Development of Therapeutics Based on RNA Interference Technology

The development of therapeutics based on RNA interference technology

Abstract

RNA interference (RNAi) is a biological process in which RNAi molecules are used to inhibit gene expression. Historical, RNAi was known as RNAi, co-suppression, Post-transcriptional Gene Silencing (PTGS), quelling. Three primary categories of RNA interference are short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) and microRNAs (miRNAs). Since the discovery of RNAi, it has become one of the most important advances in biomedical research to investigate gene function and has been used as a potential therapeutic approach to treat various diseases in human (e.g., cancer, eye diseases, infections, cardiovascular diseases, metabolic disorders).

The first part of this review is focused on the discovery of RNAi, the RNAi mechanism and its therapeutic application. In addition, we also mention two applications of this technology in clinical medicine as the treatment for Huntington’s disease and breast cancer. Finally, the future perspective of RNAi technology is discussed.

Keywords: RNA interference (RNAi), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), gene silencing, Dicer, RNA-inducing silencing complex (RISC), Huntington’s disease (HD), mutant HTT (mtHTT), adeno-associated viral serotype 5 vector (AAV5),  triple negative breast cancers (TNBCs), Forkhead Box M1(FoxM1), nanoparticle.

Introduction

In recent years, lots of genome sequencing of organisms have been being identified and this poses a huge challenge for scientists in understanding the function of thousands of genes in the genome. Many new techniques have been developed to address this problem. Reverse genetics is now the most effective way to assess the function of a gene, which based on sequence information in the genome to determine the function of the genes. RNA interference (RNAi) is one of the “Reverse Genetics” technique that inhibits the gene expression by the double strands RNA (dsRNA) that can cause the degradation of target messenger RNA (mRNA). It has been widely used as a knock-down technology to analyze gene function in various organisms. RNA interference (RNAi) is a general name given to a gene-silencing process, it is also known as ‘quelling’ in fungi, ‘PTGS’ in plants and ‘RNAi’ in animals [1].

RNAi was first discovered in plants by R. Jorgensen and his colleagues during experiments connected with changes in pigmentation in the Petunia in 1990. They were working on Petunia to deepen the purple color of Petunia flowers by introducing exogenous transgenes. Instead of getting the intensify the color, most of the resulting flowers had lost their color. This result led them to hypothesize that the introduced transgene was co-suppressing the endogenous gene [2].

In 1995, RNA silencing was first documented in animals by Guo and Kemphues, who observed that the sense and antisense RNA were equally effective in inhibition of gene expression in Caenorhabditis elegans (C. elegans) [3].

In 1998, Andrew Fire and Craig C. Mello described the RNA interference process in C. elegans. They did not only use sense or anti-sense RNA but also used dsRNA to inhibit gene expression in C.elegans. This experiment showed that the use of dsRNA for gene silencing was more effective than using only sense or anti-sense RNA 10 times. This novel phenomenon has given the RNAi era [3].

After the report of Andrew Fire and Craig C. Mello, RNA interference has been reported in a majority of the organisms which include plants, fungi, zebrafish, planaria, insects (Drosophila), and mice. This suggests an ancient evolutionary origin of this phenomenon [2].

Mechanism of RNAi

In general, at the beginning of the process, the double-stranded RNA (dsRNA), which is either introduced into cells naturally or artificially, is processed into small pieces (23–25 nucleotide)-siRNAs- by an RNase-III-like enzyme called Dicer. These siRNAs then bind to another protein complex RNA-inducing silencing complex (RISC), which contains the “slicing” protein Argonaute 2, and which unwinds the small fragments RNAs into single-stranded molecules. The passenger strand is degraded as a RISC complex substrate. The other strand, called the guide strand, is loaded into the RISC complex and links the complex to the mRNA strand by specific base-pairing. Finally, mRNA is cleaved and destroyed, and by that prevent the formation of the proteins that they code for [4]. This is called the gene silencing. A schematic overview of this cellular process is given in Figure 1.

Figure 1. Mechanism of RNAi. [5]

There are at least three small RNAs in the RNAi pathway: small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) and microRNAs (miRNAs) that can be classified based on their origin and function [6].

