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 adeno-associated virus. 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.
Highlights
Gene transfer for therapeutic purposes is an established and promising treatment strategy in disease paradigms where conventional treatments are unavailable or inadequate
ultrasoundassisted gene transfer (UAGT) to the salivary gland results in global but heterogeneous gene transfer and does not result in cellular infiltration or tissue disruption In our initial report describing UAGT to the mouse salivary gland,[8] we reported that the process appeared to be stochastic and without preference to cell type, but our analysis was somewhat confounded by autofluorescence of the salivary gland tissue
We found >25% of all proteins were altered 24 hours after UAGT relative to naive gland. These results indicate that plasmid-mediated gene transfer exerts a profound effect upon the mouse salivary gland, approaching in magnitude that seen with virus-mediated gene transfer
Summary
Gene transfer for therapeutic purposes is an established and promising treatment strategy in disease paradigms where conventional treatments are unavailable or inadequate. Nonviral gene transfer overcomes one of the most vexing challenges to clinical implementation of gene therapy, namely the introduction of viral vector antigens into host cells and tissues.[4,5,6] due to the vanguard role viral vectors have historically played in advancing gene therapy from the theoretical to clinical reality, research examining host response to nonviral vectors has understandably lagged. Because nonviral vectors lack the protein antigens necessary to initiate classical humoral or cell-mediated extracellular immunity, these vectors have often been assumed to be modestly or negligibly immunogenic provided they encode a therapeutic protein that is native to the host.[7] This assumption is reasonable as far as it goes, but intracellular host response to nonviral vectors has not been well studied and may present an additional challenge for gene therapy
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