miRNAs are often associated with extracellular vesicles (EVs), RNA-binding proteins, lipoproteins, or lipid derivatives, along with nanoparticles.
Ongoing research continues to uncover new insights into the molecular mechanisms that drive the dysregulation of miRNA biogenesis and aberrant expression in cancer.
2. miRNAs
Following the discovery of lin-4 as the first miRNA in 1993 in
Caenorhabditis elegans [85][86], it became evident that miRNAs are widespread in the animal and plant kingdoms, some of which exhibit high levels of conservation across species
[87][88][89].
miRNAs, short non-coding RNA molecules typically about 22 nucleotides long, are naturally encoded in the genomes of diverse species
[87][88][89][90].
They play pivotal roles in regulating gene expression at both transcriptional
[5][6][7] and post-transcriptional
[8][9][10][11][91] levels of their target mRNAs
[8][10], influencing mRNA stability and translation
[92] across a wide array of biological processes
[93], impacting activities such as cell differentiation, proliferation, angiogenesis, and apoptosis.
Additionally, miRNAs demonstrate distinct expression patterns in various tissues
[12][13] and during different developmental stages
[14][15][16].
There are currently estimated to be more than 2588 mature human miRNAs present in human cells
[94], each with a unique temporal and tissue-dependent expression pattern. These miRNAs are estimated to control over 60% of human gene expression, showcasing their significant regulatory roles in diverse physiological processes. Because a single microRNA can regulate multiple genes, many miRNAs can contribute to the development of many human diseases when they become dysfunctional
[2][8][18][20][21][22][23][24][25][26][28][30][31][32][33][34][35][37][66][95][96][97] including many types of cancer
[39][41][42][43][44][47][50][51][53][55][56][57][58][59][60][61][62][63][64][65][66][67][69][98][99][100][101][102][103].
However, determining the precise relevance of individual miRNAs has been challenging, despite their evident significance as regulatory molecules
[104]. Studies investigating miRNA functions through either suppression or overexpression of specific miRNAs have generated data that sometimes conflict with findings from loss-of-function models
[104]. For example, studies in
Caenorhabditis elegans involving systematic miRNA deletions suggest that fewer than 10% of the miRNAs are individually essential for normal development or viability
[105] and this trend appears consistent in mice as well
[96].
As illustrated in
Figure 1, miRNAs are primarily transcribed from DNA sequences into primary miRNAs (pri-miRNAs), which undergo an initial processing step by Drosha within the nucleus to yield precursor miRNAs (pre-miRNAs)
[8][68][106]. It is important to note that up to 40% of miRNA genes might be located within either the introns or exons of other genes
[107]. After their transportation from the nucleus to the cytoplasm by exportin 5 (XPO5), pre-miRNAs undergo additional processing by endoribonuclease Dicer, leading to the formation of miRNA duplexes characterized by distinct 3′ overhangs of 2 nucleotides. Subsequently, these miRNA duplexes are loaded onto the Argonaute (AGO) protein, which retains one miRNA strand while discarding the other
[10]. The AGO-miRNA complex, along with co-factors like GW182 (TNRC6A), forms the RNA-induced silencing complex (RISC)
[91] responsible for cognate mRNA degradation and hence inhibition of translation through interaction with complementary mRNA sequences, typically located within the 3′-untranslated region (3′-UTR) of mRNAs (
Figure 1)
[108][109][110][111].
Figure 1. Illustration of miRNA biogenesis, processing, and mechanisms of translational suppression or degradation of target RNA. miRNAs are a class of small, single-stranded non-coding RNAs that function as a guide molecule in RNA silencing and hence modulate the expression of most mRNAs. The miRNA: target–mRNA interaction usually occurs at the 5′ end of the miRNA (i.e., ‘seed’ region). However, recent evidence suggests that there is a special class of target mRNAs which bind the miRNA not only through the ‘seed’ region, but also through a second region of complementarity at the 3′ end of the miRNA. The extended complementarity forces the miRNA out of Ago2, where it becomes accessible to enzymatic degradation. This phenomenon is referred to as the target-directed miRNA degradation mechanism (TDMD).
