Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Summary

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Abstract

Prion diseases result from the misfolding of the normal, cellular prion protein (PrP^C) to an abnormal protease resistant isomer called PrP^Res. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrP^Res isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrP^Res formation in brain tissue, do not specifically target neuronal PrP^C, and are often too toxic to use in animal or human subjects.

We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrP^C is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrP^C expression of neurons in the CNS.

Introduction

Prions are severe neurodegenerative diseases that affect the CNS. Prion diseases result from the misfolding of the normal cellular prion protein, PrP^C, by an infectious isomer called PrP^Res. These diseases affect a wide variety of species including bovine spongiform encephalopathy in cows, scrapie in sheep, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease in humans^1-3. Prions cause neurodegeneration that starts with synaptic loss, and progresses to vacuolization, gliosis, neuronal loss, and plaque deposits. Eventually, resulting in the death of the animal/individual⁴. For decades, researchers have investigat....

Protocol

All mice were bred and maintained at Lab Animal Resources, accredited by the Association for Assessment and Accreditation of Lab Animal Care International, in accordance with protocols approved by the Institutional Animal Care and Use Committee at Colorado State University.

1. Preparation of LSPCs

1. Use a 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium-propane):cholesterol ratio for LSPCs. For a 4 nmole liposome preparation, mix 2 nmoles of DOTAP and 2 nmoles of cholesterol into 10 ml of a 1:1 chloroform:methanol solution in a flask.
2. Evaporate 9 ml of the chloroform:methanol solution using N₂ gas in a fume hood. E....

Results

To increase the efficiency of siRNA encapsulation within anionic PALETS, the siRNA was mixed with protamine. To determine the best protamine concentration for the siRNA, the siRNA was mixed with different concentrations of protamine, from 1:1 to 2:1 (Figure 3A). There was a 60-65% siRNA encapsulation efficiency in anionic liposomes without the use of protamine. Samples with protamine:siRNA molar ratios from 1:1 to 1.5:1 (133-266 nM) had 80-90% siRNA encapsulation. Molar r.......

Discussion

This report describes a protocol to create two targeted delivery systems that efficiently transports siRNA to the CNS. Previous methods of delivering siRNA to the CNS included injecting siRNA/shRNA vectors directly into the brain, intravenous injection of targeted siRNA, or intravenous injection of non-targeted liposome-siRNA complexes. Injection of siRNA/shRNA vectors into the CNS does cause a decrease in target protein expression levels. However, the siRNA/shRNA does not diffuse freely through the CNS. Furthermore, the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DOTAP lipid	Avanti Lipids	890890
Cholesterol	Avanti Lipids	700000
DSPE	Avanti Lipids	850715
DSPE-PEG	Avanti Lipids	880125
Chloroform	Fisher Scientific	AC268320010
Methanol	EMD Millipore	113351
N2 Gas	AirGas
Sucrose	Fisher Scientific	S5-500
Extruder	Avanti Lipids	610023
1.0, 0.4, and 0.2 μm filters	Avanti Lipids	610010, 610007, 610006
PBS	Life Technologies	70011-044
Protamine sulfate	Fisher Scientific	ICN10275205
EDC	Thermo Scientific	22980	Aliquoted for single use
Sulfo-NHS	Thermo Scientific	24510	Aliquoted for single use
40 μm Cell Strainer	Fisher Scientific	08-771-1
Rat anti-mouse CD16/CD32 Fc block	BD Pharmigen	553141
Anti-PrP antibody (PRC5)	Proprietary - PRC