We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

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
<ol>
	<li>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...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216 (4542), 136-144 (1982).</li><li>Jeffrey, M., McGovern, G., Sis&#242;, S., Gonz&#225;lez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+sub-cellular+pathology+of+prion+diseases:+relationship+between+morphological+changes,+accumulation+of+abnormal+prion+protein+and+clinical+disease.">Cellular and sub-cellular pathology of prion diseases: relationship between morphological changes, accumulation of abnormal prion protein and clinical disease.</a> Acta Neuropathologica. 121 (1), 113-134 (2011).</li><li>B&#252;eler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+devoid+of+PrP+are+resistant+to+scrapie.">Mice devoid of PrP are resistant to scrapie.</a> Cell. 73 (7), 1339-1347 (1993).</li><li>Pfeifer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivector-mediated+RNAi+efficiently+suppresses+prion+protein+and+prolongs+survival+of+scrapie-infected+mice.">Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.</a> J Clin Invest. 116 (12), 3204-3210 (2006).</li><li>White, M., Farmer, M., Mirabile, I., Brandner, S., Collinge, J., Mallucci, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+treatment+with+RNAi+against+prion+protein+rescues+early+neuronal+dysfunction+and+prolongs+survival+in+mice.">Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice.</a> Proc Natl Acad Sci. 105 (29), 10238-10243 (2008).</li><li>Gallozzi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prnp+knockdown+in+transgenic+mice+using+RNA+interference.">Prnp knockdown in transgenic mice using RNA interference.</a> Transgenic Research. 17 (5), 783-791 (2008).</li><li>Mallucci, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cellular+prion+protein+reverses+early+cognitive+deficits+and+neurophysiological+dysfunction+in+prion-infected+mice.">Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice.</a> Neuron. 53 (3), 325-335 (2007).</li><li>Pulford, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-siRNA-peptide+complexes+cross+the+blood-brain+barrier+and+significantly+decrease+PrPC+on+neuronal+cells+PrPRes+in+infected+cultures.">Liposome-siRNA-peptide complexes cross the blood-brain barrier and significantly decrease PrPC on neuronal cells PrPRes in infected cultures.</a> PLoS One. 5 (6), (2010).</li><li>Malone, R. 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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...

Prions are severe neurodegenerative diseases that affect the CNS. Prion diseases result from the misfolding of the normal cellular prion protein, PrPC, by an infectious isomer called PrPRes. 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 humans1-3. 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 investigated compounds meant to slow or stop the progression of prion disease. However, researchers have not found either a successful therapy or an effective systemic delivery vehicle.
Endogenous PrPC expression is required for the development of prion diseases5. Therefore, decreasing or eliminating PrPC expression may result in a delay or amelioration of disease. Several groups created transgenic mice with reduced levels of PrPC or injected lentivectors expressing shRNA directly into murine brain tissue to investigate the role of PrPC expression levels in prion disease. These researchers found reducing the amount of neuronal PrPC resulted in halting the progressive neuropathology of prion diseases and extended the life of the animals6-9. We have reported that PrPC siRNA treatment results in clearance of PrPRes in mouse neuroblastoma cells10. These studies suggest that the use of therapies to decrease PrPC expression levels, like small interfering RNA (siRNA), which cleaves mRNA, may sufficiently delay the progression of prion diseases. However, most therapies investigated for prion diseases were delivered in ways that would not be practical in a clinical setting. Therefore, a siRNA therapy needs a systemic delivery system, which is delivered intravenously and targeted to the CNS.
Investigators have studied the use of liposomes as delivery vehicles for gene therapy products. Cationic and anionic lipids are both used in the formation of liposomes. Cationic lipids are more widely used than anionic lipids because the charge difference between the cationic lipid and the DNA/RNA allows for efficient packaging. Another advantage of cationic lipids is that they cross the cell membrane more easily than other lipids11-14. However, cationic lipids are more immunogenic than anionic lipids13,14. Therefore, researchers have started to shift from using cationic to anionic lipids in liposomes. Gene therapy products can be efficiently packaged into anionic liposomes using the positively charged peptide protamine sulfate, which condenses DNA/RNA molecules15-19. Since anionic lipids are less immunogenic than cationic lipids they may have increased circulation times, and may be more tolerated in animal models13,14. Liposomes are targeted to specific tissues using targeting peptides that are attached to the liposomes. The RVG-9r neuropeptide, which binds to nicotinic acetylcholine receptors, has been used to target siRNA and liposomes to the CNS17-20.
This report outlines a protocol to produce three siRNA delivery vehicles, and to package and deliver the siRNA to neuronal cells (Figure 1). Liposome-siRNA-peptide complexes (LSPCs) are composed of liposomes with siRNA and the RVG-9r targeting peptide electrostatically attached to the outer surface of the liposome. Peptide addressed liposome encapsulated therapeutic siRNA (PALETS) are composed of siRNA and protamine encapsulated within the liposome, with RVG-9r covalently bonded to lipid PEG groups. Using the below methods to generate LSPCs and PALETS, PrPC siRNA decreases PrPC expression up to 90% in neuronal cells, which holds tremendous promise to cure or substantially delay the onset of prion disease pathology.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DOTAP lipid</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">700000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">850715</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">880125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">AC268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:104px;">EMD Millipore</td>
			<td style="width:119px;">113351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Gas</td>
			<td style="width:104px;">AirGas</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">S5-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extruder</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.0, 0.4, and 0.2um filters</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610010, 610007, 610006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Life Technologies</td>
			<td style="width:119px;">70011-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">ICN10275205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDC</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">22980</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sulfo-NHS</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">24510</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40um Cell Strainer</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rat anti-mouse CD16/CD32 Fc block</td>
			<td style="width:104px;">BD Pharmigen</td>
			<td style="width:119px;">553141</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-PrP antibody (PRC5)</td>
			<td style="width:104px;">Proprietary - PRC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. 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 PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC 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 PrPC expression of neurons in the CNS.

