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Identifier 344661
Title Ανάπτυξη νέων παραμέτρων στις κατασκευές ιογε΄νων φορέων για τη βελτίωση της γονιδιακής μεταφοράς και έκφρασης σε αιμοποιητικά κύτταρα
Author Φράγκος, Μιχάλης
Abstract Oncoretrovirus vectors for human γ-globin are being developed for gene therapy of the β-chain hemoglobinopathies. Progress toward this goal has been hampered by problems with vector expression and silencing. These problems have been overcome in the case of transgenic mice and lentiviral gene transfer vectors, by the inclusion of elements from the human β-globin Locus Control Region (LCR). However, inclusion of such elements in oncoretrovirus vectors result in vector instability and insufficient titers. For this reason, others and we turned to the HS-40 element from the human β-globin locus, as an alternative erythroid enhancer, and the cHS4 chromatin insulator from the chicken β-globin locus, as a means of reducing silencing position effects. Even with these improvements, γ-globin expression still averaged at a level that is less than half of that predicted to be clinically therapeutic. In order to address these problems, we turned to elements defined from patients with hereditary persistence of fetal hemoglobin (HPFH), a genetic condition that results in elevated levels of γ-globin gene expression in adults. HPFH is caused either by single point mutations in the promoter of the γ-globin genes or by large chromosomal deletions, which remove the β-globin gene and distal sequences. In the latter case, known as deletional HPFH, the γ-globin gene is often activated due to the juxtaposition of enhancer elements located in the adjoining sequences. One of these enhancer elements is contained within a 722 bp bridging fragment downstream of the Aγ-globin gene in HPFH-2. This element, called HPFH-2 in this study, has been shown to have enhancer activity in transgenic mice. In the studies described here, we wanted to test the use of the HPFH-2 enhancer in oncoretroviral vectors, in order to increase the expression of the γ-globin transgene. Initially, the HPFH-2 enhancer was studied functionally, by performing in vitro experiments. The interaction between the enhancer with the LCR and the enhancer with the truncated (-382) or the complete (-1350) promoter of the Aγ-globin gene was studied. Towards this goal, MEL585 cells were stably transfected with 8 different plasmid vectors that contain either the truncated or the complete Aγ-promoter in the presence or absence of the LCR and the HPFH-2 enhancer. The cells were co-transfected with a plasmid that contains the neomycin resistance gene. At least 50 Neo-resistant MEL colonies were isolated and pooled together to create 5-9 pools for each vector. The cells were then induced to express γ-globin and RNase protection assay was performed to study transgene expression. The copy number of the transgene in each pool was determined by Southern analysis and results were corrected based on the copy number of the vector in each pool. All constructs lacking the LCR gave no or very low expression, both in the presence and absence of the HPFH-2 enhancer. The vectors that contain the complete Aγ-promoter with the LCR gave high levels of expression, both in the presence and absence of the enhancer. Finally, the vectors with the truncated Aγ-promoter and the LCR gave low expression in the absence of the HPFH-2 enhancer, whereas in the presence of the enhancer, expression increased significantly. These results show that the HPFH-2 enhancer is able to significantly increase the expression levels of embryonic γ-globin in an adult environment, when the gene is transcribed from the truncated Aγ-promoter. For this reason, the HPFH-2 enhancer was decided to be cloned in oncoretrovirus vectors in combination with the truncated (-382) Aγ-promoter, which seems to interact satisfactorily with the HPFH-2 enhancer. The HPFH-2 enhancer was also used in luciferase expression experiments, in order to locate and investigate the functional part of the enhancer. A series of plasmid vectors was constructed, which contain the luciferase reporter gene under the control of the γ-globin promoter, and different parts of the HPFH-2 enhancer downstream of the reporter gene. The vectors were used to transiently transfect hematopoietic K562 cells and HeLa cells. The results of this experiment show that the HPFH-2 enhancer is erythroid-specific and that the functional region of the enhancer includes the GATA-1 and CCAAT sites, which are located in the AccI-AccI part of the enhancer. Using the knowledge from the MEL stable transfection experiments, we used a modified coding cassette for γ-globin, transcribed from a 382 bp Aγ-globin promoter in an oncoretrovirus vector that transcribes the neomycin resistance gene from the virus LTR. This vector was flanked with the cHS4 chromatin insulator from the chicken β-globin LCR to minimize silencing position effects. To this base vector was added either the α-globin HS-40 enhancer (vector AγHS40) or the HPFH-2 enhancer (vector AγHPFH2), and ecotropic producer clones were generated. Vector AγHS40 is genetically stable