Long - Term Amplification Regulation Of Transgenic Hematopoietic Cells And Establishment Of Cell Culture Model Of Transgenic Blood Malignant Tumor

BackgroundMany of the cells critical functions, such as cell division, devleopment into speicalized cell types, response to stimui, and even cell death, typically take place through the processes of cell signaling and gene activation. Research over the last decade has revealed that many of these key cellular signaling functions take place through a series of induced interactions between proteins. When two proteins are brought within close proximity, their signaling activities are activated. This molecular phenomenon called dimerization. Dimerization is a heart of many signaling pathway in activation of intracellular proteins. We have exploited this finding to develop gene regulated expression technology, a powerful technology for controlling cellular events using small molecules, and make possible the activation of specific cell signaling, gene transcription in cultured cells or in whole animals with exquisite precision and dose responsive control.Research has identified an essential role for the JAKs-STATs pathway in stem cell self-renew, and a large amount of experimental data has accumulated to indicate that most cytokines transmit their signal via a new family of tyrosine kinases termed JAK kinases. JAK2is one of the members play a pivotal role in the self-renewal of hematopoietic stem/multipotential hematopoietic progenitor cells (HSC/MHPCs). Upon above the principle of biology, we constructed a retrovirus vector that contains a JAK2functional domain linked a protein that provides a high-affinity binding site for a drug called a chemical inducer of dimerization (AP20187). AP20187is a novel gene-targeted drug that is being developed a small molecule on the basis of the structure-based drug design and protein engineering. This dimerizer drug can make JAK2dimerization and activates its signaling pathway. We have previously shown that multipotenial hemopoietic progenitor cells transduced with the JAK2vector can expand dramatically in response to the AP20187-induced JAK2signal combined with either ckit lignand or flt3lignand. In conclusion, the application of our gene regulated expression technology has overcome several of the major limitations to the development of stem cell therapy--the inability to efficiently transfer genes into these rare cells, to control their growth, and direct their differentiation towards the desired cell type and tissue.ObjectiveThe studies described herein were performed to explore the feasibility of regulated expansion and committed differentiation potential of JAK2gene modified hematopoietic cells in vivo.MethodsA murine stem cell virus (MSCV) based retroviral vector MGI-2F36vJAK2(GFP-F36vJAK2), which encodes a green fluorescent protein (GFP) and a fusion protein contains two copies modified FK506binding protein (F36v) linked tyrosine kinase JAK2was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GPE+86packaging cell line was generated by transfection with this vector. This vector was first transfected into Ba/F3cells and the engineered Ba/F3cells expressing JAK2were treated with AP20817for MTT assay and RT-PCR. Then the murine primary bone marrow cells from Ly5.1female mice were isolated and transduced by co-cultivated with irradiated (1500cGy) GPE+86producer clone for48h. After retroviral transduction, the transduced bone marrow cells were injected via the tail vein into lethally irradiated (1050cGY)10female C57BL/6recipents. Beginning17weeks posttransplantation, The experimental group of7mice were intraperitoneal (IP) injection with three successive course of AP20187(10ng/kg/day), The first given for28days and each of the last two course for7days. The control group of3remained mice with daily IP injections of carrier alone (without AP20187). During the all experiment processing, Complete blood counts (CBC) and Flow cytometry for measuring GFP positive cells were performed for the peripheral blood sample. In other10mice, AP20187was administered in combination with human Flt-3ligand for a coadministration experiment. For the JAK2and MPL in vivo competition experiment, cells were transduced with either GFP-F36vJAK2or dsRed-F36vMPL and then an equal number of each transduced cell type was transplanted into lethally irradiated Ly5.1recipents.Results1. MTT assay showed Ba/F3cell clones exhibited a dose-dependent proliferative response to AP20187. Real-time PCR of JAK2transduced and non-transduced Ba/F3cells indicated that JAK2transcripts are expressed at levels approximately25-fold higher than those of endogenous JAK2mRNA,2. a) In the first course of AP20187, GFP-positive red cells rose in all experimental mice from baseline frequencies of21～38%to peak levels of84～92%by weeks4and5. Thereafter, the frequency of GFP-positive red cells gradually fell, reaching nadir values of between3and28%by11～15weeks. b) In the second course of AP20187(7days), six mice survied, responded with rises in GFP-positive red cells from baseline values of4to28%to peak values of36～65%. c) In the third course of AP20187(7days), three mice survived, responded with rises in GFP-positive red cells from4-25%to peak of25～53%.3. In all cases, rise in GFP-positive red cells were reversible upon AP20187withdrawl. In contrast to the AP20187-treated mice, no spontaneous rise in GFP-positive cells in any of the non-AP20187treated controls.4. GFP-positive platelets failed to rise in response to AP20187administration. Furthermore, AP20187produced no discernible rise in GFP-marked neutrophils, B cells, or T cells.5. Spleen weights were significantly increased in AP20187-treated mice relative to control group (p=0.05).6. The coadministration of FL and AP20187showed:AP20187plus FL group that had the greatest red cell response also exhibited significant rises in the frequency of GFP-positive granulocytes.7. The direct comparison of AP20187-mediated activation of dsRed-F36vMPL versus GFP-F36vJAK2confirmed the conclusion that GFP-F36vJAK2is a relatively less potent red cell expander and has no capacity to expand platelets. 8. The causes of died mice apparently unrelated to AP20187administration.Conclusions1. This study demonstrates that AP20187-induced dimerization and activation of a cytoplasmic JAK2signaling domain, can trigger expansion of genetically modified red cells in mice. This expansion was only restricted to red cells, failed to expand granulocytes, B cells, or T cells and platelets.2. This in vivo selection strategy gives genetically modified red cells a preferential growth or survival advantage relative to other unmodified cells.3. This approach meets two criteria that are essential for gene therapy applications: the proliferative stimulus is restricted to the genetically modified cell population, and it is reversible. The activation of JAK2signal is absolutely dependent on the presence of dimerizer (AP20187).4. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy. Especially, the erythroid-restricted growth response suggests that this system may be well suited to gene therapy applications in sickle cell anemia or (3-thalassemia. Background and ObjectiveRecent studies have found, most leukemia has a chromosomal translocation, translocation can produce new fusion gene, encoding a fusion protein. So far, at least more than50chromosome aberrations have been reported involving the blood disease, these anomalies have become molecular specific mark for different types of leukemia. Using these markers can diagnosis of different types of leukemia. A large body of evidence indicates that cooperating factors, in addition to the oncogenic fusion, are necessary to generate a leukemic phenotype. We evaluated a series of leukemia-associated fusion proteins in primary murine bone marrow cells. The aim of this study is to develop an ex vivo cell culture system for establishing the hematological malignancy model and evaluated its biological characterization.MethodsMouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing10%FCS without growth factors, or with one of the following growth factor combinations:1) murine c-kit ligand (KL) plus human flt3ligand (FL);2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6(3/T/G/H6);3) KL/FL plus3/T/G/H6. The combinations of growth factors were tested for the ability to complement the oncogene fusion protein to support self-renewal of the transfected cells. The transfected and expanded cells were further confirmed by Flow cytometry analysis, RT-PCR, Western blotting and Southern blotting.ResultsOur findings indicate that the transfected cells could be amplified sustainably in the logarithmic growth way. c-Kit ligand (KL) in combination with flt-3ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK. In addition to KL/FL, the self-renewal of p210BCR-ABL transfected-marrow cells also required IL-3. All the above oncogene transfecred and expanded cells were confirmed by flow cytometry, RT-PCR, Western blotting and Southern blotting. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy.ConclusionThis system provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.