Death switch for gene therapy: application to erythropoietin transgene expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)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 mu g/50 g ptet-mEpoD and 0.5 mu g/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 mu g/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.437634644Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Red blood cell indices, cation content, and membrane cation transports

In sickle cell disease, in the homozygous state, the increased heterogeneity of erythrocytes results mainly from membrane defects secondary to Hb S polymerization and the increased survival of F cells. The density distribution curve, using phthalate esters or the red blood cell indices measured with the H*3 system, are useful methods for the hematological follow-up of patients under specific therapies. The methods evaluating the red blood cell cation contents and the abnormal membrane potassium transport pathways are also described, in order to evaluate agents which can restore normal hemoglobin concentration and water content in dehydrated sickle cells

Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse

The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD