[Transgenic human thymopoiesis from retrovirally transduced umbilical cord blood hematopoietic stem cells: experimental studies in the SCID-hu mouse]. / Thymopoïèse humaine transgénique à partir des cellules souches hématopoïétiques du sang de cordon ombilical génétiquement modifiées par voie rétrovirale: étude expérimentale dans la souris SCID-hu.

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.

A cell surface marker gene transferred with a retroviral vector into CD34+ cord blood cells is expressed by their T-cell progeny in the SCID-hu thymus.

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T-cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.