Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

Abstract

In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of AAV. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis.

Advances in salivary gland gene therapy – oral and systemic implications

“…Recent studies by Passineau and colleagues have carefully examined the utility of two different methods of gene transfer to salivary glands: use of pseudotyped AAV vectors and use of UAGT [76–78]. Their results provide important methodological information that should be valuable to all investigators planning to use salivary gland gene transfer for intended clinical applications.…”

“…UAGT with a 15% microbubble solution led to robust and fairly stable transgene expression for up to 28 days [76]. Very recently [78], they compared UAGT efficiency and host response in murine submandibular glands using a conventional plasmid versus a minicircle, both encoding luciferase. Minicircles are miniaturized, episomal covalently closed, circular gene expression vectors, generally biosynthesized in recombinant bacteria, with long-lasting gene expression and a safe clinical profile [79].…”

“…Minicircles are miniaturized, episomal covalently closed, circular gene expression vectors, generally biosynthesized in recombinant bacteria, with long-lasting gene expression and a safe clinical profile [79]. The minicircle vector led to more efficient gene transfer [78]. Next, they characterized physiological responses of the targeted glands to UAGT using global proteomic profiling.…”

“…Sonoporation alone, in the absence of a gene transfer vector, was without significant effect on the salivary gland proteome. While a plasmid vector profoundly perturbed the proteome, to a level comparable to that seen with high doses of AAV, UAGT with a minicircle vector resulted in minor proteomic changes, similar to sonoporation alone [78]. Thus, using the same expression cassette and equimolar amounts of gene, the authors found that UAGT with minicircles eliminated >95% of the protein changes associated with plasmid vectors.…”

Radiation‐induced salivary hypofunction may become a thing of the past

“…Non‐viral gene transfer to salivary glands has previously been shown to be relatively inefficient, despite its generally non‐immunogenic nature (e.g., Baccaglini et al , ). Passineau and colleagues have “focused on UAGT to the salivary gland, which combines the use of a non‐viral DNA [plasmid] vector and lipid/perflutren microbubbles with a low‐frequency acoustic field to create a ‘sonoporation’ effect, allowing gene transfer to the cells of the salivary gland without the introduction of viral antigens.” (Wang et al , ) Impressively, their studies in mice have shown much improved efficiency of gene delivery using UAGT, as well as a dramatic reduction in host immune responses (Geguchadze et al , ). However, many potential therapies have been shown to be successful in mice and rats, but are unable to be extended to large animals for diverse reasons.…”