Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Death switch for gene therapy: application to erythropoietin transgene expression.

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Early proliferation of umbilical cord blood cells from premature neonates.

BACKGROUND AND OBJECTIVE: Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates. MATERIAL AND METHODS: Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting. RESULTS: Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells. CONCLUSION: These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.

The association of cytokine gene polymorphisms with febrile non-hemolytic transfusion reaction in multitransfused patients.

Cytokines are associated with inflammatory responses including febrile non-hemolytic transfusion reactions (FNHTR). Moreover, there are some polymorphisms of these cytokine genes associated with different levels of gene expression. The aim of the present study was to investigate the association of inflammatory cytokine gene polymorphisms with the occurrence of FNHTR in multitransfused patients. We studied two groups of transfused patients: one presenting FNHTR before 20 transfusions of red blood cells concentrates and the other which never presented FNHTR even after 20 transfusions. The gene polymorphisms studied were IL1B-511C/T and +3953C/T, IL1RN (intron 2, variable number tandem repeat), IL6-174G/C, IL10-1082G/A and -819C/T, TNF-308G/A and LTA+253G/A using polymerase chain reaction and restriction digestion or sequencing methods. An association of IL1RN*2.2 genotype with the occurrence of precocious FNHTR (P < 0.025) was detected. This allele and this genotype have been related with higher serum levels of interleukin (IL)-1beta in vivo and higher promoter activity. No other association was demonstrated. The association of gene polymorphisms related with the increase of inflammatory cytokine gene expression may be a relevant factor in FNHTR and requires confirmation.