Diseases and Disorders

RNA Interference in Treatment of Neurological Conditions

Marta Luterek


RNA interference (RNAi) is one means of reducing gene expression. Interference effects take place after the transcription stage by degrading a corresponding sequence of messenger RNA (mRNA). Since the discovery of RNAi, for which A. Fire and C.C. Mello were awarded the Nobel Prize, it has become evident that RNAi holds great potential to be used as a treatment for many types of diseases, including those of neurological origin. In this article, research is presented on the topic of RNAi in treatment of Huntington’s disease, spinocerebellar ataxia, Parkinson’s disease, and Alzheimer’s disease. Moreover, a discussion on both the potential and risks of using RNA interference as a therapeutic agent is included. 


The Discovery of RNA Interference

RNA interference (RNAi) is a means of silencing a gene after its sequence has been transcribed from DNA into mRNA. It was discovered by Fire and Mello, who were awarded the 2006 Nobel Prize in Physiology or Medicine for their findings [1].

They injected double-stranded RNA (dsRNA) directly into the body of their model organism – the nematode worm Caenorhabditis elegans. The dsRNA they used corresponded to a 742-nucleotide sequence of the unc22 gene, which encodes an abundant but nonessential myofilament protein. The decrease in unc22 activity leads to a phenotype of severe twitching movements [2].

The injected nematodes showed strong twitching, while the control group and the worms injected with single-stranded RNA remained phenotypically normal. This led them to conclude that injection of dsRNA inhibited expression of the unc22 gene while single strands of RNA (sense or antisense) did not inhibit genes with the same efficiency. They also stated that the silencing effect was specific for the mRNA sequence homologous to the dsRNA because other mRNA sequences were not affected. In a follow-up paper, they stated that interference by dsRNA causes no changes in the original DNA sequence [3]. This was surprising at the time because interference effects were hypothesized to result from a simple mechanism of hybridization between the injected single-stranded RNA and the mRNA within the cell [1].

Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi technology is precise, efficient, and stable and that it has potential in inhibiting expression of desired genes [4].


The Mechanism of RNAi

Protein synthesis requires the cell to perform transcription – the copying of the DNA sequence into mRNA, which is then transported into the cytoplasm. After post-transcriptional modifications, the mRNA sequence is translated into a protein. RNAi interrupts gene expression after the transcription stage. The process of RNAi is initiated by double-stranded RNA molecules (dsRNA). They can be either exogenous – introduced into the cell via vectors or by laboratory manipulations – or endogenous – in the form of microRNAs coming from introns – genes that are not translated into proteins [5].

When the dsRNA is in the cell, it is recognized by a protein called Dicer, which functions as a ribonuclease – an enzyme that catalyzes the degradation of RNA into smaller fragments [5]. The dsRNA is cleaved into small pieces (20-25 nucleotides). These fragments are called small interfering RNAs (siRNAs). They are then separated into single strands and integrated with proteins to form the RNA-induced silencing complex (RISC) [6]. The single strand of RNA integrated with the complex binds to a complementary sequence in the mRNA and degrades it [6]. The mRNA is disabled and no protein is made. Thus, the expression of a gene is silenced.

The Therapeutic Potential of RNAi

Since the discovery of RNAi, the idea of RNAi therapeutics has developed into a creative and competitive field that has attracted intense interest and is one of the most highly investigated fields in biotechnology research [7]. Understanding the mechanism of RNAi leads to a conclusion that due to its specificity and effectiveness, RNAi holds the potential to be used as a therapeutic agent for dominantly inherited disorders and disorders in which a protein is a key element to its pathogenesis. The possibility of engineering siRNA to silence specific sequences of a mutant allele without interfering with the expression of the normal allele has been demonstrated in multiple studies [8-12]. Diseases with a genetic component that could potentially be treated using RNAi include many neurological disorders, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD) and spinocerebellar ataxia (SCA) [13].


RNAi in Treatment of Huntington’s Disease

Neurogenetic diseases caused by nucleotide repeat expansion are often fatal. Such diseases include Huntington’s disease and spinocerebellar ataxia, which are induced by a repetition of CAG sequences, coding for the amino acid glutamine [14].

Huntington’s disease is an autosomal dominant disorder, which means that a person needs to inherit only one copy of the defective gene to develop the disorder [15]. However, the offspring of a person affected by HD  may have more rapid progression or severity of symptoms due to the relative increase in the length of the CAG repeat during meiosis and the subsequent expression of proteins with longer polyglutamine tracts [14]. The molecular defect that causes the disease is a somatic mutation found on chromosome 4 in a gene that codes for a protein called huntingtin [15]. The disease causes neurons to die in various areas of the brain, such as the neostriatum, substantia nigra, cerebral cortex, and hippocampus [15]. The hallmark symptom of HD is uncontrolled movement of the arms, legs, face, and upper body [16]. Psychological symptoms include changes in behavior, emotion, judgement, and cognition [15]. Treatments available for HD patients are only able to lessen symptoms of involuntary movements and psychiatric disorders using antipsychotic drugs and antidepressants [15]. However, there is currently no cure for HD and no way to slow progression [16]. 


RNAi in Treatment of Spinocerebellar Ataxia

Spinocerebellar ataxia is a term referring to a group of ataxias that are characterized by neurodegeneration in the cerebellum and the spinal cord – areas involved in movement control [17]. Most types of SCA are caused by trinucleotide CAG repeat expansions [17]. Like in HD, the severity of symptoms depends on the genetic recombination of the expanded CAG repeats.