The interaction between miRNA and target mRNA typically takes place at the 5′ end of the miRNA, referred to as the ‘seed’ region. Yet, recent evidence points to a unique group of target mRNAs that bind the miRNA, not just through the seed but also via a complementary region at the 3′ end of miRNAs. This extended complementarity displaces the miRNA from Ago2, rendering it vulnerable to enzymatic degradation. This process is referred to as the target-directed miRNA degradation mechanism (TDMD)
[112][113].
miRNAs are regarded as master regulators of the genome because of their capability to bind to and modify the expression of numerous protein-coding RNAs
[114]. Because of this, a single miRNA can potentially regulate distinct mRNAs (anywhere from 10 to 100 protein-coding RNAs) due to their ability to bind to target mRNAs even when the pairing is not perfect
[55][115]. As a result, a single miRNA can regulate a range of targets involved in similar cellular processes and pathways, thereby amplifying the cellular response potentially making miRNAs powerful therapeutics to restore perturbed cell functions seen in disease phenotypes. Conversely, a specific messenger RNA can become the target of many miRNAs, whether concurrently or in a context-dependent manner
[116], leading to a collaborative repression effect
[117][118]. Bioinformatic analyses indicate that a single miRNA can potentially bind to as many as 200 distinct gene targets with various functions, such as transcription factors, receptors, and more (https://bitesizebio.com/24926/mysterious-mirna-identifying-mirnas-and-their-targets/) (accessed on 20 December 2023).
3. miRNAs’ Role in Cancer
Cancer, a complex and heterogeneous disease, is characterized by a sequence of genetic and genomic abnormalities that promote tumorigenesis
[119]. These alterations in the genome influence gene function, frequently resulting from genomic aberrations such as chromosomal translocations, amplifications, deletions, insertions, single-nucleotide mutations, or epigenetic modifications. These genetic and epigenetic alterations often result in the activation of oncogenes and the suppression of tumor suppressor genes
[120]. In addition, miRNAs have been identified as additional genomic regulators that also play a crucial role in various aspects of organismal development, normal physiological processes, and the development of disease, including many types of cancers
[68]. It has been shown that miRNAs play a pivotal in all the known processes involved in cancer, such as proliferation, survival, metastasis, and apoptosis
[114]. Data suggest that dysregulation of miRNA function, either through its loss or gain, contributes to cancer development by either upregulating or silencing specific target genes. As a consequence, utilizing miRNAs either as miRNA mimics or antagomirs could present a potent therapeutic strategy to interfere with key molecular pathways associated with cancer as such miRNAs have the capacity to regulate all the recognized hallmarks of cancer, either acting as tumor suppressors or promoting oncogenic processes. Several of these cancer hallmarks influenced by miRNAs are discussed in detail in the literature
[65][66].
It is widely accepted that alterations in miRNA genes and their expression are influenced by genetic deletions or amplifications, epigenetic methylation of miRNA gene locations, and modifications affecting pri-miRNA regulation by transcription factors as well as factors involved in miRNA biogenesis, often alter miRNA expression and function across various cancer types
[66].
In addition, changes in the miRNA biogenesis process can also impact the availability of target mRNAs, including those associated with the development of cancer
[121]. When miRNAs or the machinery involved in miRNA processing are altered or dysregulated this often leads to the loss of normal homeostatic state, leading to malignant transformation, including various types of cancer
[51][52][56][65][66][67][122].
Due to their pivotal role in regulating the expression of numerous genes implicated in cellular responses to environmental stressors like hypoxia, oxidative stress, DNA damage, and nutrient deprivation, miRNAs can serve either as oncogenes (oncomirs) or tumor suppressors (onco-suppressor miRs). This is supported by recent findings that have identified miRNAs with oncogenic and tumor-suppressing roles in a range of neoplastic malignancies, and the dysregulation of miRNA expression is closely linked to the initiation, progression, and metastasis of cancer
[43][45][103].
Moreover, dysregulated circulating miRNAs have demonstrated associations with disease origin, progression, treatment response, and patient survival
[123][124]. For example, the distinctive tissue specificity of miRNAs
[13], crucial for the maintenance of normal cells and tissues
[40], renders them promising candidates for potential biomarkers in diagnosing cancers of unknown primary
[125][126].
Furthermore, with the frequent genetic and epigenetic changes identified in particular miRNAs and the elements involved in miRNA biogenesis across diverse cancer types, oncogenic and tumor suppressor miRNAs have emerged as promising candidates as miRNA-based therapeutics.
4. RNA Therapeutics
As detailed in the literature
[127][128], over the past few years, more than 50 siRNA-based drugs have progressed into phase I–III clinical trials. Of those, around 15 programs based on siRNA therapeutics are currently being investigated in phase I, II, and III trials for the treatment of different cancer types
[128].
