1. Introduction

RNA interference (RNAi) is an intracellular mechanism for regulating gene expression by targeting and degrading specific mRNAs with small molecule RNAs, thereby preventing protein synthesis[1].This process gives RNAi the potential for a wide range of gene regulation and therapeutic applications, and has shown a strong impact in disease treatment particularly. The highly selective nature of RNAi makes it particularly valuable in therapy. Unlike traditional therapeutic treatments, RNAi can target specific gene sequences and precisely silence disease-related genes without affecting the expression of other normal genes. Moreover, RNAi has excellent safety and specificity. By engineering suitable siRNA or miRNA sequences, RNA interference can selectively target mRNAs, significantly minimizing adverse effects during therapy. Compared to small molecule drugs or chemotherapy, RNAi does not directly interfere with proteins or cell surface receptors, thus reducing the risk of non-specific side effects. Discovered in Caenorhabditis elegans in 1998 by Fire and Mello, who later received the Nobel Prize in Physiology or Medicine for their groundbreaking work [2]. This discovery revolutionized the field of gene regulation by providing a tool to selectively silence genes with remarkable precision. RNAi operates through two main pathways: the small interfering RNAs (siRNAs) and microRNAs (miRNAs) pathways, both of which guide the RNA-induced silencing complex (RISC) to complementary mRNA sequences, resulting in either mRNA cleavage or translation inhibition [3]. This mechanism has provided researchers with a precise and efficient method for studying gene function by selectively silencing genes, a crucial step in identifying their roles in biological processes and diseases.

In recent years, RNAi has shown tremendous potential in therapeutic applications, particularly in the treatment of genetic disorders, viral infections, and cancer. The ability of RNAi to selectively target and silence disease-related genes has driven significant advancements in drug development. A landmark achievement in RNAi therapeutics the approval of the FDA-approved drug ‘Patisiran’—an RNAi-based therapy used to treat hereditary transthyretin amyloidosis[4]. Furthermore, several siRNA drugs (fitusiran, nedosiran, teprasiran, tivanisiran, and vutrisiran, among others) have advanced to phase III clinical trials, while many other RNAi-based treatments are progressing through preclinical or early-stage clinical studies. However, the primary obstacle to clinical success has long been the delivery of RNAi therapies. Many pharmacokinetic and physical barriers restrict the efficacy of RNAi treatments once administered, limiting their ability to achieve the intended therapeutic outcomes[5]. Ongoing research is exploring improved delivery methods and the development of next-generation RNAi-based treatments, highlighting the potential of this technology in addressing a wide range of diseases [6]. This review will examine the mechanisms of RNAi, its role in gene regulation, and its growing potential in therapeutic applications.

2. Basic Mechanisms of RNAi

RNA interference (RNAi) is a natural cellular process that regulates gene expression at the post-transcriptional level, leading to the silencing of specific genes. This mechanism is driven by small RNA molecules, such as small interfering RNA (siRNA) and microRNA (miRNA), which guide the RNA-induced silencing complex (RISC) to complementary target messenger RNA (mRNA) for degradation or translational repression[1, 4]. Despite sharing a similar function, miRNA andsiRNA are distinct in their structure, origin, and methods of controlling gene expression. This section provides a detailed overviews of the key components involved in RNAi, the stepwise process, and its biological significance in gene regulation.

2.1. Small Interfering RNA (siRNA)

Typically, siRNA molecules are exogenous, originating from sources outside the cells, including viruses or experimental introduced RNA. siRNAs are double-stranded RNA molecule, 21 and 23 nucleotides in length, with two-nucleotide overhangs at 3' ends. They are processed from longer double-stranded RNA precursors by the enzyme Dicer, an RNAse III-like enzyme that cleaves the precursor RNA into smaller fragments[7].

Once processed, the siRNA duplex is loaded onto the RNA-induced silencing complex (RISC), where one strand (the guide strand) is retained, and the other strand (the passenger strand) is degraded. The guide strand directs RISC to a complementary sequence on the target mRNA[8]. Upon binding, the Argonaute (Ago) protein, a key component of RISC, cleaves the target mRNA at the complementarity site. This cleavage leads to mRNA degredation, preventing translation and effectively silencing the gene.

