Investigating RNA Interference as an Emerging Treatment for Prion Diseases

By Neelesh Sathish

Abstract

Prion diseases, such as Creutzfeldt-Jakob disease in humans, are dangerous neurodegenerative diseases due to the misfolding of the prion protein (PrP). This essay investigates the possibility of RNA interference (RNAi) to treat prion diseases by targeting and degrading the mRNA molecules that are responsible for the production of PrP. RNAi occurs when double-stranded RNA (dsRNA) is induced into cells to degrade the intended mRNA molecules. Scientists suggest using recombinant viral vectors to deliver short hairpin RNAs (shRNAs) that would be processed into small interfering RNAs (siRNAs) within the brain. These siRNAs would then target and degrade the mRNA molecules, potentially reducing PrP production and slowing disease progression. The research investigates the viability of this process through in vitro and in vivo experiments. In vitro studies presented a significant decrease in PrP levels in cells transfected with siRNA plasmids. In vivo experiments using chimeric mice notably demonstrated that anti-PrP shRNA increased the survival time after prion injection. However, studies also convey the important role of PrP and how depleting PrP levels could produce unintended consequences. The Emerging RNAi Treatment

RNA interference has surfaced as a powerful biological process that serves as a treatment for various neurological diseases. In this process, double-strand ribonucleic acid (dsRNA) leads to the silencing of specific genes. RNAi was first discovered in the worm species Caenorhabditis elegans where dsRNA-targeted gene products disappeared from the worm and its offspring [1]. RNAi has offered promise in the treatment of prion diseases.

Prion Diseases and RNAi Treatment

Prion diseases include various neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy ("mad cow disease") [2]. One prion disease, Kuru, was widespread in the Eastern Highlands of Papua New Guinea during the early 1900s and is still prevalent now. During this time, the people of New Guinea practiced a form of cannibalism in which they would consume the bodies of deceased relatives as a sign of showing respect which led to brain degeneration [3]. The main cause of prion diseases is the misfolding of the prion protein (PrP), converting it from its normal cellular form (PrPC) into a fatally infectious, misfolded form (PrPSc) [4]. As the PrPSc accumulates, they can clump up within nerve cells which can block the nerves from sending out signals, causing severe cognitive impairment, motor dysfunction, and ultimately death. The body cannot get rid of PrPSCs by itself.

The process of RNAi begins with dsRNA being introduced into the cells of the brain. Inside the cell, the enzyme Dicer cuts up dsRNA into smaller bits that are known as small interfering RNA duplexes (siRNA duplexes). After this, the siRNA duplexes activate a group of proteins called the RNA-induced silencing complex (RISC). Within RISC, the siRNA duplex undergoes an unwinding process, leading to the formation of an active form of RISC (referred to as RISC*). This separates the duplex into single-stranded siRNAs, where one of these strands, known as the antisense strand, remains bound to RISC*. The RISC*, guided by the antisense strand of the siRNA, specifically recognizes and degrades the target mRNA [5].

Mechanism of RNAi Action

The expression of prion proteins depends entirely on mRNA molecules so Dr. Melanie White and Dr. Giovanna Mallucci believed that by targeting and degrading the mRNA molecules within the brain they would be able to halt prion protein production, reducing further damage done by PrPSc. To treat neurodegenerative diseases, repeated delivery of siRNAs directly into the central nervous system was necessary. Researchers believed that they could use recombinant viral vectors (genetically engineered viruses) to deliver the siRNAs into the brain. In this approach, siRNAs would need to be expressed as short hairpin RNAs (shRNAs). Once shRNAs are induced into the cells of the brain, they would be processed by Dicer and the formation of siRNAs would occur. A highly effective type of recombinant virus would be retroviruses as they are less likely to trigger a strong immune response by the body. Retroviruses are also of interest as they can insert their genetic material into the DNA of their intended host cells. This indicates that the shRNA would be induced into the cell’s genetic material which allows the shRNA to be stably and continuously expressed over a long period [7].

To further validate this approach, Dr. Zhao-Yun Wang et al. also investigated the direct construction of recombinant plasmids containing siRNAs. To construct the recombinant plasmids that have siRNA, two conservative motifs were selected as potential targeting areas for the PrP-specific siRNAs. The sequences for the siRNAs were chemically synthesized to create two plasmids. After transfecting these plasmids to cells containing PrPC, the health of the cells was tested using a method called Western blot analysis. Through this process scientists came to the result that as the amount of transfected siRNA plasmids increased, the signal intensity of cellular PrP decreased, showcasing that siRNAs were successful in reducing the amount of PrP in the cells [8].

Implications of RNAi Treatment

Dr. Zhao-Yun Wang et al. removed PrP from chimeric mice and found that mice that lacked PrP suffered from extensive oxidation damage, unlike mice with PrP, showcasing the benefit prion proteins produce for the body. Also, neurons with PrP are significantly more resistant to copper toxicity than cells that lack PrP [8]. The advantages PrP provides for the body showcases the necessity of a siRNA that only partially knocks down PrP and would be enough to slow aggregation but still permit the therapeutic benefits of PrP. Along with this, RNAi poses the risk of unintended silencing of other crucial genes which could propose severe consequences to the body [8]. Dr. Jayakrishna Ambati and colleagues suggest that siRNA can potentially cause adverse effects on blood vessel growth in multiple organs as well [12].

RNAi, being a novel and complex process, may be prone to skepticism by the public. To avoid this it would be necessary to set up educational initiatives that address the common misconceptions and provide clear information on the benefits and risks of the RNAi process. This would increase trust and build up a supportive public perspective. Different parts of the world produce different legal opinions on RNAi technology. In the United States, under the Myriad decision, significant challenges will arise for RNAi technologies seeking patent protection while in the European Union, there is a higher focus on how RNAi technologies can solve immense problems and their practical uses in medicine [11]. As well as all of this, the cost of RNAi treatment is approximately $450,000 for annual treatment. This price is only after typical discounts [13]. This presents that the process of RNAi is largely inaccessible to people living in underrepresented countries. To help solve this issue, it would be vital to implement a tiered pricing strategy where the price of the drug is significantly lower in underrepresented countries and higher in wealthier nations. This would ensure that RNAi would be more accessible in underrepresented countries. References

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