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Adenoviral vector cytotoxicity depends in part on the transgene encoded
C Zheng1, C M Goldsmith, B C O'Connell
1Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, Bethesda, Maryland, 20892-1190, USA.
Abstract:
First-generation adenoviral vectors induce G(2)/M arrest and cell death at high multiplicities of infection (m.o.i.'s) in vitro. It is unclear whether this cytotoxicity is entirely adenoviral gene related or influenced in part by the encoded transgene. We examined this question in epithelial cells using seven vectors at relatively low (50) or higher (200) m.o.i.'s. The vectors contained no transgene (+/-promoter), transgenes encoding a cytoplasmic reporter protein (two luciferase constructs; beta-galactosidase), or transgenes encoding a secretory protein (alpha1-antitrypsin; growth hormone). After 24 h with a m.o.i. of 50, vectors encoding cytoplasmic reporter proteins led to greatest cytotoxicity (approximately 35-40% cells in G(2)/M). Vectors without a transgene resulted in lower cytotoxicity (approximately 15%, minus, or 23%, plus promoter, cells in G(2)/M). Vectors encoding secretory proteins led to approximately 22-25% cells in G(2)/M. A similar pattern resulted when cell number was measured. Results were unrelated to the steady-state levels of transgene product. At the higher m.o.i., all vectors caused substantial growth retardation. This is the first demonstration that adenoviral vector-induced cytotoxic effects are in part related to the transgene encoded.
Insights
First-generation adenoviral vectors cause cell death, with cytotoxicity influenced by the encoded transgene. This study demonstrates that the specific transgene impacts adenoviral vector-induced toxicity in epithelial cells.
Area of Science:
- Gene therapy
- Molecular biology
- Cell biology
Background:
- First-generation adenoviral vectors are known to induce G(2)/M arrest and cell death.
- The contribution of the encoded transgene to this cytotoxicity has remained unclear.
Purpose of the Study:
- To investigate whether adenoviral vector cytotoxicity is solely related to the vector or also influenced by the transgene.
- To compare the cytotoxic effects of vectors with different transgenes at varying multiplicities of infection (m.o.i.).
Main Methods:
- Epithelial cells were infected with seven adenoviral vectors at low (50) and high (200) m.o.i.
- Vectors included those with no transgene, cytoplasmic reporter proteins (luciferase, beta-galactosidase), or secretory proteins (alpha1-antitrypsin, growth hormone).
- Cell cycle progression (G(2)/M arrest) and cell number were measured after 24 hours.
Main Results:
- Vectors encoding cytoplasmic reporter proteins induced the highest cytotoxicity (35-40% G(2)/M arrest) at a m.o.i. of 50.
- Vectors without transgenes showed lower cytotoxicity (15-23% G(2)/M arrest).
- Vectors encoding secretory proteins resulted in intermediate cytotoxicity (22-25% G(2)/M arrest).
Conclusions:
- Adenoviral vector-induced cytotoxicity is, in part, dependent on the specific transgene being encoded.
- This finding is crucial for optimizing adenoviral vector design in gene therapy applications.