and capable of generating virus at 1x105 cfu/ml, while vector AγHPFH2 is moderately stable and capable of generating virus at 5x103 cfu/ml. These vectors were used to transduce MEL cells and Neo-resistant clones were isolated under G418 selection. These clones were then induced and analysed for γ-globin expression by immunofluorescence and flow cytometry. For vector AγHS40, 4 of 12 clones expressed γ-globin in a uniform pattern, while the remaining clones either expressed γ-globin in a variegated pattern (6 of 12) or failed to express γ-globin completely (2 of 12). For vector AγHPFH2, 8 of 10 clones that contain intact provirus expressed γ-globin in a uniform pattern, while the remaining clones expressed -globin in a variegated pattern (2 of 12). Preliminary RNase protection analysis indicated that vectors AHS40 and AHPFH2 expressed -globin at an average of 42% and 126% per copy of mouse -globin in all clones that have intact provirus, respectively. These results suggest that the HPFH-2 element can function to increase both the likelihood and the level of expression of a linked -globin cassette in an oncoretrovirus vector, and that this activity is at least as strong as that observed for the α-globin HS-40 enhancer. The above results permitted the design of new studies in order to improve the retroviral vectors, such as the possible interaction of the HS-40 and HPFH-2 enhancers, as well as the interaction of the HPFH-2 enhancer with the β-globin promoter, which is less prone to silencing in an adult setting. Towards this goal, retroviral vector AHS40-HPFH2 was constructed, which contains the truncated -382 A-globin promoter and both HS-40 and HPFH-2 enhancers. This vector was genetically unstable, due to a deletion between the HPFH-2 enhancer and the γ-globin promoter. For this reason, a series of new vectors was constructed that contain either a shorter Aγ-globin promoter or a different promoter, in order to overcome the genetic instability problem. Vector Aγ201HS40-HPFH2, which contains the wild-type -201 bp A-globin promoter, was found to be genetically stable with a titer of 6x104 cfu/ml. Vector Aγ117HS40-HPFH2, which includes a 201 bp Aγ-globin promoter with the -117 Greek HPFH mutation, was also genetically stable and capable of generating virus titers at 1x105 cfu/ml. Vector AγβHS40-HPFH2, which contains a 127 bp A-globin promoter, is genetically stable with a titer of 7x104 cfu/ml. These vectors were used to transduce MEL cells and Neo-resistant clones were isolated under G418 selection and were then induced and analysed for γ-globin expression by immunofluorescence and flow cytometry. Vector Aγ201HS40-HPFH2 expressed low γ-globin levels, vector Aγ117HS40-HPFH2 resulted in high and uniform expression, while vector AγβHS40-HPFH2 expressed the transgene at remarkably high levels in a variegating pattern. RNase protection analysis was also performed and documented expression at an average of 28.4% for vector Aγ201HS40-HPFH2, 248% for vector Aγ117HS40-HPFH2, and 191.9% for vector AγβHS40-HPFH2, per copy of mouse α-globin. These results suggest that the -117 HPFH mutation in combination with the two enhancers, increases the expression of the transgene and prevents silencing of the fetal A-gene, a beneficial effect for β-thalassemia gene therapy. Finally, vector Aγ117HS40-HPFH2 was used to transduce murine bone morrow cells, which were then transplanted into lethally irradiated syngenic mice. The efficiency of transduction was measured by counting CFU-GM colonies that were resistant to G418. These experiments showed that the transfer rate of the virus in the bone marrow cells was relatively low. The insertion of the γ-globin transgene in the hematopoietic cells of the transplanted mice was investigated by PCR, which confirmed the insertion of the vector in the majority of the animals. The expression of the transgene was then studied in a monthly basis by immunofluorescence and flow cytometry. The analysis showed that the percentage of cells that express γ-globin is low and that it decreases 5-6 months after transplantation. 6 months after transplantation the mice were sacrificed, in order to obtain DNA from the spleen and perform Southern blotting, to determine the copy number of the transgene in the genome of the hematopoietic cells of the mice. This experiment showed that the transfer of the transgene was successful, but inefficient in most mice. Based on the copy number of the transgene, the flow cytometry results were corrected, so as to calculate the percentage of the hematopoietic cells that contain and express the transgene. This percentage reached 10.21% of the cells of the peripheral blood of the transplanted mice, which shows that if the transfer rate of the retroviral vector into the hematopoietic stem cells is improved, vector Aγ117HS40-HPFH2 can be used as an effective means of transfer and expression of the γ-globin gene into hematopoietic cells for gene therapy purposes.
Language Greek
Issue date 2004-12-01
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/d/2/f/metadata-dlib-2004DIS1201.tkl Bookmark and Share
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