Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation.

The transcription factor hypoxia inducible factor 1 (HIF1), an HIF1alpha-aryl hydrocarbon receptor nuclear translocator (ARNT) dimeric factor, is essential to the cellular response to hypoxia. We described a t(1;12)(q21;p13) chromosomal translocation in human acute myeloblastic leukemia that involves the translocated Ets leukemia (TEL/ETV6) and the ARNT genes and results in the expression of a TEL-ARNT fusion protein. Functional studies show that TEL-ARNT interacts with HIF1alpha and the complex binds to consensus hypoxia response element. In low oxygen tension conditions, the HIF1alpha/TEL-ARNT complex does not activate transcription but exerts a dominant-negative effect on normal HIF1 activity. Differentiation of normal human CD34+ progenitors cells along all the erythrocytic, megakaryocytic and granulocytic pathways was accelerated in low versus high oxygen tension conditions. Murine 32Dcl3 myeloid cells also show accelerated granulocytic differentiation in low oxygen tension in response to granulocyte colony-stimulating factor. Interestingly, stable expression of the TEL-ARNT in 32Dcl3 subclones resulted in impaired HIF1-mediated transcriptional response and inhibition of differentiation enhancement in hypoxic conditions. Taken together, our results underscore the role of oxygen tension in the modulation of normal hematopoietic differentiation, whose targeting can participate in human malignancies.

Using lentiviral vectors for efficient pancreatic cancer gene therapy.

Pancreatic cancer (PC) remains a life-threatening disease. Efficient therapeutic gene delivery to PC-derived cells continues to present challenges. We used self-inactivated lentiviral vectors to transduce PC-derived cells in vitro and in vivo. We showed that lentiviral vectors transduce PC-derived cell lines with high efficiency (>90%), regardless of the differentiation state of the cell. Next, we transferred human interferon beta (hIFN-beta) gene. Expression of hIFN-beta in PC cells using lentiviral vectors resulted in the inhibition of cell proliferation and the induction of cell death by apoptosis. In vivo, lentiviral administration of hIFN-beta prevented PC tumor progression for up to 15 days following gene therapy, and induced tumor regression/stabilization in 50% of the mice treated. Again, hIFN-beta expression resulted in cancer cell proliferation inhibition and apoptosis induction. We provide evidence that human immunodeficiency virus (HIV)-1-based lentiviral vectors are very efficient for gene transfer in PC-derived cells in vitro and in vivo. As a consequence, delivery of hIFN-beta stopped PC tumor progression. Thus, our approach could be applied to the 85% of PC patients with a locally advanced disease.

Successful transduction of human multipotent, lymphoid (T, B, NK) and myeloid, and transplantable CD34+CD38low cord blood cells using a murine oncoretroviral vector.

Hematopoietic stem cells (HSC) are subject to great interest because of their medical importance and their biological properties. Therefore, the possibility of genetically modifying human HSC is a major concern in several inherited pathologies. In this study, we aimed to demonstrate that a murine oncoretroviral vector can transduce multipotential cord blood (CB) stem cells. Sorted CB CD34(+)CD38(low) cells were transduced with a Moloney-based MFG retroviral vector containing the coding sequence of the murine CD2 (mCD2). CD34(+)mCD2(+) cells were sorted by flow cytometry and cultured either in bulk or at one cell per well in culture conditions that allow differentiation along lymphoid (T, B, and NK) and myeloid (M) lineages. Phenotypic analysis of cells generated in culture showed that CD34(+)mCD2(+) cells could give rise to all lymphoid and myeloid progeny, indicating that the MFG/mCD2 vector had transduced progenitors of all tested lineages. Moreover, clonal cultures of 660 CD34(+)mCD2(+) cells showed that approximately 5% of these cells were able to generate both myeloid and lymphoid (B + NK) progenies; for 25% of them, this included the production of lymphoid T cells. We also demonstrate that transduced CD34(+)CD38(low) CB cells with lymphoid and myeloid potentials were capable of engraftment into the bone marrow (BM) of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice during several months. These results show that MFG retroviral vectors can transduce multipotent (T, B, NK, M) human hematopoietic progenitors with in vivo repopulating activity.

Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.