Ad5/F35-GFP Chimeric Adenovirus

Biosafety of gene therapy products remains a major challenge for their clinical application. The immunogenicity of viral vectors is of particular concern. Large animals, such as pigs, have advantages in testing vector systems because of their anatomical and physiological characteristics similar to those of humans. Here, researchers conducted comprehensive in vitro and in vivo studies to evaluate the biosafety of a chimeric adenoviral vector carrying the green fluorescent protein gene (Ad5/35F-GFP) in a mini-pig model. Transcriptome and secretome analysis of miniature pig leukocytes transduced with Ad5/35F-GFP revealed alterations in the suppression of proinflammatory processes and cytokine production. Clinical, instrumental, laboratory, and histological examinations performed within one week after direct or autologous leukocyte-mediated administration of Ad5/35F-GFP to miniature pigs revealed no adverse effects. The decrease in cytokine levels in the blood of experimental animals was also consistent with the in vitro data, confirming the immune tolerance of miniature pigs to Ad5/35F-GFP. These studies demonstrate the safety of Ad5/35F in the miniature pig model and provide evidence that Ad5/35F is a promising gene therapy vector.

Fluorescence microscopy showed the presence of GFP-positive cells in the spleen, thymus, submandibular lymph nodes, and bone marrow after delivery of Ad5/35F-GFP chimeric adenovirus in vivo and ex vivo (Figure 1A, C). No GFP-specific green fluorescence was observed in the heart, lungs, liver, or kidneys. Morphological examination of immunocompetent organs showed only moderate changes in the lymph nodes in both the in vivo and in vitro groups (Figure 1B, D). These changes included signs of paracortical hyperplasia, disorganized lymphoid follicle architecture, and sinusoidal histiocytosis. No pathological changes were detected in the heart, lungs, liver, or kidneys in either group.

Figure 1. Fluorescent (A,C) and morphological (B,D) examination of the spleen, thymus, submandibular lymph nodes, and bone marrow in the mini-pigs after 7 days of direct (in vivo group) and cell-mediated (ex vivo group) delivery of Ad5/35F-GFP. (Izmailov A, et al., 2024)