Engraftment of genetically modified bone marrow cells in sensitized hosts

Expression of retrovirally transduced genes in bone marrow-derived cells can be used to establish stable long-term B- and T-cell tolerance. To determine whether preexisting antibodies may prohibit the use of gene therapy to establish tolerance, we examined the extent to which preexisting antibodies specific for the carbohydrate antigen Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) could affect engraftment and development of bone marrow progenitors expressing the enzyme UDPgalactose: beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 2.4.1.151), or simply alphaGT, which synthesizes the alphaGal epitope. Groups of alphaGT knockout mice (GT(o) mice) lacking alphaGal were presensitized to alphaGal by immunization and then lethally irradiated and reconstituted with varying numbers of alphaGT-transduced syngeneic bone marrow cells. Whereas unimmunized controls were reconstituted with as few as 2 X 10(6) transduced cells, a significant fraction of immunized mice reconstituted with 2 x 10(6) or 4 x 10(6) alphaGT-transduced cells failed to undergo bone marrow engraftment and died. Immunized mice in which radiation protection was achieved failed to express alphaGal. However, radiation protection and expression of alphaGal on bone marrow-derived cells, resulting in tolerance, could be achieved by increasing the number of transduced cells used to reconstitute immunized mice. Thus, although high levels of preexisting antibodies can be a significant barrier to engraftment, this barrier can be overcome by increasing the number of transduced cells used for reconstitution.