Two siRNA-based drugs, Patisiran and Givosiran (both developed by Alnylam Pharmaceuticals (Cambridge, MA, USA)), obtained approval from the Food and Drug Administration (FDA, Washington, DC, USA) in 2018 and 2019. Patisiran was approved for hereditary transthyretin-mediated amyloidosis, while Givosiran gained approval for acute hepatic porphyria
[129][130].
The first example of an FDA-approved RNA-based drug, a siRNA-based therapy developed by Alnylam Pharmaceuticals, is Patisiran, sold under the brand name Onpattro™ for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults. Based on the completion of a successful phase III APOLLO trial, Onpattro™ was approved by the US FDA in August 2018. Onpattro™ contains patisaran, which comprises a siRNA targeting transthyretin (TTR) mRNA conjugated with a lipid complex which leads to a decrease in TTR protein levels in the liver, thus resulting in a reduction in amyloid deposits. Patisiran targets and binds to a genetically conserved sequence found in the 3′UTR of both mutant and wild-type TTR mRNA
[131]. Findings from the APOLLO trial, a placebo (77 patients)-controlled phase III trial which enrolled 225 patients showed that 51% of patients receiving Onpattro™ (148 patients, once every three weeks (0.3 mg/kg body weight)) experienced an enhanced quality of life (measured using the Norfolk Quality of Life Diabetic Neuropathy (QoL-DN)), as compared to only 10% of patients in the control group, which received a placebo drug
[131][132].
During the COVID-19 pandemic, mRNA technology became instrumental, notably in the development of highly effective mRNA vaccines. These vaccines have played a crucial role in controlling the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The groundbreaking science behind mRNA vaccines earned Katalin Karikó and Drew Weissman the prestigious 2023 Nobel Prize in Physiology or Medicine for their pioneering work on nucleoside base modifications, enabling the development of these impactful COVID-19 vaccines.
The evolution of cap analogs has vastly improved mRNA translation, while advancements in purification, packaging, and delivery methods have revolutionized medicine. Visionaries like Katalin Karikó, Drew Weissman, Edward Darzynkiewicz, Robert Rhodes, Ugur Sahin, and Ozlem Tureci made pivotal early contributions to mRNA research, deserving recognition for their pioneering efforts. This narrative around mRNA charts a remarkable journey showcasing breakthroughs in a field holding immense promise for the future of medicine.
Consequently, the success of mRNA vaccines has paved the way for integrating mRNA-based technology into personalized neoantigen vaccines, seamlessly incorporating them into the standard oncological workflow
[133][134]. These mRNA-based vaccines can be tailored and manufactured as individualized vaccines with multiple neoantigens
[135], and can effectively stimulate antigen-presenting cells
[136][137][138][139] and be delivered using clinical-stage delivery formulations
[140]. The studies and insights from the mRNA-based COVID-19 vaccines highlight the promise of RNA therapeutics as an innovative class of treatments.
However, the effectiveness of miRNA and other nucleic acid-based therapies hinges on a reliable delivery method with minimal adverse events and drug- or treatment-related toxicity.
Delivering miRNA therapeutics to cells poses challenges, requiring precise targeting of diseased cells while sparing healthy ones. In contrast to mRNA COVID-19 vaccines, which are taken up by scavenging immune cells such as dendritic cells and other professional antigen-presenting cells, thus inducing a specific immune response through the processing and expression of the translated mRNA molecules
[141], miRNA therapeutics must effectively bypass immune recognition to reach their target cells without triggering an immune response.
5. Advances in the Delivery of miRNA Therapeutics
While a handful of phase 1 and 2 clinical trials have explored miRNA-based therapeutics, there are currently no miRNA-based therapeutics undergoing phase III human clinical trials. This is partly attributed to challenges associated with precisely delivering miRNAs to specific cell types, tissues, and organs.
While several approaches, such as antibodies, nanoparticles, or ligands, have been documented to enhance the effectiveness of miRNAs and decrease off-target effects (like immunotoxicity
[142]) when directing miRNAs to specific cells of interest, limitations and challenges persist in the field of miRNA therapeutics.
As illustrated in
Figure 2 and discussed in detail in the literature
[77][84][143][144], there are various strategies being explored as mechanisms to deliver miRNA therapeutics (mimics and antagomirs) to the indented tissue and to improve pharmacokinetic mechanisms, and avoid off-target effects.
