Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Summary

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Abstract

Previous studies showed that knockdown of ITSN-1s (KD_ITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition¹. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNA_ITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce². Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration³Here, efficient and specific KD_ITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNA_ITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KD_ITSN throughout the study⁴. Mouse tissues collected at several time points post-siRNA_ITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KD_ITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Introduction

Naked siRNA cannot penetrate the cell membrane, being negatively charged, and it is easily degraded by enzymes in blood, tissues, and cells. Even with recently structural modifications to improve stability, siRNA accumulation at the target site after administration is extremely low and requires an efficient intracellular vehicle⁵. Cationic liposomes emerged as safe nucleic acids carriers with the potential to transfer large pieces of DNA/RNA into cells by encapsulating and protecting the nucleic acids from enzymatic degradation⁶. Also, cationic liposomes spontaneously interact with DNA/RNA, thus promoting gene transfer to the cells². More recently liposomes have been applied to deliver vaccines and low-molecular-weight drugs⁷. Liposomal delivery of microRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells⁸. When targeting the vascular endothelium, the intravenous delivery is essential because the complexes are unlikely to cross the endothelial barrier and extravasate in to the interstitium⁹. By comparison to other organs, ECs from the microvasculature of the lung have the greatest uptake and avidly internalize cationic liposome and DNA/RNA complexes, followed by the lymph nodes and Peyer’s patches⁹. Intravenous delivery in rodent models of siRNA via retro-orbital or tail vein injections has been proven harmless even in high concentrations, such as 50 mg/kg¹⁰. In the published literature the composition of cationic liposomes differs, based on the lipids formulation and their equimolar ratio^{11, 12}. There is a wide array of potential applications using cationic liposomes for gene delivery in vivo, whether targeting down-regulation/over-expression of the proteins, delivery of vaccines or anti-tumor therapies^13-15. Important to remember is that the efficiency of DNA/RNA-cellular membrane interaction is regulated by the shape coupling between generated complexes and membrane lipids. This suggests that tailoring the lipids composition to the targeted cellular membrane profiles may be beneficial and result in high rates of transfection in a particular cell type⁶.

Protocol

1. Cationic Liposomes Preparation Autoclave a clean round-bottom flask to be used for lipososmes preparation. Prepare stock solutions: – Dissolve 200 mg of dimethyl dioctadecyl ammonium bromide (DOAB) in 10 ml chloroform using a small glass bottle. – Dissolve 200 mg of cholesterol in 10 ml chloroform using a small glass bottle. – Prepare and autoclave to sterilize 5% glucose in 100 ml of RNA/DNA-ase free distilled water. Rinse …

Representative Results

ITSN-1s protein and mRNA levels were monitored at several time points after siRNAITSN delivery by Western blot and conventional and quantitative PCR as in4. ITSN-1s protein and mRNA levels in siRNAITSN-treated mouse lungs were about 75% lower by reference to controls during continuous knockdown of ITSN-1s for 21 days. Without ITSN-1s, dynamin-2, a major interacting partner of ITSN-1s and essential player in detachment of caveolae from the plasma membrane is not efficiently recruited to th…

Discussion

Based on previous studies published by others⁹ and us^{1, 18} we developed this methodology for long term knock down of ITSN-1s in vivo by repeated intravenous administration (every 72 hr, for 24 consecutive days) of specific siRNA/liposome complexes. This experimental approach is efficient, can be used safely and repeatedly and it can be easily extended to study the involvement of a gene encoding any protein of interest in lung endothelium and pulmonary homeostasis. Under the experimen…

Materials

Name of Reagent/Material	Company	Catalog Number
Dimethyl Dioctadecyl Ammonium Bromide (DOAB)	Sigma-Aldrich	D2779
LiposoFast stabilizer (mini-extruder)	Avestin Inc.	LF-STB
Polycarbonate membrane, 19 mm diameter, 50 nm pore	Avestin Inc.	LFM-50
Cholesterol	Sigma-Aldrich	C-8667
Chloroform	Mallinckrodt Chemicals	4432-04
Hank’s balanced salts	Sigma-Aldrich	H1387
Round-bottom flask	Fisher Scientific	FHB-275-030X
Mouse siRNAITSN – on target	Dharmacon/ThermoScientific	J-046912-11
Peristaltic pump	Ismatec	REGLO-CDF digital with RHOO pump head
Ventilator	Hugo Sachs Electronik	NA
Barbital	Sigma-Aldrich	B-0500
Uranyl acetate	EM Sciences	22400
Epon812	EM Sciences	Discontinued by the manufacturer
Propylene oxide	EM Sciences	20400
Embedding molds	EM Sciences	69923-05
Tannic acid	EM Sciences	21710
8 nm gold-albumin tracer		Prepared in the laboratory as in16
			Comments
			Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)
			Pyrex, 100 ml
			Modified siRNA, in vivo
			Part of IDEX corp.
			D-79232 March, Germany
			Replaced with Embed812, cat. # 14120