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...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

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

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.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

The overall goal of this procedure is to design a delivery system that can deliver small molecule drugs like siRNA to the brain. This method can answer key questions in the neurodegeneration field such as Is siRNA a viable therapeutic option for protein misfolding diseases? and Can we design a reliable drug delivery system to the brain?The main advantage of creating a liposomal drug delivery system is that we can deliver our siRNA intravenously or through the blood stream rather than directly through neuronal tissue which results in neuronal damage. The implications of this technique extends towards therapeutics and diagnostics of neurodegenerative diseases because this technique allows for specific delivery across the blood-brain barrier, specifically to acetylcholine-expressing neurons. Visual demonstration of this technique is critical as mouse tail vein injections are difficult to learn due to the small size of the mouse tail vein.Begin by preparing a one-to-one DOTAP to cholesterol ratio. For a foreign animal liposome preparation, mix two nanomoles of DOTAP and two nanomoles of cholesterol into ten milliliters of a one-to-one chloroform to methanol solution. Then, evaporate nine milliliters of the chloroform and methanol solution using nitrogen gas.Evaporate the last one milliliter of solvent in a fume hood overnight without gas. The next day, a thin lipid film should be visible on the bottom of the flask. Heat both this flask with the lipid film and ten milliliters of a ten percent sucrose solution to 55 degrees Celsius.Then, slowly pipette the heated sucrose solution onto the thin lipid film while gently swirling the flask. Next, assemble an extruder with one micron filters according to the manufacturer's instructions. Then, add one milliliter of the heated liposome suspension to one of the syringes on the extruder.Then pass the suspension between the two syringes 11 times. While keeping the liposome suspension heated for easier sizing, size the liposomes through 45 micron and a 2 micron filter To generate large, unilamellar vesicles, next add 20 microliters of the four nanomole liposome suspension to 200 microliters of a 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature. Then add 80 microliters of a 500 micromolar stock of RVG-9R targeting peptide to the siRNA liposome solution and incubate for ten minutes at room temperature.Prepare a foreign animal lipsomoe suspension by mixing 2.2 nanomoles of either DSPE or DOTAP into ten milliliters of a two-to-one chloroform to methanol solution with 1.6 nanomoles of cholesterol and 2 nanomoles of DSPE peg. Afterwards, apply nitrogen gas and when most of the solvent has been evaporated, leave the flask in the fume hood overnight. Then, slowly add ten milliliters of 1X PBS heated to 75 degrees Celsius to the thin lipid film.Allow the lipids to rehydrate at 75 degrees Celsius for at least one hour before sizing. After sizing the liposome suspension using an extruder as before, pipette 20 microliters of each of the four nanomole liposome suspensions into single use aliquots and lyophilize for 30 minutes using a bench top freeze dryer. Next, add 1.4 microliters of a 26.6 micromolar solution of protamine sulfate to 200 microliters of 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature to condense the siRNA.Following the incubation, rehydrate DSPE palets with 201.4 microliters of siRNA protamine solution. Rehydrate the DOTAP palets with 200 microliters of a foreign animal stock solution of siRNA. Incubate each liposome siRNA solution for ten minutes at room temperature.Next, add ten microliters of a 60 millimolar