Figure 2. Examples of miRNA delivery systems. miRNA therapeutics can be administered orally or intranasally or through venous (intravenously) or muscle (intramuscularly) or skin (subcutaneously) injections, or via cell-/tissue-directed approaches, or adoptive cell transfer, or the implantation of 3D matrices that release miRNA therapeutics, or other extracorporeal miRNA delivery strategies. Other modes of delivery of miRNA therapeutics include vector based and non-vector-based delivery systems including (A) adeno-associated virus (B) Lentivirus; (C) bacterial nanocells; (D) bacteriophages; liposomes, including monovalent and multivalent lipids such as (E) cationic lipid-based ligand-targeted nanoparticles; (F) natural polymer-based nanoparticles; (G) polymer-based nanoparticles (natural, green and synthetic, blue) conjugated with polyethylene glycol (PEG); (H) extracellular vesicles or exosomes; (I) gold nanoparticles; (J) carbon nanotubes; (K) quantum dot nanoparticles; (L) core–shell magnetic nanoparticles; (M) mesoporous silica nananoparticles and others such as polymeric micelles, and mesoporous silica nanoparticles are the examples of nanocarriers as drug-delivery systems. Moreover, there have been efforts to improve the serum stability, pharmacokinetics, and tissue specificity by targeted delivery of miRNA mimics, miRNA inhibitors, and other nucleic acid therapeutics through the incorporation of various chemical modifications and/or conjugation of these RNA and nucleic acid therapeutics to biomolecules to facilitate receptor-mediated uptake such as N-acetylgalactosamine (GalNAc), 2′-O-methyl nucleotide, phosphorothioate, cholesterol, locked nucleic acid (LNA), and aptamer moieties.
These methods include both vector and non-vector approaches, ranging from lipid-based nanoparticles, polymeric vectors, dendrimer-based vectors, cell-derived membrane vesicles, 3D scaffold-based delivery systems, to various biodegradable and biocompatible nanoparticles derived from polymers and metals
[77].
Other strategies for the delivery of RNA-based therapeutics, as discussed in the literature
[145], include adeno-associated virus, lentivirus, bacterial nanocells
[146], bacteriophages, cationic lipid-based liposomes (including monovalent and multivalent lipids), natural polymer-based nanoparticles, polymer based nanoparticles conjugated with polyethylene glycol (PEG), extracellular vesicles (EVs) or exosomes, nanocomplex-forming functionalized metals such as gold nanoparticles, and carbon nanotubes, polymeric micelles, and mesoporous silica nanoparticles
[147], and many others that are engineered to contain biomolecule conjugates for improved stability and pharmacokinetics and target delivery to the intended cell or tissue type
[84][144][145][148][149]. A recent study showcased the increased antitumor effectiveness of STING agonists through the covalent attachment of cyclic dinucleotides (CDN) to polymer nanoparticle (poly(β-amino ester) formulation for intravenous delivery
[150].
Non-pathogenic recombinant viral vectors, such as retroviruses and lentiviruses (which pose a genomic integration risk), adenoviruses, and adeno-associated viruses (which remain transiently stable in an episomal form within the host cell’s nucleus)
[151][152], are under investigation for their capacity to encode the desired RNA transgene. These vectors are being explored for intracellular delivery of miRNA-based therapeutics, making them a significant area of interest
[153].
A phase II trial is currently testing an adeno-associated viral vector for the delivery of the miRNA drug AMT-130 for the treatment of Huntington’s disease (ClinicalTrials.gov identifier NCT04120493)
[154][155][156]. Despite their potential for the delivery and expression of miRNAs, there are various side effects with the use of viral vectors such as immunogenicity and transgene-related immune responses
[157].
The packaging of the negatively charged nucleic acids in liposome nanoparticles masks their negative charge and also protects against serum nuclease degradation
[149][158]. Delivery of miRNAs using liposome nanoparticles has already been applied in several clinical studies, such as MRX34 (NCT01829971, NCT02862145)
[82][83].
Likewise, bacterial minicells loaded with miRNAs were employed to deliver miR-16 mimics during a phase 1 trial involving patients with recurrent malignant pleural mesothelioma (MesomiR 1, NCT02369198)
[159][160]. However, the study also reported several side effects including dose-limiting toxicities, decreased lymphocyte counts, or cardiac events
[160].