Symptoms of SCA include loss of balance, poor coordination, and slurred speech, though patients usually retain full mental capacity [18]. There is currently no way to fully cure SCA. The clinical care of patients focuses on managing the symptoms through physiotherapy, occupational therapy, and speech therapy [18]. 

There is considerable debate about how the proteins with expanded CAG sequences cause disease, what causes the selective neurodegeneration by mutant proteins, and how their expression causes the specific symptoms of each nucleotide repeat disease [14]. However, there is no doubt that each of the CAG-induced diseases is caused by expression of the mutant gene [14]. This leads to a conclusion that RNAi could serve as a method of inhibiting expression of the mutant genes in some of the diseases, thus inhibiting onset of the disease. However, it has been demonstrated that expanded CAG repeats, although accessible to RNAi, are not preferential targets for silencing [18]. Nevertheless, an associated single-nucleotide polymorphism can be exploited to achieve silencing provided that it is placed centrally in the siRNA. Researchers were successful in inhibiting the SCA3 gene using this method, and it is thought that this method should extend to HD [18].


RNAi in Treatment of Alzheimer’s Disease

RNAi is also researched as a possible treatment for Alzheimer’s Disease (AD). AD is the most popular type of elderly dementia characterized by irreversible neurodegeneration. Symptoms of AD include trouble recalling very recent events, loss of verbal memory, and decline in cognitive function. Doctors are currently unable to cure AD, but research in this field continues while patients receive treatments meant to slow the progression of symptoms and improve their quality of life [17]. There are two key proteins in the pathogenesis of AD: tau protein and amyloid precursor protein (APP). Developing ways to inhibit accumulation of these proteins is of great interest when it comes to developing potential treatments for AD. Researchers were successful in developing siRNAs that allele-specifically suppressed the most widely studied tau protein and APP mutations, and they delivered the siRNAs into cells via short hairpin (shRNA) plasmids [19]. They also found that successful engineering of effective siRNAs required mutations to be placed centrally in the guide strand of the siRNA [19]. Thus, they concluded that in mammalian cells, it is possible to silence a single disease allele without activating pathways that result in the spread of silencing signals (such pathways are present in plants and worms). However, delivering the siRNA to the correct target cells in the brain still poses a major challenge. Viral vectors have shown potential in delivering genes into neurons of the central nervous system [19]. Nevertheless, the long-term consequences of triggering the RNAi pathway are still unknown, but future RNAi studies in transgenic animal models of AD and other diseases may help to answer these questions [19].


RNAi in Treatment of Parkinson’s Disease

Parkinson’s disease is a neurodegenerative disorder that predominantly affects dopaminergic (dopamine-producing) neurons in the substantia nigra – a basal ganglia structure located in the midbrain [21]. Symptoms of PD usually develop slowly and differ among patients but generally include tremor (involuntary shaking of the hands, arms, legs, or jaw), bradykinesia (slowed movement), rigid muscles, speech problems, and balance problems [21]. As for previously mentioned diseases, PD cannot currently be fully cured, and treatment is aimed at controlling the symptoms.

A rare, inherited form of PD is connected with a mutation of the alpha-synuclein protein, which is a component of Lewy bodies – abnormal structures composed of protein that develop inside nerve cells [22]. Alpha-synuclein is encoded by the SNCA gene and overexpression of the gene as well as nucleotide variations appear to be involved in the pathogenesis of PD [23]. Scientists developed a RNAi-based treatment method called ExCont-RNAi – expression-control RNAi – which they used to moderately silence the overexpressed SNCA genes to return to a normal level [22]. They introduced the siRNA into the fibroblasts from a PD patient in whose cells SNCA was expressed approximately twofold more than that in normal fibroblasts [23]. Fibroblasts treated with the siRNA reduced the expression of SNCA approximately by half, similar to the level found in healthy fibroblasts [23]. These findings demonstrated that normalization of overexpressed SNCA by RNAi is possible in PD cells. Furthermore, no significant off-target effects of the therapy were found.



The discovery of RNAi expanded the possibilities of gene therapy for many diseases and changed the way we think about inhibiting gene function. As discussed in this article, RNAi holds the potential to be used in therapy of multiple neurological disorders. Since the discovery of RNAi, genetic therapy has become one of the most quickly advancing fields in medicine. Companies devoted solely to developing RNAi-based medications have emerged, such as Alnylam Pharmaceuticals. However, despite the promising potential, the long-term consequences of chronically triggering the RNAi pathway in vivo, as may be required to treat neurodegenerative conditions, are unknown [10]. Another problem connected with RNAi as a therapeutic agent is that data has shown that genes can differ widely in their susceptibility to inhibition with no obvious sequence features predicting the success or failure of a given siRNA. For example, every siRNA designed against ataxin-3 displayed significant activity (7/7, 100%), whereas tau proved to be more difficult to inhibit with only a single region centered on the V337M mutation yielding effective siRNAs (3/7, 43%) [11]. Furthermore, it is difficult to directly predict the siRNA concentration required for therapeutic effects in vivo, where the disease protein is expressed at much lower levels than in experimental systems used in the studies and siRNA delivery could be less efficient [17]. Despite the uncertainties connected with RNAi therapeutics, it is definitely an area with great therapeutic potential, so it continues to be studied as a means to develop ways for use as a treatment for neurological diseases of genetic origin or with a genetic component.




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    Marta Luterek

    Marta Luterek

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