MicroRNAs (miRNAs), represent endogenously encoded small RNAs, are non-coding single-stranded RNA molecules of about 21–23 nucleotides long, which regulate gene expression by bind to target mRNAs in a sequence-specific manner. The transcription of miRNAs genes is done by RNA polymerase II produce primary miRNA (pri-miRNA). These pri-miRNAs molecules are cleaved by Drosha/DGCR8, forming the precursor miRNA (pre-miRNA). The pre-miRNAs form a double strand stem-loop structure. Exportin-5 protein transports the pre-miRNAs into the cytoplasm from the nucleus. The pre-miRNAs are processed by enzyme Dicer to miRNAs duplex. These miRNAs are incorporated into a RISC where it binds to its target mRNA through complementary base pairing, induces degradation of target mRNAs.

Short hairpin RNAs (shRNAs) are synthesized in the nucleus of transfected cells and form a hairpin structure which is a similar structure as miRNAs.  The shRNAs is introduced to the nucleus of target cells using either bacterial or viral vectors. This shRNAs is cleaved by Dicer enzyme, remove hairpin to form the siRNAs and then bound to RISC. This complex bind to target mRNA and leads to the degradation of mRNA.

Small interfering RNA (siRNA) is a short segment of RNAs (21-25 nucleotides) siRNAs can be exogenously introduced into cells by various transfection methods. siRNAs designed to specifically target a particular mRNA for knock-down gene expression. These molecules have become a powerful tool for gene functions studies.

Therapeuticapplication of RNAi

There have been extensive investigations made in vitro and in vivo to understand the role of RNAi to possibilities of treating various diseases and conditions. The major targets of RNAi in medical therapeutics are viral infections, cancer, cardiovascular and cerebrovascular Diseases, and neurodegenerative disorder [7] [8] [9].

Viral infection

The ability to inhibit the replication or cellular uptake of viruses and other infectious agents of RNAi has been shown clearly in cell culture studies, thus promise for the treatment of human patients. Human immunodeficiency virus (HIV), hepatitis, and influenza are prominent examples of RNAi-based therapies [10].

Because of the well understand of lifecycles and pattern of gene expression of HIV, HIV was the first infectious agent targeted by RNAi. The HIV-encoded RNAs in cell lines (Tar element, tat, rev, nef, vif, env, and gag genes) or reverse transcriptase have been silenced, resulting in inhibition of viral replication in cultured cells through the using of synthetic siRNAs and expressed shRNAs [10].

Secondly, a reduction in hepatitis B virus RNA and replicative intermediates has been demonstrated upon introduction of siRNAs or shRNA vectors [11]. And finally, the accumulation of influenza viral mRNAs was arrested following the addition of siRNAs specific for nucleocapsid or a component of the RNA transcriptase [11].

Cancer

There are two general abnormalities in cancer cells:

First, they accelerate cell division rates by creating a harmonic disturbance in the cell cycle.

Second, they death fail to undergo programmed cell death or apoptosis.

The aim of using RNAi in cancer therapy is to knock out the expression of a cell cycle gene or an anti-apoptotic gene in cancer cells. As a result, it can stop tumor growth and kill the cancer cells. To selectively eliminate cancer cells without damaging normal cells, RNAi would target a gene specifically involved in the growth or survival of the cancer cell, or the siRNAs would be selectively delivered into the cancer cells by transfection (Figure 2) [10] [12].

Figure 2. Designing siRNA therapeutics for cancer treatment [12]

Thanks to the recent advances in RNAi technology, cancers in any stage of its progression can be treated.

For example, the formation of tumor vasculature can be promoted by the Angiopoietin-1 factor. The tumor growth almost depends on the blood supply by tumor vasculature. Thus, to inhibit the growth of the tumor, we have to block tumor vasculature. This treatment strategy has been shown to be effective in a recent study where an adenovirus-based Angiopoietin-1 siRNA expression system was tested in esophageal cancer cells line Eca109 in vitro and in the mice model for esophageal cancer. this study is reported a reducing tumor growth and also reduce blood vessel formation [13].

RNAi therapy can also be used in combination with a drug therapy in which the drug action can potentiate in various ways as sensitizing cancer cells toward the drug or by suppressing the ability of cancer cells to develop resistance towards the drug. Table 1 provides detailed information about the current status of RNAi-based drugs [14].

Table 1. Current Status of cancer siRNA-based drug. [14]

Cardiovascular and Cerebrovascular Diseases

The cardiovascular disease is most commonly resulting from the progressive occlusion of arteries in a process called atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke, resulting in the death of cardiac muscle cells or neurons. Although some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis; data from animal studies shown that such cardiac myocytes and brain neurons are saved from death by apoptosis. RNA interference technology can be used in the process of atherosclerosis or to reduce the damage to heart tissue and brain cells that patients suffer from cardiovascular disease) [10].

Neurodegenerative Disorders 

Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and polyglutamine (polyQ) repeat diseases are neurodegenerative disorders. Aberrant accumulation of misfolded proteins appears to play a central role in disease onset and progression. Misfolded protein is usually sticky and glued to itself or glues itself to other proteins. This makes it useless for the cell and the dangerous plaques formed. This can lead to neurodegenerative disorder diseases. By using RNAi-based approaches, the levels of neurotoxic proteins are modest reduced.  It had succeeded in cell and animal models.

For example, amyloid plaques protein is composed of the misfolded Aβ peptide, which is produced by proteolytic processing of the amyloid precursor protein (APP). The abnormal of this protein within the brain is considered as the reason for Alzheimer’s disease. RNAi has been used to reduce Aβ peptides in vivo by targeting the expression of enzymes required for the proteolytic processing of APP, or by directly targeting the expression of APP [7].

Example of successful application of RNA interference technique

 Example 1, Triple negative Breast Cancer Treatments by siRNA Nanoparticles

Breast cancer presents the most commonly occurring cancer in women.  In 2017, an estimated 252,710 new cases of invasive breast cancer will be diagnosed among women and approximately 40,610 women are expected to die from breast cancer in 2017 in the United States [15].

In breast cancer, hormone receptors are the proteins which signal cells to grow (healthy and cancerous) located in and around breast cells. In cancer cells, when the hormone receptors tell these cells to grow uncontrollably which results in the tumor formation. The main receptors in breast cancer are estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2). Based on the level of receptor expression, breast cancer can be classified into three major subtypes, which include hormone receptor positive, HER2 positive and triple negative breast cancers (TNBCs). The overexpress of only hormonal receptors (ER+ and/or PR+ and HER2−) is luminal A cancers while the overexpress all three receptors (ER+ and/or PR+ and HER2+) is luminal B cancers. Breast cancers that overexpress HER2 only (i.e., HER2+/ER−/PR−) are referred to as HER2-enriched. The negative of three receptors (i.e., ER−, PR−, and HER−) is TNBCs that means the growth of the cancer is not supported by the hormones estrogen and progesterone, nor by the presence of too many HER2 receptors. Therefore, TNBCs breast cancer does not respond to hormonal therapy (such as tamoxifen or aromatase inhibitors) or therapies that target HER2 receptors, such as Herceptin. It is also frequently resistant to chemotherapy. Various targeted treatment approaches are currently being explored as siRNA nanoparticles to overcome the challenges associated with TNBCs treatment [16].

The clinical success of siRNA nanoparticles for the treatment of various types of solid cancer has laid the foundation for its application in TNBCs. A number of recent preclinical studies have uncovered different delivery vehicles for siRNAs to silence a target gene that is associated with poor prognosis in TNBCs. There are many siRNA nanoparticle application areas in TNBCs, this review will be focused on the inhibition of cell cycle components of siRNAs therapy specifically FOXM1-specific small interfering RNA [17].

siRNA has more advantages than small molecule drugs based on its specificity to inhibit target gene expression in the cytoplasm with low toxicity, providing an efficient way to silence the expression of many oncogenes [18].

In an individual breast tumor, there are nearly 80 genetic mutations can be found, a dozen in these mutations is thought to be actively driving oncogenesis. The oncogene usually mentioned breast cancer that is v-myc myelocytomatosis viral oncogene homolog (c-Myc), murine double minute clone 2 (MDM2) and Forkhead Box M1(FoxM1) [18].

FOxM1 is a member of the FOX protein family, which is characterized by a conserved winged-helix DNA binding domain. FOxM1 has an important role in cell proliferation and cell cycle transitions – inducing the transition from G1 to S phase and from G2 to M phase so increasing expression of FOxM1 was detected in numerous cancers like pancreas, stomach, liver, lung, brain, breast, nervous system and blood [18].  In general, FOxM1 overexpression in tumors is linked to late tumor stage, high proliferation rate, and poor prognosis, suggesting that FOxM1 could serve as a prognostic marker for cancer patients [18]. Moreover, since serve as a master regulator of the cell cycle, its suppression inhibits the transcription of genes associated with proliferation and tumor growth. Recognizing the important role FOxM1 plays in breast cancer biology, this transcription factor may be a beneficial target for cancer treatment.

