Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model.

Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x10⁷ T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns.

Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2).

Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels.

Dave:Vitara Biomedical: Ended employment in the past 24 months.