Advancing cancer treatments: The role of oligonucleotide-based therapies in driving progress

Although recent advancements in cancer immunology have resulted in the approval of numerous immunotherapies, minimal progress has been observed in addressing hard-to-treat cancers. In this context, therapeutic oligonucleotides, including interfering RNAs, antisense oligonucleotides, aptamers, and DNAzymes, have gained a central role in cancer therapeutic approaches due to their capacity to regulate gene expression and protein function with reduced toxicity compared with conventional chemotherapeutics. Nevertheless, systemic administration of naked oligonucleotides faces many extra- and intracellular challenges that can be overcome by using effective delivery systems. Thus, viral and non-viral carriers can improve oligonucleotide stability and intracellular uptake, enhance tumor accumulation, and increase the probability of endosomal escape while minimizing other adverse effects. Therefore, gaining more insight into fundamental mechanisms of actions of various oligonucleotides and the challenges posed by naked oligonucleotide administration, this article provides a comprehensive review of the recent progress on oligonucleotide delivery systems and an overview of completed and ongoing cancer clinical trials that can shape future oncological treatments.


INTRODUCTION
Up to 10 million deaths and 19.3 million newly diagnosed cancer cases were reported worldwide in 2020, according to the World Health Organization 1 and recent statistical data have suggested that the global incidence of cancer is expected to double in the coming decades. 2 Nevertheless, significant advancements in early detection methods and surgical procedures have fueled progress in the battle against cancer. 3Over the last 20 years, oncological research has been focused on addressing the limitations of traditional treatments such as drug resistance and cancer recurrence.Therefore, there has been a remarkable paradigm shift in cancer treatment, transitioning from treatment strategies based on broad-spectrum cytotoxic drugs to targeted therapies.Unlike conventional chemotherapeutics, targeted drugs exhibit the ability to selectively recognize cancer cells while preserving normal cells, resulting in potent efficacy and minimal toxicity. 4,5nce understanding of the molecular mechanisms of tumor development is progressively advancing, and numerous molecular targets have been identified, oligonucleotides (ONs) such as antisense ONs (ASOs), RNA interference (RNAi) molecules, aptamers, DNAzymes, and transcription factor decoys (TFDs) addressed different therapeutic applications across cancer.Thus, ONs, due to their various mechanisms of action, including gene silencing, 6 splice modulation, 7 and protein interaction, 8 offer a versatile platform for oncological drug development.Nevertheless, the clinical development of naked ON therapeutics faced challenges given by their physico-chemical properties (size, charge), off-target effects, interactions with the immune system, rapid clearance, and nuclease degradation.0][11][12] Thus, in tight connection with recent findings, this review brought insight into the oncological treatment approaches based on different types of ONs and their mechanisms of action (Figure 1; Table 1).Moreover, the second part of the review presented the most relevant delivery systems proposed to improve the tumor delivery of ONs and the current state of clinical trials testing the antitumor efficacy of ON therapeutics (Table 2).

THERAPEUTIC CLASSES OF ONs
The understanding of DNA's role in heredity in 1944 62 and the description of its helical structure in 1953 63 provided the essential knowledge and tools to discover and utilize the properties of ONs for future oncological therapies.Thus, the primary mechanism of action of ONs is based on recognizing and binding to specific messenger RNA (mRNA) via Watson-Crick base pairing, leading to gene silencing, steric block, or modified splicing patterns. 64Alternatively, aptamers identify their targets (small molecules, peptides, and proteins) 65 based on their unique three-dimensional structures. 66Below, a condensed overview of different types of therapeutic ONs (Table 1) and their mechanism of action (Figure 1) is presented.