A study of Hamurcu et al. demonstrated that FoxM1 overexpressed in multiple TNBCs cell lines and FoxM1 expression impairment inhibited cyclin D1 expression and Src (Y416) and activated Erk. Functionally, these changes have combined to reduce the formation, proliferation, invasion, and migration of colonies. Traditionally, transcription factors are often considered ‘indisputable’ due to the lack of enzymatic activities [19]; however, these limitations are raised by the nanoparticles siRNA. Two weeks after the nude mice were transplanted with MDA-MB-231 tumors, a significant reduction in FoxM1 expression in the primary tumor is reported, which was associated with a reduction in primary tumor burden (Figure 3) [17] [20].

Figure 3. siRNA nanoparticles targeting cell cycle. siRNA nanoparticles delivering siRNA against FoxM1, CDK11/CK2, and CDK1 are demonstrated to significantly reduce primary tumor burden. [17]

There are two main roadblocks in delivery siRNA to the cells:  transport across the cell membrane and escape from the endocytic pathway. Nanoparticles are the most common choice to address these challenges. there are several advantages to the use of nanoparticles. using nanoparticles to deliver siRNA will protect siRNA from plasmatic nucleases and immune responses thus assisting in endocytosis. Furthermore, ligand-linked nanoparticles increase the selectivity of the nanoparticle’s siRNA distribution to tumor cells.

Even though nanoparticles are the promising therapeutic strategy for cancer treatment, nanoparticle carriers still possess disadvantages that limited the technology. First, synthesis of nanoparticles in larger amounts may compromise the function of the nanoparticles, for example through aggregation. The cost associated with synthesis and quality control could be prohibitively high. Additionally, the large volume of nanoparticles transferred into a patient could introduce additional challenges with immunogenicity and toxicity [21].

Example 2, RNAi therapy for Huntington’s disease

Huntington’s disease (HD) is an incurable, dominant neurodegenerative disorder, caused by the repeated expansion of CAG trinucleotide in the huntingtin (HTT) gene (>36 CAGs). the repeat of CAG is known as polyglutamine (polyQ). Reducing the expression of mutated HTT (mtHTT) is the potential way for treatment of HD [22].

Tetrabenzene is the only approved drug used in HD treatment which palliates motor abnormalities. There are no disease-modifying therapies currently available to patients [22].

Silencing of transgenic mutant HTT (mtHTT) may provide a cure for Huntington’s disease. This can be achieved by designed micro (mi)RNAs to target HTT transcripts and delivered by an adeno-associated viral serotype 5 vector (AAV5) (AAV5-miHTT). AAV vectors deliver miRNAs directly to the brain to non-selective knock-down the huntingtin gene.

In an acute HD mouse model, the AAV5-miHTT construct was found to be most effective in preventing the formation of mutant HTT and eventually leading to inhibition of DARPP-32 associated neuronal dysfunction [23].

Based on this technique, uniQure company is developing a gene therapy for HD (AMT-130).

Adeno-associated viral (AAV) vectors are the most common vehicles of choice in clinical trials because of their low immunogenicity, numerous engineered tissue-specific serotypes, and long-term transgene expression through chromosomal integration [22].

Future perspective of RNAi technology

The RNAi technique has been widely used for drug development and several phase I and II clinical trials are underway. However, there are still some disadvantage and challenges in developing RNAi [24] [25].

(1) The potential for off-target effects that can lead to the silencing of gene expression of non-pathogenic genes results in hinder the cellular normal function.

(2) Triggering innate immune responses by high levels of siRNA restricted its therapeutic efficacy.

(3) Another important issue is the stability. siRNAs can be easily degraded in the physiological condition.

(4) and most importantly that is the delivery systems. These challenges being both intracellular and extracellular are unavoidable. (Figure 4) [21] [26].

Figure 4. Schematic illustration of delivery barriers in extracellular (A) and intracellular (B) regions. [26]

Because of these challenges, the use of RNAi technique with its full potential is still a long way off. To overcome the delivery barrier, researches on liposomal and various nanoparticle-based siRNA delivery systems are underway that have proven their worth and are widely recognized as the best means to achieve safe and targeted delivery of siRNA [21]. The nanoparticles have limited by their physicochemical properties and there are still other barriers to using RNAi. Thus, future research to understand the barriers that are still not fully known is one of the first steps that must be undertaken.

The very first therapeutic small interfering RNA (siRNA, Onpattro (Patisiran) has recently been approved by the Food and Drug Administration for the treatment of nerve damage caused by a rare disease called hereditary transthyretin-mediated amyloidosis (hATTR) [27].

RNA drug provides a new weapon in the arsenal to treat the disease by preventing the production of specific cellular proteins. With the approval of Onpattro, it is possible to predict the application of RNA therapy to other genetic diseases.

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