Long-term genetic modification of rhesus monkey hematopoietic cells following transplantation of adenoassociated virus vector-transduced CD34+ cells.

We have explored the potential of recombinant adenoassociated virus (AAV) vectors for gene transfer of the human beta-globin gene and the genetic modification of primate pluripotent hematopoietic stem cells (P-PHSCs). Transduction of P-PHSCs was tested in a preclinical bone marrow transplantation model in rhesus monkeys. CD34+ cells were transduced ex vivo and autologously transplanted without prior selection into irradiated rhesus monkeys. Vector-transduced peripheral blood mononuclear cells and granulocytes were present in the circulation for more than 15 months after transplantation. Approximately 1 in 10(5) cells in the circulation was vector modified. The vector was detected in the bone marrow, in granulocytes, and in purified populations of B and T cells, thus demonstrating multilineage repopulation by vector-transduced stem cells. Comparison of transduction protocols suggested that short-term culture of P-PHSCs enhances transduction and subsequent repopulation by rAAV-transduced cells. These results demonstrate that rAAV vectors can be used for the permanent genetic modification of a rhesus monkey hematopoietic system in the absence of selective pressure.

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.

Gene therapy for adenosine deaminase deficiency.

In the last decade, gene transfer into hematopoietic cells has evolved from an experimental procedure which resulted in successfully transduced in vitro hematopoietic colonies to the first clinical trials in patients suffering from severe combined immunodeficiency disease caused by the absence of functional adenosine deaminase. Significant in vivo expression of the newly introduced gene encoding human adenosine deaminase has been observed in descendents of murine and rhesus monkey hematopoietic stem cells following retrovirus mediated gene transfer. So far, 10 patients have received genetically repaired T-cells, hematopoietic stem cells or both without the appearance of any side effect. The clinical bone marrow gene transfer studies differ largely from the monkey studies with respect to myeloablation, which was applied in the monkey studies, but not in the patient studies. Ongoing studies in patients show that the introduced gene is present in circulating blood cells. In the initial phase of the trial, the frequency of transduced circulating blood cells is lower than in rhesus monkey studies. This difference may be contributed to the fact that conditioning was not performed in the patients.

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.

Bone marrow gene therapy for adenosine deaminase deficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency disease (SCID). The cause for this is believed to be the accumulation of one of the substrates for ADA, 2'-deoxyadenosine to which especially T cells are hypersensitive. This disease can be treated successfully with bone marrow transplantation if a suitable donor is available. Alternatively, the human ADA gene could be introduced into the autologous bone marrow. We have generated a retroviral vector containing the human ADA gene. With this vector we were able to restore human ADA-activity in ADA-SCID T cells to normal levels resulting in a sensitivity to 2'-deoxyadenosine that is also found for T cells from a healthy donor. In murine studies we have shown that our retrovirus can infect pluripotent hemopoietic stem cells resulting in long-term (> 6 months) expression of human ADA in the hemopoietic system of transplanted animals. These results were confirmed in rhesus monkeys where we were able to detect the provirus in both peripheral blood mononuclear cells and granulocytes for as long as the animals were analyzed, i.e. up to more than 1 year post bone marrow transplantation. On the basis of these results we have proposed a clinical protocol for the treatment of ADA-SCID patients with bone marrow gene therapy.

Toward gene therapy in haemophilia A: retrovirus-mediated transfer of a factor VIII gene into murine haematopoietic progenitor cells.

To study and evaluate the potential of the haematopoietic system as a target for gene therapy in haemophilia A, we have infected murine bone-marrow cells with a recombinant retrovirus encoding blood-coagulation factor VIII and the bacterial enzyme neomycin-phosphotransferase. After transplantation of the infected bone marrow into lethally irradiated mice, the presence of intact vector could be demonstrated in DNA isolated from individual haematopoietic progenitor-cell-derived spleen colonies. About 8% of the spleen colonies were shown to contain the intact vector. Selection for resistance to the neomycin analogue G418 prior to transplantation specifically killed the uninfected bone-marrow cells and, as a result, over 90% of the spleen colonies contained the factor VIII vector. However, expression of factor VIII in vivo, either at the RNA or at the protein level could not be demonstrated. From these data we conclude that: 1) retroviral vectors can be used to transfer factor-VIII cDNA into haematopoietic progenitor cells; 2) the vector sequences are expressed immediately after integration; and 3) transcription of the vector is repressed in the progenitor-cell-derived cells.

Construction and expression of an adenosine deaminase::lacZ fusion gene.

A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of beta Gal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.

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.