Gene transfer into nonhuman primate CD34+CD11b- bone marrow progenitor cells capable of repopulating lymphoid and myeloid lineages.

We investigated whether rhesus monkey CD34+CD11b- hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b- cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marrow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b- bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency.

Retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells: the effect of viral titers on transduction efficiency.

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

Factors affecting the transduction of pluripotent hematopoietic stem cells: long-term expression of a human adenosine deaminase gene in mice.

An amphotropic retroviral vector, LgAL(delta Mo + PyF101) containing a human adenosine deaminase (ADA) cDNA was used to optimize procedures for the lasting genetic modification of the hematopoietic system of mice. The highest number of retrovirally infected cells in the hematopoietic tissues of long-term reconstituted mice was observed after transplantation of bone marrow (BM) cells that had been cocultured in the presence of both interleukin-1 alpha (IL-1 alpha) and IL-3. A significantly lower number was detected when IL-1 alpha was omitted from such cocultures. The yield of cells that generate spleen colony-forming cells (CFU-S) in the BM of lethally irradiated recipients (MRA-CFU-S) significantly improved on inclusion of the adherent cell fraction of cocultures in the transplant. Retroviral integration patterns in MRA-CFU-S-derived spleen colonies showed that an MRA-CFU-S can produce many CFU-S during BM regeneration. Expression of hADA was detected in the circulating white blood cells of long-term reconstituted animals, demonstrating that the LgAL(delta Mo + PyF101) vector is capable of directing the sustained expression of hADA, and in approximately 35% of the transduced MRA-CFU-S-derived spleen colonies. These results should facilitate the development of gene therapy protocols for the treatment of severe combined immunodeficiency caused by a lack of functional ADA.

IL-6 production by retrovirus packaging cells and cultured bone marrow cells.

Retrovirus integration into the host cell genome occurs most efficiently in replicating cells. In agreement with this notion, it was observed that the efficiency with which hemopoietic stem cells (HSC) can be transduced is greatly enhanced when the hemopoietic growth factor (HGF) interleukin 3 (IL-3) is added to co-cultures of bone marrow cells with retrovirus-producing cells. The HGF IL-6, which enhances the IL-3-induced formation of blast cell colonies in vitro, is also believed to improve the transduction of HSC. Because IL-6 can be produced by a number of different cell types, we investigated whether IL-6 was present in the culture supernatant of retrovirus packaging cells and bone marrow cells. We found that the six retrovirus packaging cells tested produced large amounts of IL-6. Bone marrow cells cultured with IL-1 alpha and IL-3 also make IL-6, and, following co-cultivation of both cell types, the concentration of IL-6 in the medium is even up to 10-fold higher than the sum of the concentrations obtained when both are cultured separately. Considering that IL-6 is produced in large amounts during co-cultivations, we believe that its effect on the transduction of HSC cannot be measured by adding extra growth factor to the co-culture medium.

Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses.

Amphotropic recombinant retroviruses were generated carrying sequences encoding human adenosine deaminase (ADA). Transcription of the human ADA gene was under control of a hybrid long terminal repeat in which the enhancer from the Moloney murine leukemia virus was replaced by an enhancer from the F101 host-range mutant of polyoma virus. Hemopoietic stem cells in murine bone marrow were infected with this virus under defined culture conditions. As a result, 59% of day-12 colony forming unit spleen (CFU-S) stem cells became infected without any in vitro selection. Infected CFU-S were shown to express human ADA before transplantation and this expression sustained upon in vivo maturation. Mice transplanted with infected bone marrow exhibited human ADA expression in lymphoid, myeloid, and erythroid cell types. Moreover, human ADA expression persisted in secondary and tertiary transplanted recipients showing that human ADA-expressing cells were derived from pluripotent stem cells. These characteristics of our amphotropic viruses make them promising tools in gene therapy protocols for the treatment of severe combined immunodeficiency caused by ADA deficiency. In this respect it is also relevant that the viral vector that served as backbone for the ADA vector was previously shown to be nonleukemogenic.

Retrovirus-mediated gene transfer into embryonal carcinoma and hemopoietic stem cells: expression from a hybrid long terminal repeat.

Retroviral vectors can be used as an efficient gene delivery system in a wide variety of cell types. However, in some cell types, such as embryonal carcinoma (EC) cells or normal bone-marrow cells the expression of genes introduced by retroviral vectors has been very inefficient. This expression block has severely hampered the application of retroviral vector systems in those cell types. The enhancer sequences present in the long terminal repeat (LTR) of retroviruses are known to be responsible for the tissue specificity of viral expression. Therefore, we set out to construct a vector in which this enhancer element has been replaced. A recombinant retrovirus was constructed in which the enhancer from the Moloney murine leukemia virus LTR was replaced by the enhancer of a mutant polyoma virus (PyF101) that was selected to grow on EC cells. A neomycin-resistance marker (neoR) was placed under the transcriptional control of the hybrid LTR. Following infection with this virus, neoR was expressed in EC cells, as well as in the hemopoietic progenitor cells present in normal murine bone marrow. Moreover, upon transplantation of infected bone marrow cells into lethally irradiated mice, neoR expression was sustained in hemopoietic cells of the engrafted recipients.