2.2. MicroRNA (miRNA)

Unlike siRNAs, miRNAs are endogenous small RNA molecules that regulate gene expression in a sequence-specific manner. miRNAs are transcribed from the genome as long primary miRNA (pri-miRNA) molecules and undergo a series of processing steps. First, pri-miRNAs are cleaved by the RNAse III enzyme Drosha in the nucleus to form precursor miRNA (pre-miRNA) molecules, which are then exported to the cytoplasm. In the cytoplasm, Dicer further processes the pre-miRNA into mature miRNA duplexes, similar to siRNA processing. Like siRNAs, miRNAs are loaded onto RISC complex, where only one strand—the guide strand—is retained. However, miRNAs bind to target mRNAs with partial complementarity, especially in the 3' untranslated region (3' UTR). This imperfect pairing leads to translational repression rather than mRNA cleavage. By blocking translation, miRNAs regulate a wide array of cellular processes, including development, differentiation, and apoptosis.

2.3. Pathway Overview

RNAi can lead to either mRNA degradation or translational repression, depending on the type of small RNA involved and the degree of complementarity between the small RNA and its target mRNA.

The RNA interference (RNAi) pathway begins when the enzyme Dicer processes double-stranded RNA (dsRNA) molecules into small RNA duplexes. For siRNAs, exogenous dsRNA is cleaved into shorter duplexes, whereas for miRNAs, Dicer cuts precursor miRNAs (pre-miRNAs) into mature miRNA duplexes after their export from the nucleus. This cleavage generates small RNA fragments with 2-nucleotide 3' overhangs, crucial for subsequent gene silencing.

These processed RNA duplexes are then loaded onto the RNA-induced silencing complex (RISC). RISC, which contains an Argonaute (Ago) protein, is pivotal in the silencing mechanism. In humans, Ago2 specifically facilitates the cleavage of target mRNA. During RISC assembly, one strand of the RNA duplex (the guide strand) is retained, while the other strand (the passenger strand) is degraded. The guide strand directs RISC to complementary mRNA sequences, determining the silencing outcome.

Gene silencing occurs based on the complementarity between the guide RNA and the target mRNA. siRNAs, which exhibit nearly perfect complementarity, enable Ago2 to cleave the mRNA, leading to rapid degradation and prevention of translation. In contrast, miRNAs usually bind imperfectly to the 3' untranslated region (3' UTR) of target mRNAs, resulting in translational repression rather than cleavage. These miRNA-bound mRNAs are often sequestered in P-bodies, where they can be either stored for future translation or degraded, depending on cellular conditions.

Biological Significance of RNAi

RNA interference is fundamental in regulating gene expression and serves multiple biological functions crucial for maintaining cellular homeostasis. Its natural role in gene regulation is multifaceted, involving the control of development, differentiation, and responses to stress and infection. MicroRNAs (miRNAs) are keys to post-transcriptional regulation, controlling the expression of approximately 30% of human genes and influencing essential processes such as cell differentiation, proliferation, and apoptosis.

For instance, the miRNA family miR-17-92 is critical in regulating cell cycle progression and has been implicated in cancer when dysregulated. Similarly, miR-21 plays a important role in apoptosis and is often upregulated in tumors, making it a promising target for cancer therapies. In addition to its regulatory role, RNAi functions as an antiviral defense mechanism, particularly in plants and invertebrates. In response to viral infection, cells produce siRNAs derived from viral RNA, which guide RISC to target and degrade viral mRNA, thereby limiting viral replication. Some viruses, however, have evolved suppressors of RNAi to evade this defense system, highlighting the evolutionary arms race between host organisms and pathogens.

RNAi also contributes to the maintenance of genome integrity by silencing transposable elements and repetitive sequences in the genome. In organisms like C. elegans and plants, small RNAs derived from transposon sequences can guide RISC to these elements, leading to their silencing and preventing their potentially harmful mobilization.

3. Applications in Gene Regulation

RNA interference (RNAi) has profound impact on gene regulation by providing a means to selectively silence specific genes. This technology has been employed across a wide range of biological applications, including functional genomics, model organism studies, and disease research. By enabling precise gene silencing, RNAi not only clarifies gene function but also supports large-scale genetic screening and provides a powerful tool for use in model organisms. This section examines the role of RNA interference (RNAi) in functional genomics, presents examples of gene silencing across different model organisms, and compares RNAi with contemporary gene regulation methods, including CRISPR and traditional knockout approaches.

