Development of Efficient Transient Transfection Systems for Introducing Antisense Oligonucleotides into Human Epithelial Skin Cells

Abstract

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)₅ random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 µg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene β-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.

3D‐model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

“…Other positively charged surfaces, such as poly-L-lysine, but not PLO, have been used previously to facilitate the formation of hepatocyte aggregates exhibiting liver-specific functions [Tzanakakis et al, 2001]. PLO, similar to other polycations especially poly-L-lysine, has been used mostly in combination with other molecules for various applications such as non-viral gene delivery [Fimmel et al, 2000;Ramsay and Gumbleton, 2002;Brown et al, 2003], packaging system (in alginate beads) for cellular grafts [Tobias et al, 2001;Calafiore, 2003], or as attachment matrix/ scaffold for neuronal and embryonic stem cell cultures [Murashov et al, 2005;Flanagan et al, 2006;Morizane et al, 2006]. Polyamino acids, such as PLO, have also been shown to activate cell signaling pathways [Haber et al, 1991].…”

An update on the role of the sebaceous gland in the pathogenesis of acne

“…35 AR is a member of the steroid superfamily of ligand-dependent transcription factors. 5α-DHT binds to the AR with greater affinity than testosterone and the 5α-DHT/AR complex appears to be more stable 36 and therefore, more effective.…”

Advantages of Antisense Drugs for the Treatment of Oral Diseases

“…Because the cellular uptake of nonconjugated oligonucleotides in the absence of such carriers as lipids or other delivery agents is limited (Akhtar and Juliano, 1992;Akhtar, 1998;Redenti et al, 2001), many attempts have been made in recent years to increase uptake and cell tropism using different delivery strategies (Redenti et al, 2001;Fimmel et al, 2000;Bijsterbosch et al, 2001;Lou et al, 2001;for review, see Akthar et al, 2000). At present, however, no carrier substances, formulations, or nonviral vectors are available for application in humans, and delivery of the drug and uptake by the target cells generally are left to largely unknown or uncontrollable mechanisms.…”