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Crossing the blood-brain barrier: siRNA treatment for prion diseases

Date

2017

Authors

Bender, Heather Rose, author
Zabel, Mark, advisor
Telling, Glenn, committee member
Gustafson, Daniel, committee member
Bamburg, James, committee member
Dow, Steve, committee member

Journal Title

Journal ISSN

Volume Title

Abstract

Protein misfolding diseases such as prion diseases, Alzheimer's disease, and Parkinson's disease, are fatal neurodegenerative diseases caused by a misfolded protein. There are no known therapeutics that extend survival times of afflicted individuals with these diseases. An attractive therapeutic option for protein misfolding disorder is RNA interference, which uses either short hairpin RNA or small interfering RNA (siRNA) to target a specific mRNA for degradation that results in a reduction of protein levels. The reduction of a target mRNA/protein can result in a decrease of misfolded protein in the central nervous system (CNS). However, crossing the blood-brain barrier remains the main challenge for developing RNA interference therapeutics to the CNS. Liposomes are commonly utilized to deliver siRNA to peripheral sites and are being investigated for their ability to deliver siRNA to the brain. We have previously reported on a new liposome delivery system that delivered siRNA targeted towards the cellular prion protein, PrPC, to mouse neuroblastoma cells. PrPC is a normal host cellular protein that misfolds into a protease resistant isomer, PrPRes, which leads to the development of prion diseases. We call these siRNA delivery vehicles: liposome-siRNA-peptide complexes (LSPCs). LSPCs are targeted towards the CNS using a small peptide from the rabies virus glycoprotein, called RVG-9r. In the second chapter of this dissertation, we show that an intravenous injection of LSPCs results in a 40-50% reduction of neuronal PrPC. Upon injection of LSPCs, we observed that half of all treated mice had PrPC siRNA targeted towards the area of the brain several hours after injection. However, we also observed a clearance of PrPC siRNA by the kidneys in the other half of LSPCs-treated mice. Therefore, we designed two other liposomal delivery vehicles that would allow us to encapsulate the siRNA in the liposome and covalently link RVG-9r to the outside of the liposome. We also added PEG lipids to these new delivery vehicles to extend the circulation half-life of the liposomes. We call these additional delivery vehicles peptide-addressed liposome-encapsulated therapeutic siRNA (PALETS). The two PALETS formulations include one cationic (DOTAP [1,2-dioleoyl-3-trimethylammonium-propane]) PALETS and one anionic (DSPE [1,2-Distearoyl-sn-glycero-3-phosphoethanolamine]) PALETS. We have utilized the cation protamine sulfate to encapsulate the siRNA within the anionic PALETS. The addition of protamine sulfate to the siRNA resulted in an encapsulation efficiency of 80-90% in DSPE PALETS. Four days after treatment with LSPCs and PALETS, LSPCs have the biggest decrease in neuronal PrPC on the cellular surface, while DOTAP PALETS have the greatest reduction of PrPC-positive cells. DSPE PALETS showed no statistical difference between the treated and untreated mice at this time point; however, two of the three treated mice did have a decrease in their neuronal PrPC, indicating that this delivery vehicle is able to deliver PrPC siRNA to the brain. There was no reduction in mRNA levels of any of the treated mice in the brain but the DOTAP LSPCs and DOTAP PALETS resulted in a 2-fold decrease of PrPC mRNA levels in the kidney, while DSPE PALETS resulted in a 2-fold increase of PrPC mRNA levels in the same organ. The first therapeutics for prion diseases targeted the mechanism of conversion between PrPC and PrPRes. These therapeutics were successful in decreasing the amount of PrPRes in vitro but they had limited success in vivo. Challenges of these therapeutics included toxicity, inability to cross the blood-brain barrier, strain specificity, and/or failure to affect survival times. PrPC became an attractive therapeutic option when it was shown that PrP-null mice did not develop any outward phenotypic differences from the removal of PrPC. Our LSPCs, with PrPC siRNA, reduced the amount of PrPC protein and PrPC mRNA levels in mouse neuroblastoma cells. This reduction in PrPC resulted in a