3.1. Functional Genomics: RNAi as a Tool to Study Gene Function

Functional genomics is concerned with understanding the roles of genes within the genome, especially through high-throughput technologies. RNAi has proven to be a powerful tool because it enables gene-specific silencing without the need for permanent genome editing. By reducing the expression of specific genes, RNAi facilitates the identification of their roles in biological processes and disease mechanisms.

3.2. Genome-Wide RNAi Screens and Pathway Discovery

RNAi has revolutionized functional genomics through its use in genome-wide screens, allowing researchers to simultaneously assess the roles of thousands of genes. By employing libraries of siRNAs or shRNAs to systematically reduce gene expression, these screens have uncovered genes critical for processes such as cell proliferation, apoptosis, and stress responses. Notably, RNAi-based approaches have been pivotal in identifying genes involved in diseases like cancer, viral infections, and neurodegenerative disorders. For instance, RNAi screens have revealed key regulators of apoptosis and pathways responsible for drug resistance in cancer cells. Similarly, they have identified host factors necessary for viral replication, paving the way for new antiviral strategies.

In addition to the activity of individual genes, RNA interference is crucial for the identification of biological pathways. By inhibiting several genes, researchers can delineate genetic pathways and comprehend intricate gene connections that regulate biological processes. This approach has been used to dissect signaling pathways related to cell differentiation, immune responses, and DNA repair, leading to the identification of promising therapeutic targets. Additionally, RNAi studies have advanced our understanding of the tumor microenvironment, revealing genes that mediate interactions between cancer cells and surrounding stromal or immune cells. These findings have inspired novel therapies aimed at modifying the tumor microenvironment to enhance the body's immune response against cancer. Together, the flexibility and precision of RNAi make it a powerful tool for elucidating gene function and discovering potential treatment avenues across diverse biological systems.

3.3. Gene Silencing in Model Organisms

RNAi has been widely utilized in various model organisms to study gene function and regulation in a more physiological context. Model organisms such as Caenorhabditis elegans (C. elegans) and Drosophila melanogaster (fruit flies) offer systems where RNAi can be applied with high specificity and efficiency, prividing insights into developmental biology, aging, and disease.

3.3.1. RNAi in C. elegans

The nematode C. elegans was one of the first organisms in which RNAi was discovered, and it remains a gold standard for RNAi studies. RNAi in C. elegans is straightforward, with gene silencing achieved by feeding the worms bacteria that express double-stranded RNA (dsRNA) corresponding to the target gene. This simplicity allows for genome-wide RNAi screens to be conducted in living organisms.

RNAi has been used extensively in C. elegans to study developmental processes, such as cell fate determination and tissue patterning. It has also been applied to investigate the aging process, leading to the identification of key genes involved in longevity and stress resistance. Moreover, RNAi in C. elegans has facilitated research on neurodegenerative diseases by enabling the silencing of genes associated with neuronal function and survival.

3.3.2. RNAi in Drosophila melanogaster

In Drosophila, RNAi is primarily achieved by expressing dsRNA or siRNA in specific tissues or developmental stages. This approach allows researchers to study gene function in tissues such as the nervous system or immune system, and enables tissue-specific knockdowns of essential genes. RNAi in Drosophila has been particularly valuable in studying cell signaling pathways that govern development and disease.

For example, RNAi has been used in Drosophila to explore the role of genes in synaptic function and neurodevelopment, providing insights into how neuronal circuits are formed and maintained. Additionally, RNAi has been applied to cancer research in Drosophila, where tumor suppressor genes are silenced to model human cancers.

3.3.3. RNAi in Vertebrate Models

RNAi has also been applied in vertebrate models, such as zebrafish and mice, though delivery methods are more complex in these organisms. In vertebrates, RNAi has been instrumental in studying developmental biology, immune responses, and disease pathways. In mice, RNAi-based knockdown of genes has been crucial in understanding neurodegenerative disorders such as Alzheimer’s disease, where specific genes are silenced to observe their role in disease progression.

