OSTEOPROGENITOR CELLS AS TARGETS FOR EX-VIVO GENE-TRANSFER

We transduced osteoprogenitor cells with recombinant retrovirus and an alysed proviral integration patterns into chromosomal DNA to detect fo r the first time the clonal and cellular fate of osteoprogenitor-deriv ed progeny cells, Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with th e vM5neolacZ recombinant retrovirus, and selected in the neomycin anal og, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells wer e injected into the ablated site. These rats were killed 5-6 days late r. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determine d. High numbers of beta-galactosidase colonies with an osteoblast phen otype-were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, bet a-galactosidase colonies derived from diaphyseal transplants were dete cted in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than met aphyseal transplants, Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integrati on fragment analysis showed a single integration site for recovered me taphyseal cell clones, consistent with their origination from a common single progenitor, This is one of the first demonstrations of success ful transplantation of clonal osteoprogenitors to their site of origin in bane, It may be possible to use these cells to target genes to bon e for therapeutic use in skeletal and hematopoietic diseases.