concomitant decrease of PrPRes and a 'curing' of the prion-infected cells. In the third chapter of this dissertation, we have treated two different mouse models with our LSPCs at different time points to assess the pharmacodynamics of the treatment. In vivo live imaging followed by flow cytometry revealed delivery of PrPC siRNA to the brain one hour after intravenous injection. The LSPCs resulted in a decrease of neuronal PrPC in a C57Bl/6 mouse model at 24, 48 hours, and 4 days after treatment. A decrease in neuronal PrPC was also observed in a CD1 mouse model at 4 and 15 days after treatment. Surprisingly, mRNA levels did not always concur with the protein level data. At certain time points, the mice with the biggest decline in PrPC protein had the greatest increase of PrPC mRNA. Off-target effects were observed in the kidney, which might have been caused non-specifically by LSPCs treatment and not by the PrPC siRNA. We also show that PrPC protein levels decrease by 70% in prion-infected mice after three consecutive LSPCs treatments spaced two weeks apart. Analysis of mRNA levels of these mice after three treatments revealed a simultaneous reduction in PrPC mRNA levels. Several researchers have shown a reversal in prion neuropathology that results after decreasing the amount of PrPC, either by a Cre/loxP system or short hairpin RNA. Therefore, we treated prion-infected mice with our LSPCs treatment targeting PrPC. Two treatment studies were conducted to determine the optimal dosing regimen of LSPCs treatment. The first study treated prion-infected mice with LSPCs every two weeks starting at 120 days post inoculation and the second study treated mice with LSPCs every 3-5 weeks starting at 120 days post inoculation. The mice were intraperitoneally inoculated with a low dose of RML-5 prions to simulate a more natural prion infection. Unfortunately, in the fourth chapter of this dissertation we show that neither of the dosing regimens resulted in an increase in survival times of prion-infected mice. The mice in these two dosing studies were also subjected to burrowing and nesting behavioral tests to determine if LSPCs treatment improves behavioral outcomes. We show that LSPCs treatment every two weeks improves behavior scores at 141 and 169 days post inoculation in some treated groups. This improvement in behavior indicates that, while the LSPCs treatment are not affecting survival times, they are improving behavioral outcomes of prion-infected mice. Surprisingly, three of the uninfected, treated controls died immediately after LSPCs treatment of an apparent Type III hypersensitivity. Therefore, we performed ELISAs to measure the immune response towards the RVG-9r peptide. Several groups of treated mice in the terminal dosing studies had increased levels of IgG against RVG-9r compared to the infected, untreated control. In another study, it was revealed that three total IgG levels against RVG-9r increased after three subsequent LSPCs treatments spaced two weeks apart. We also assessed the amount of PrPRes in the brains and spleens of LSPCs-treated mice. Using the protein misfolding cyclic amplification assay, we determined that LSPCs treatment causes an increase in PrPRes levels in the brain after one to six LSPCs treatments. No trends can be seen in the spleen. Taken together these results indicate that the current LSPCs formulation using RVG-9r and PrPC siRNA result in an immune response that may interfere with any benefits of the treatment. Another explanation for these results is that PrPC may be tightly regulated at the transcriptional level, so the cell may try to return the mRNA/protein levels to normal by increasing PrPC mRNA when it detects a decrease in PrPC mRNA or protein levels. The increase in PrPC may be the cause of the increase of PrPRes observed in these studies. Therefore, transiently decreasing PrPC via siRNA may not be the best therapeutic option available. It is recommended that more studies are undertaken to further elucidate the transcriptional regulation and immune response towards the LSPCs treatment. LSPCs will need to be further modified to become a viable therapeutic option for prion diseases.

Description

2017 Fall.
Includes bibliographical references.

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Subject

prions
siRNA
PrPC
blood brain barrier

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