Rescue of Hemostasis-deficiency States

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Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term, but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting liver cells from FX+/+ Rosa26 fetuses into midgestation embryos-derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinataily, similar to untreated FX-/- mice. The plasma levels of FX in the rescued 16-week genotypically FX-/- mice were approximately 2-5% of age-matched wild-type levels. Beta-galactosidase-staining cells, derived from the donor Rosa26 fetal liver cells, were detected in approximately 45% of the livers of adult mice. The observations suggest that this rescue approach can be used to treat potential hemophiliacs detected in utero. The aims of this proposal are to better define and modify the procedure to develop: 1) a therapeutic strategy to treat deficiencies of liver-expressed proteins; 2) a research protocol for conditional rescue of critical hemostatic factors. This latter aim involves characterizing the donor cell population to identify the cell subpopulations possessing liver-colonizing properties. It is also proposed to develop cultured cell lines with colonizing activity, thus eliminating the need for primary cells. Furthermore, the donor cells will be modified genetically to increase their efficacy for this strategy. This will involve increasing the levels of expression of the factor of interest, or increasing the liver-colonizing potential of the donor cell lines. The introduction of negative selection markers into the donor cell population would enable the eradication of the donor cell population at a chosen time. This permits the approach to be used as a conditional rescue strategy to study the physiological consequences of the deficiency of particular genes in adult animals, when the same deficiency would normally result in embryonic or perinatal lethality. In particular, this strategy will be used to study the pathophysiological consequences of FX deficiency in vivo in older mice, a feat that cannot be accomplished at present.