Extracellular vesicles (EVs), including exosomes, are under exploration as potential drug delivery systems, capable of delivering specific genetic cargo for cellular transfer within the body
[161]. For example, EVs derived from mesenchymal stromal cells obtained from human adipose tissue were modified to carry miR-125b. This modification resulted in the inhibition of human hepatocarcinoma cell proliferation
[162].
In addition, different modalities of drug delivery systems have been explored for the delivery of miRNA-based drugs such as core–shell magnetic nanoparticles, quantum dot nanocrystals, polymeric micelles, and mesoporous silica nanoparticles are among the other examples of nanocarriers as drug-delivery systems to improve the therapeutic effectiveness and specificity, and tissue targeting of miRNA and other nucleic acid therapeutics
[147].
An encouraging strategy involves the covalent conjugation of miRNAs, along with other nucleic acid-based drugs and biomolecules, to lipids, peptides, or sugars. These compounds function through receptor-mediated endocytosis mechanisms
[149].
Likewise, a lipophilic cholesterol conjugate was employed to deliver an miR-29-based mimic (remlarsen/MRG-201) to human skin fibroblasts, irrespective of cell type via skin injection in a phase II trial for keloid disorder. The aim was to suppress the expression of extracellular matrix and fibroplasia within the skin (NCT02603224, NCT03601052)
[163].
Another approach involves the coupling of aptamer conjugates to specific miRNA therapeutics using a straightforward sticky-end annealing method
[164]. This method serves as a strategy for delivering miRNAs to targeted cell types.
Aptamers are single-stranded nucleic acids that are developed as high-affinity ligands specific to cell surface receptors to facilitate the delivery of therapeutic cargo including miRNAs through receptor-mediated transport
[149][164]. In ongoing preclinical investigations, researchers are currently investigating aptamer-linked miRNAs, such as the Aptamer-miR-34c conjugate (known as GL21.T-miR-34c) in non-small-cell lung cancer cells
[165].
Additional efforts have been made to enhance the serum stability, pharmacokinetics, and tissue specificity of miRNA mimics, miRNA inhibitors, and other nucleic acid therapeutics by incorporating various chemical modifications to miRNA and nucleic acid therapeutics or attaching various biomolecule conjugates to these therapeutic miRNAs to facilitate receptor-mediated uptake such as N-acetylgalactosamine (GalNAc), 2′-O-methyl nucleotide, phosphorothioate, cholesterol, locked nucleic acid (LNA), and aptamer moieties are also shown as examples
[77][84][144][166][167].
For example, biomolecule conjugates, such as N-acetylgalactosamine (GalNAc), have been investigated in clinical trials. GalNAc facilitates the targeted delivery of nucleic acid therapeutics through endocytosis by activating liver cell-specific asialoglycoprotein receptors
[168][169]. GalNAc linked to a miR-122 inhibitor (RG-101) and a miR-103/107 inhibitor (RG-125/AZD4076) are in clinical trials for chronic HCV [EU Clinical Trials Register (clinicaltrialsregister.eu) EudraCT numbers 2015-001535-21, 2015-004702-42, 2016-002069-77] and steatohepatitis (NCT02612662, NCT02826525), respectively
[166][169][170]. However, due to reported side effects such as cases of jaundice, the clinical trial for RG-101 was halted, and investigations into the cause of these effects are ongoing
[171][172][173].
Recent preclinical investigations have also explored other examples of GalNAc-conjugated LNA, anti-miR-122 antisense oligonucleotides, or nano-carrier vehicles in combination with cell type-specific biomolecule conjugates or miR-155 inhibitors by gold nanoparticles formulated with antagomir and AS1411 aptamer
[174][175].
In addition, the 3D matrices for delivering nucleic acid-based therapeutics and conventional drugs are currently undergoing optimization with diverse design features. This encompasses various application routes, such as edible or injectable carriers
[77][176][177][178].
One potential method of administering miRNAs is orally
[75]. Data demonstrate that miRNAs are commonly associated with EVs, lipoproteins, or lipid derivatives, and RNA-binding proteins. These associations, along with the use of nanoparticles, shield miRNAs from the harsh conditions in the gastrointestinal tract. This includes protection against salivary and pancreatic RNases, the stomach’s acidic pH, digestive enzymes, peristaltic activity, and microbial enzymes. This safeguarding mechanism is thought to aid in the absorption of miRNAs from the digestive tract
[75]. However, there is ongoing debate surrounding the absorption, stability, and physiological impact of these edible or food-derived miRNAs.