Zebrafish, due to their transparency during early development, have been used to study organogenesis through RNAi-mediated knockdown. These studies have provided valuable insights into heart, brain, and vascular development, highlighting the conservation of gene function between zebrafish and humans.

3.3.4. Small Conclusion

RNA interference (RNAi) is essential in model organisms for advancing our understanding of gene function, development, and disease pathways. In Caenorhabditis elegans, RNAi enables efficient genome-wide screens to uncover genes that regulate processes like cell differentiation, aging, and stress resistance, many of which have human counterparts. This has contributed foundational knowledge, particularly in neurodegenerative disease research, by identifying genes crucial for neuronal survival that may inform therapeutic targets for human treatments[9].

In Drosophila melanogaster (fruit flies), RNAi has been instrumental in studying tissue-specific functions, including in the nervous and immune systems. This approach allows researchers to investigate cancer pathways, as silencing genes like tumor suppressors in flies can mimic aspects of human cancer biology. This work supports preclinical research by pinpointing gene pathways that may be targeted for cancer treatments in humans[10].

For vertebrate models like zebrafish and mice, RNAi facilitates insights into developmental biology and disease progression, especially for organogenesis. The transparency of zebrafish embryos aids real-time observation of organ development, which has direct implications for understanding congenital diseases in humans. In mice, RNAi helps examine complex diseases such as Alzheimer’s, enabling the silencing of genes involved in neurodegeneration. Such models bridge basic research and human health applications by offering a platform to test gene-specific therapies[9, 11].

These RNAi studies in model organisms accelerate the identification of disease-related genes and inform the design of RNAi-based therapies, directly contributing to human precision medicine and gene-targeted treatment strategies.

4. RNAi in Disease Treatment

4.1. Genetic Disorders: Targeting Mutations with RNAi

RNAi-based therapeutics have shown great promise in treating genetic disorders by targeting the faulty genes or transcripts responsible for disease. One prominent example is the use of “Patisiran” (Onpattro), an FDA-approved siRNA therapeutic used to treat hereditary transthyretin-mediated amyloidosis (hATTR). Patisiran silences the transthyretin (TTR) gene, which accumulates mutant proteins leading to nerve and organ damage. Clinical trials demonstrated significant improvements in patients treated with Patisiran, marking a milestone in RNAi-based therapeutics[12].

Beyond Patisiran, other genetic disorders, such as acute hepatic porphyria (AHP), are being targeted with siRNA drugs like “Givosiran”[12]. Givosiran targets the gene encoding “ALAS1” (aminolevulinic acid synthase 1), the enzyme responsible for producing neurotoxic intermediates in patients with AHP. By silencing the ALAS1 gene, Givosiran reduces the accumulation of aminolevulinic acid (ALA) and porphobilinogen (PBG), toxic byproducts that cause the severe neurovisceral attacks in AHP patients[13, 14].

Givosiran effectively blocks the initial steps of the heme biosynthesis pathway, preventing the buildup of these harmful intermediates. Clinical trials like the “ENVISION Phase 3” study, demonstrated that patients treated with Givosiran experienced a significant reduction in attack frequency and an overall improvement in quality of life[15].

Table 1: Comparison between Givosiran Treatment and Placebo Group

Moreover, Givosiran's subcutaneous delivery ensures that the therapeutic reaches hepatocytes, offering a targeted approach with fewer off-target effects[16]. These RNAi therapies silence genes responsible for the production of toxic metabolites, providing a targeted approach for conditions that were previously considered untreatable.

4.2. Cancer: Targeting Oncogenes and Pathways

The use of RNAi in cancer treatment focuses on silencing oncogenes or key signaling pathways involved in tumorigenesis. By targeting specific genes that drive cancer progression, siRNA-based therapies offer precision treatments with fewer off-target effects compared to traditional chemotherapy. RNAi has successfully targeted the “KRAS” gene, which is mutated in many cancers, as well as other cancer-promoting pathways like “PI3K/AKT/mTOR”. The “PI3K/AKT/mTOR” pathway is crucial to cellular growth, survival, and metabolism. When deregulated, it can lead to uncontrolled cell proliferation, a hallmark of many cancers. Activation of this pathway begins when phosphoinositide 3-kinase (PI3K) phosphorylates lipids on the cell membrane, recruiting and activating AKT. AKT then phosphorylates downstream proteins that promote cell survival by inhibiting apoptosis and stimulating cellular growth[12, 17].

