These findings attest to the successful introduction of the retroviral vector into long-lived haematopoietic progenitors present in the umbilical cord blood. The ability of haematopoietic stem cells to engraft in vivo
after clinical bone marrow transplantation without ‘making space’ with cytoablative therapy has long been controversial. Recent experiments with murine bone marrow transplantation models indicate that haematopoietic stem cell engraftment is possible without the use of cytoablative therapy15–17
. Our results demonstrate that long-lived human haematopoietic progenitor cells present in umbilical cord blood are capable of engrafting in neonates without the administration of cytoablative therapy.
The absolute frequency of circulating leukocytes containing the LASN vector (0.03–0.001%) is in accord with theoretical expectations. In preclinical studies of gene transfer/bone marrow transplantation in large animals and retroviral-mediated gene marking of human bone marrow from patients with cancer, a maximum of 1% of the haematopoietic stem cells have been transduced18-20
. The efficiency of gene transfer into the umbilical cord haematopoietic stem cells was, therefore, most probably in the 1% range. Because the infants did not receive cytoablative therapy, the endogenous bone marrow mass was still present. Assuming that the infused umbilical cord blood cells represent 1% of the endogenous marrow mass, dilution of the infused cord blood cells would result in a final frequency of gene-transduced haematopoietic stem cells in the range of 0.01%. To achieve higher levels of vector-containing stem cells, it would be necessary to increase gene transfer efficiency and/or to administer cytoablative therapy before transplantation of the transduced cells.
Evaluation of the patients’ bone marrow one year after treatment was highly informative. The frequency of vector-containing progenitor cells exceeds by 100-fold the frequency of vector-containing cells in the mature haematopoietic cell compartments. The explanation for this dichotomy is unknown. Potentially, the expression of the ADA gene is beneficial for progenitor cell proliferation and allows expansion of the committed CD34+ progenitor pool in a fashion similar to that expected for T-Iymphoid progenitors. However, the relatively high frequency of progenitor cells containing the vector is not reflected in mature leukocytes. In studies in which normal mice were transplanted with bone marrow without prior cytoablation, higher frequencies of donor cells were seen in the early haematopoietic progenitor cells than in the mature peripheral leukocytes17
. This observation suggests that although primitive progenitor cells . may engraft without cytoablative therapy, they fail to undergo complete maturation in vivo.
Alternatively, the presence of the vector may interfere with mature haematopoietic cell production. Some reports have suggested that the neomycin phosphotransferase gene (neo
) may impair haematopoietic cell function21,22
. The high level of ADA expression seen in progenitor-derived colonies could also be harmful to developing cells.
There have been no adverse effects from the administration of gene-modified umbilical cord blood cells. The continued administration of ADA enzyme replacement therapy has allowed the patients to develop normal immune function and to remain free of infections. To be successful, the PEG-ADA therapy must lower the levels of toxic deoxyadenosine metabolites, and would, therefore, blunt any selective survival advantage afforded to T-lymphocyte precursors by vector-derived ADA expression. Based on the stable presence of vector-containing cells, we have recently decreased the dosage of PEG-ADA administered from 60 U kg–1 per week to 30 U kg-1 per week.
ADA deficiency has been the focus for the initial attempts of gene therapy with haematopoietic stem cells, because transduced T-lymphoid progenitors are expected to have a selective survival advantage in vivo, similar to that seen in allogeneic BMT for ADA deficiency. The present studies demonstrate that primitive haematopoietic cells present in umbilical cord blood can be genetically modified and engrafted without the administration of cytoablative therapy. A similar approach may be used in the future to treat other genetic diseases. Clinical benefits may be expected even with the present inefficient gene transfer techniques if the progeny of the transduced progenitor cells have a selective advantage in vivo. The successful application of gene therapy to other haematologic disorders where the transduced progenitors do not have a selective advantage, such as haemoglobinopathies, lysosomal storage disorders, and AIDS, will require more efficient gene transfer. These advances will require better understanding of the biology of haematopoietic stem cells and the cytokines that regulate their proliferation.