In vitro and in vivo effects of glucocorticoids on gene transfer to skeletalmuscle

Abstract

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.

Mechanisms of unprimed and dexamethasone‐primed nonviral gene delivery to human mesenchymal stem cells

“…While DEX and other steroids have been used to increase nonviral gene delivery efficiency in several cell types before this study, other cell types generally show only moderate transgene expression increases (i.e. 2-4-fold increases relative to VC; Bernasconi et al, 1997;Braun et al, 1999;Chen et al, 2011;Choi & Lee, 2005;Jain et al, 1999;Koster et al, 2002;Köster et al, 2006;Lin et al, 2003;Nair et al, 2002) compared to those seen here in hMSCs. Although DEX increased transgenic luciferase activity normalized to total protein by about 10-fold in transfected hMSCs, DEX only increased the amount of transgenic luciferase protein by about two-fold, as quantified by western blot analysis normalized to total protein (Figure 1c,d), suggesting that DEX-priming somehow increases transgenic enzyme activity downstream of transgenic protein synthesis.…”

“…In hMSCs, derived from bone marrow stromal cells (BMSCs) of multiple donors, we showed that 90 to 360 nM dexamethasone (DEX), a Gc drug, delivered 0-30 min before delivery of pDNA lipoplexes increased the transgenic luciferase activity about 10-fold, increased transgenic enhanced green fluorescent protein (EGFP) mean fluorescence intensity of transfected cells about two-fold, increased transfection efficiency about three-fold (i.e., percent of EGFP + transfected cells), increased duration of transgene expression, and ameliorated transfection-induced metabolic decline, all while retaining differentiation capacity (Kelly, Plautz, Zempleni, & Pannier, 2016). glucocorticoids, estrogens, and progesterone) has generally shown moderate increases in gene delivery to a wide variety of cell types (i.e., 2-4-fold transgene expression increases; Bernasconi et al, 1997;Braun et al, 1999;Chen, Shank, Davis, & Ziady, 2011;Choi & Lee, 2005;Jain, Seth, & Gewirtz, 1999;Koster et al, 2002;Köster et al, 2006;Lin et al, 2003;Nair, Rodgers, & Schwarz, 2002), with a few exceptions (i.e., 10-fold increase in myoblasts; Braun et al, 1999) and 900-fold increase in bronchial epithelial cells (Wiseman, Goddard, & Colledge, 2001). For example, priming of various cell types with steroids (i.e.…”

“…Our previous work has sought to improve and better understand the biology of nonviral gene delivery by pharmacologically 'priming' cells for increased transgene expression (Nguyen, Beyersdorf, Riethoven, & Pannier, 2016) through modulation of relevant molecular pathways that are important to the biological processes involved in gene delivery (Martin, Plautz, & Pannier, 2015a;Martin, Plautz, & Pannier, 2015b). Specifically, we investigated anti-inflammatory glucocorticoid (Gc) drugs as priming candidates in hMSCs due to reports in other cell types that Gc can increase transfection efficiency by increasing plasmid DNA (pDNA) nuclear internalization or reducing the inflammatory response to transfection (Braun et al, 1999;Kim, Kim, Bae, Choi, & Lee, 2009). In hMSCs, derived from bone marrow stromal cells (BMSCs) of multiple donors, we showed that 90 to 360 nM dexamethasone (DEX), a Gc drug, delivered 0-30 min before delivery of pDNA lipoplexes increased the transgenic luciferase activity about 10-fold, increased transgenic enhanced green fluorescent protein (EGFP) mean fluorescence intensity of transfected cells about two-fold, increased transfection efficiency about three-fold (i.e., percent of EGFP + transfected cells), increased duration of transgene expression, and ameliorated transfection-induced metabolic decline, all while retaining differentiation capacity (Kelly, Plautz, Zempleni, & Pannier, 2016).…”

“…For example, priming of various cell types with steroids (i.e. glucocorticoids, estrogens, and progesterone) has generally shown moderate increases in gene delivery to a wide variety of cell types (i.e., 2-4-fold transgene expression increases; Bernasconi et al, 1997;Braun et al, 1999;Chen, Shank, Davis, & Ziady, 2011;Choi & Lee, 2005;Jain, Seth, & Gewirtz, 1999;Koster et al, 2002;Köster et al, 2006;Lin et al, 2003;Nair, Rodgers, & Schwarz, 2002), with a few exceptions (i.e., 10-fold increase in myoblasts; Braun et al, 1999) and 900-fold increase in bronchial epithelial cells (Wiseman, Goddard, & Colledge, 2001).…”

Polymeric Delivery of Therapeutic Nucleic Acids

“…299 Dexamethasone dilates the nuclear pores, which can also promote the nuclear uptake of large plasmid DNA and increase the transfection efficiency. 302,328,329 For researchers endeavoring to improve the nuclear uptake of DNA cargo with polymer-based vehicles, it would be helpful to understand what levels of nuclear uptake are typically achieved with standard PEI-based transfections. Using quantitative polymerase chain reaction measurements, Szoka et al detected as few as 75 and as many as 50 000 plasmid copies (<5% of the applied dose) in the nuclei of transfected cells but found that levels above 3000 plasmids/nuclei yielded marginal returns in transgene expression.…”