A key downstream target of AKT is mechanistic target of rapamycin (mTOR), a protein kinase that regulates cellular metabolism and growth by promoting protein synthesis. Hyperactivation of the PI3K/AKT/mTOR pathway is implicated in numerous cancers, including breast, ovarian, and colorectal cancers[12]. RNAi therapies targeting components of this pathway aim to block excessive signaling, effectively slowing tumor growth and improving the efficacy of other cancer treatments such as chemotherapy. Preclinical trials have shown promising results with siRNAs designed to silence PI3K or mTOR, leading to reduced tumor size and improved patient outcomes[12, 17].

In preclinical models, siRNA molecules have been used to suppress the expression of oncogenes like BCL2, MYC, and VEGF, which are crucial for cancer cell survival and angiogenesis[18]. Additionally, RNAi therapies have targeted other critical players in cancer progression, including silencing epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). RNAi-mediated suppression of EGFR disrupts key signaling pathways like RAS/RAF/MEK/ERK, thereby halting tumor development. Similarly, FOXM1, a transcription factor involved in cell cycle regulation, has also been successfully targeted by RNAi, leading to decreased tumor growth and heightened sensitivity to existing treatments[18].

RNAi-based therapeutics are paving the way for more personalized cancer treatments, where genetic aberrations in tumor cells can be specifically targeted to improve patient outcomes. Clinical trials for siRNA-based cancer therapies are ongoing, aiming to translate these promising findings into treatments for aggressive cancers like pancreatic and lung cancer.

4.3. Viral Infections: Inhibiting Viral Replication

One of the most exciting applications of RNAi is in combating viral infections. siRNAs can be designed to target viral genomes or host factors essential for viral replication. For instance, RNAi has been investigated as a treatment for HIV by targeting host genes like CCR5, which is critical for viral entry into cells.[19] Similarly, in Hepatitis B and Hepatitis C infections, siRNAs have been developed to degrade viral RNA, preventing the virus from replicating within host cells[18, 20].

The effectiveness of RNAi against viral infections extends to more recent outbreaks like COVID-19, where siRNAs have been tested to inhibit SARS-CoV-2 replication by targeting essential viral proteins such as spike proteins and polymerase genes[21]. While these therapies are still in the experimental phase, they offer a rapid response mechanism for controlling viral pandemics[12, 18].

5. Conclusion

RNAi has transformed our understanding of gene regulation by providing a precise and powerful tool for gene silencing. The therapeutic potential of RNA interference is enormous, as evidenced by the approval of Patisiran and its application in targeting viral infections and silencing oncogenes. Innovations in delivery technologies, such as lipid nanoparticles and chemical modifications, have substantially broadened the scope of RNAi therapeutics, improving both their safety and effectiveness. Combination therapies and advances in next-generation RNAi molecules continue to push the boundaries of what is possible with gene silencing.

The future of RNAi therapies is incredibly promising, with ongoing research focused on overcoming current challenges, including delivery, off-target effects, and the cost of production. Advancements in nanoparticle-based delivery systems, next-generation RNA interference agents, and combination therapies are anticipated to propel further clinical achievements. Additionally, regulatory frameworks must adapt to these improvements to guarantee the safe and ethical implementation of RNAi technologies[12, 18, 22].

In today's era of booming AI, the combination of RNAi and AI holds great promise for advancing biomedical research, especially gene function analysis, drug discovery and personalized medicine. First, combining the data processing capabilities of AI with RNAi technology can greatly accelerate the identification of gene targets. RNAi experiments, especially genome-wide screens, generate large amounts of data, and AI algorithms such as machine learning and deep learning are ideally suited to quickly analyze this data. This synergy allows researchers to more efficiently identify genes associated with diseases such as cancer, neurodegenerative diseases and viral infections, supporting the development of new therapeutic targets. Second, the enhanced use of RNAi by AI can streamline the drug discovery process by optimizing siRNA and shRNA sequences to improve targeting accuracy and efficacy. In short, AI and RNAi are powerful ways to accelerate genetic research and therapeutic development. As these technologies evolve together, they are likely to play an increasingly critical role in personalized healthcare, improving the specificity and effectiveness of disease treatments.