of recipients may have to be considered to improve outcomes.