Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery.

The research examines how gene delivery methods using adenovirus 5 vectors affect different types of horse cells. It demonstrates that gene transfer can be effectively achieved in equine cells with high vector doses due to their relative resistance to cytotoxic effects.

Analysis and Methodology

• The study involved cells from different sources: cartilage, bone marrow and synovium. All were taken from 15 adult horses.
• The research team cultured several types of cells: equine chondrocytes (cartilage cells), synovial cells (from the fluid filled cavity of the joints), and BMD-MSCs (stem cells bone marrow derived mesenchymal stem cells).
• As a control, HeLa (human cervix carcinoma) cells were also cultured and infected.
• Cells were exposed to recombinant and modified adenoviral vector types that carried either a gene for beta-galactosidase enzyme or green fluorescent protein (Ad-GFP)
• The modified version of the adenovirus also contained an arg-gly-asp peptide capside insert (Ad-RGD-GFP).
• The percentage of transduced cells (cells which received the transferred gene), total number of cells (transduced and non-transduced), and overall cell viability were assessed at two time points: 2 and 7 days after the infection.

Findings

• The susceptibility of different cell types to adenoviral vector infection varied, with HeLa cells being the most susceptible, followed by BMD-MSCs, chondrocytes, and synovial cells.
• Morphological signs of cytotoxicity (indicating cell damage due to toxins) were evident in HeLa cells, but were not observed in equine cells – suggesting their resistance to cytotoxic effects.
• There was a decline in the quantity of transduced cells by day 7 across all cell types, with the exception of equine BMD-MSCs.
• There was no significant difference in the transduction efficiency between Ad-GFP and Ad-RGD-GFP vectors.

Conclusions

• The study finds that gene transfer can be efficiently carried out in equine cells using adenovirus vectors.
• Because equine cells demonstrated relative resistance to cytotoxic effects, it is suggested that high vector doses can be employed for gene delivery.
• The study further indicates that the BMD-MSCs are the most suitable target for gene therapy in horses due to their higher susceptibility to adenoviral vector infection and sustained expression of transgenes compared to other cell types.