EDC solution to each tube of rehydrated liposomes. Then add ten microliters of 150 millimolar sulfo-NHS solution to the liposome siRNA solution for both DOTAP and DSPE palets. Incubate for two hours at room temperature.Lastly, add 80 microliters of 500 micromolar stock of RVG-9R peptide to the siRNA liposome cross linking solution and incubate for ten minutes at room temperature. After anesthetizing a mouse using two to three percent isoflurane in oxygen cover the eyes of the mouse with apthalmic ointment and confirm an anesthetization by performing a toe pinch. Proceed only if a reaction is absent.While maintain anesthesia, place the mouse on its back or side to access the tail vein. Wipe the tail with 70 percent ethanol and then use a 26 gauge insulin syringe to inject 300 microliters of LSPCs or palets into the tail vein. Begin injecting distally and move proximally if the vein collapses or ruptures.Place the mouse in a clean cage until it maintains sternal recumbency. Monitor the animal for several hours after injection to ensure anesthesia wears off and there are no ill effects of the treatment. After allowing the LSPCs or palets to circulate for at least 24 hours and then euthanizing the mouse, dissect out half a hemisphere of the brain.Then place the half hemisphere and 2.5 milliliters of fax buffer onto a 40 micron mesh cell strainer and use the plunger of a syringe to press the tissue through the filter into a petri dish. Rinse the cell strainer and petri dish with an additional 2.5 milliliters of fax buffer. Then transfer the single cell suspension to a 15 milliliter conical tube and place on ice.Next, pipette 100 microliters of the cell suspension into micro centrifuge tube and centrifuge for five minutes at 350 times G.After discarding the supernatant, resuspend the cell pellet in one milliliter of fax buffer and centrifuge again. Then, add another milliliter of fax buffer and place the cell suspension on ice. Now, incubate the cells in 100 microliters of rat anti-mouse Fc block in fax buffer for 30 to 60 minutes on ice.After pelleting and washing as before, add 100 microliters of fluorescent primary anti-body solution and place on ice for 30 to 60 minutes. After a final wash, resuspend the cells in one mililiter of RVC lysis buffer for one minute. Then, pellet the cells.After discarding the buffer, resuspend in one milliliter of fax buffer and place on ice. The cells are now ready for fax analysis. Mice treated with siRNA delivered via LSPCs showed a significant decrease of cellular prion protein levels compared to a PBS control which shows while type cellular prion protein levels.The LSPCs treated mice showed cellular prion protein levels close to that of a prion protein knock out mouse. Across three experiments, mice treated with LSPCs showed significant decreases in cellular prion protein levels as compared to to PBS while type controls. While attempting this procedure, it's important to remember that the liposomes can be stored in the fridge for several months, but the complete LSPCs must be used within 24 hours after assembly.After its development, the generation of LSPCs or palets will pave the way for researchers to explore more therapeutic options in the field of neurodegenerative diseases.

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Research

JoVE Journal

Bioengineering

10.3K visualizações.  Colorado State University. The overall goal of this procedure is to design a delivery system that can deliver small molecule drugs like siRNA to the brain. This method can answer key questions in the neurodegeneration field such as Is siRNA a viable therapeutic option for protein misfolding diseases? and Can we design a reliable drug delivery system to the brain?The main advantage of creating a liposomal drug delivery system is that we can deliver our siRNA intravenously or through the blood stream rather than d...