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Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate
R P Wersto1, E R Rosenthal, P K Seth
1Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. werstor@gwgate.nhlbi.nih.gov
Abstract:
First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (DeltaE1) or lacking the Ad E3 region in addition to E1 sequences (DeltaE1DeltaE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34(cdc2) protein levels. In some instances, infection with DeltaE1 or DeltaE1 DeltaE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DeltaE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in DeltaE1 or DeltaE1 DeltaE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.
Insights
First-generation adenovirus (Ad) vectors cause cell cycle arrest by increasing specific protein levels. This G2 growth arrest, mediated by early region 4 (E4) viral products, has implications for gene therapy safety.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- First-generation adenovirus (Ad) vectors are widely used in gene therapy.
- Replication-defective Ad vectors (DeltaE1, DeltaE1DeltaE3) are designed for safety.
- Understanding vector-induced cellular responses is crucial for gene transfer applications.
Purpose of the Study:
- To investigate the impact of first-generation adenovirus vectors on cell cycle progression.
- To identify the viral components responsible for observed cellular effects.
- To assess the safety implications of Ad vector-mediated cell cycle arrest in gene therapy.
Main Methods:
- Infection of primary and immortalized human bronchial epithelial cells with DeltaE1 and DeltaE1DeltaE3 Ad vectors.
- Analysis of cell cycle progression using flow cytometry.
- Assessment of protein expression levels, including cyclins and cyclin-dependent kinases, via Western blotting.
- Comparison with a second-generation DeltaE1Ad vector lacking specific E4 regions.
Main Results:
- DeltaE1 and DeltaE1DeltaE3 Ad vectors induced G2 cell cycle arrest and inhibited G1/S transition.
- Cell cycle arrest was linked to increased expression of cyclin A, cyclin B1, cyclin D, and p34(cdc2).
- Some Ad vector infections resulted in aneuploidy and polyploidization.
- A second-generation Ad vector with E4 modifications did not induce cell cycle arrest.
Conclusions:
- Early region 4 (E4) viral gene products in first-generation Ad vectors are responsible for inducing G2 growth arrest.
- This Ad vector-induced cell cycle arrest may present unintended consequences for human gene transfer and gene therapy.
- Further research is needed to develop safer gene therapy vectors by mitigating these effects.