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Published on: May 7, 2018
Death switch for gene therapy: application to erythropoietin transgene expression
D S Souza1, D M Spencer, T S I Salles
1Centro de Hematologia e Hemoterapia, Departamento de Clínica Médica, Universidade de Campinas, Campinas, SP, Brasil.
Abstract:
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.
Insights
The inducible caspase-9 "death switch" effectively controlled erythropoietin gene therapy in mice. This safety mechanism induced apoptosis and reduced hormone levels when activated, showing promise for gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biotechnology
Background:
- Gene therapy holds promise but requires robust safety measures.
- Erythropoietin (Epo) gene therapy can enhance red blood cell production.
- Controlling therapeutic gene expression and potential side effects is crucial.
Purpose of the Study:
- To evaluate the efficacy of a caspase-9-based artificial "death switch" as a safety mechanism for Epo gene therapy.
- To assess the in vitro and in vivo performance of the inducible caspase-9 system.
- To determine if the safety switch interferes with gene transfer, expression, or regulatory control.
Main Methods:
- Utilized plasmids encoding dimeric murine Epo, tetracycline-controlled transactivator, and inducible caspase 9.
- Tested AP20187, a chemical inducer of dimerization, for apoptosis induction in modified cells.
- Administered gene therapy constructs and AP20187 to C57BI/6 mice to assess hematocrit, Epo levels, and cell apoptosis.
Main Results:
- AP20187 induced apoptosis in iCasp9-modified myoblasts in vitro.
- In vivo, Epo gene therapy increased hematocrit in most treated mice.
- AP20187 administration reduced hematocrit and Epo levels in a subset of mice, with detected apoptosis in muscle tissue.
Conclusions:
- The inducible caspase-9 system functions as a viable safety mechanism for gene therapy.
- The safety switch did not impede gene transfer, expression, or tetracycline-based regulation.
- Further research is needed to optimize the efficacy of this gene therapy safety approach, potentially using lentivirus vectors.
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