Antisense ONs
Since 1978, when Paul Zamecnik, the father of antisense ONs, and his colleague, Mary Stephenson, developed a 13-mer-oligodeoxynucleotide to inhibit Rous sarcoma virus replication and cell transformation in chicken embryos, 67 the antisense technology has shown incredible capabilities as molecular tools for in vivo cellular regulation. 68us, ASOs are short, under 30 nucleotides, synthetic single-stranded RNA (ssRNA) or single-stranded DNA (ssDNA) molecules 69,70 designed to have a sequence that complements their target, DNA or different types of RNA: precursor miRNA (pre-miRNA), mRNA, and long non-coding RNA (Table 1). 71Furthermore, ASOs can reach multiple cellular compartments, including both the cytoplasm and the nucleus (Figure 1). 72Based on their mechanism of action, ASOs can be divided into ribonuclease H-dependent (RNase H) ONs and steric blocker ONs (SBONs). 73,74RNase H-dependent ASOs are ssDNA-based ONs that produce a DNA/RNA duplex upon binding to their complementary site on a target mRNA.The newly formed duplex recruits the ubiquitous enzyme RNase H, which leads to RNA strand degradation.ASOs that work in conjunction with RNase H can significantly reduce the expression of targeted RNA, achieving a substantial downregulation of both mRNA and protein levels ranging from 80% to 95%. 74SBONs lack DNA bases in their composition and function by physically obstructing the splicing or protein translation processes 75 after targeting the AUG initiation codon. 74hree generations of modified ASOs have been developed to improve specific aspects, such as target specificity and stability against enzymatic degradation.
The first-generation ASOs were developed by introducing backbone modifications to the phosphate group connecting the nucleotides.Therefore, several chemical groups such as sulfur, methyl, amine, 71 acetate, 76 and borane 77 replaced the non-bridging oxygen atoms in the phosphodiester bond.The most used chemical modification introduced in ASOs was methylphosphonate in 1981, 78 phosphorothioate (PS) in 1987, 79 and phosphoroamidate in 1988. 80PS chemistry remains a crucial modification in contemporary ON drugs (Table 2), facilitating cellular uptake and providing protection against nuclease degradation, extending their half-life from minutes to days. 71,81ecause of the broad non-specific effects typical of first-generation ASOs, efforts to increase specificity led to the development of a new generation.In the late 1980s, 82,83 the sugar backbone was modified by the addition of alkyl groups at the 2 0 position of the ribose.Over the years, various 2 0 -O modifications have been studied, but currently, 2 0 -O-methyl (2 0 -OMe) and 2 0 -O-methoxyethyl (2 0 -O-MOE) modifications are considered the standard. 84In contrast to first-generation ONs, these ASOs have lower toxicity and a stronger binding affinity to their targets. 856][87][88] LNAs utilize a methylene bridge linking the 2 0 -oxygen and 4 0 -carbon of ribose to enhance stability, binding affinity, and inhibit backbone hydrolysis through conformational constraint. 89,90PNAs are ONs characterized by the substitution of the phosphodiester backbone with a polyamide backbone, composed of repetitive units of N-(2-aminoethyl) glycine, wherein the nucleobases are linked by a methyl carbonyl linker. 91By substituting ribose rings with morpholino rings and phosphodiester bonds with phosphorodiamidate bonds, PMOs ensured high solubility in aqueous solution. 92PNAs and PMOs showed an increased resistance to nuclease activity as well as lower binding affinity to plasma proteins, which facilitates their elimination through urine. 9,71,90oreover, to further increase their resistance against nuclease, novel versions of ASOs, such as 2 0 ,4 0 -constrained MOE, and constrained ethyl (cEt) bicyclic nucleic acids combined features of second-generation 2 0 -O-MOE and third-generation LNA. 93tably, an optimized variant of ASOs with regard to the binding affinity to targets and resistance to nuclease degradation is offered by gapmers (Table 1; Figure 1).5][96] The central DNA "gap" region can bind target transcripts via complementary base pairing, thus recruiting RNAse H to degrade the target RNA.[99]

RNAi molecules
The challenges associated with targeting oncological markers using small molecular drugs, recombinant proteins, and monoclonal antibodies have led researchers and clinicians to explore RNAi as an alternative strategy for tumor-targeted therapies (Table 2).Since its discovery, RNAi has been defined as a mechanism of gene silencing (Table 1; Figure 1) using small RNAs, such as microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or bifunctional short hairpin RNA (bi-shRNA) that target a wide range of protein-coding transcripts. 100us, miRNAs are endogenous ssRNAs (about 22 nucleotides) and siRNAs, shRNAs, and bi-shRNAs are exogenous, double-stranded RNAs (dsRNAs) comprising about 15-30 nucleotide pairs. 101,102Besides having a double-stranded stem, the shRNA molecule has a loop of at least 4 single-stranded nucleotides and a 3 0 end dinucleotide overhang. 103,104All RNAi molecules employ cellular internal processing machinery to induce gene silencing. 105Being endogenous molecules, miRNAs, after their biogenesis as precursors, primary miRNA (pri-miRNA), are then processed into pre-miRNAs by the class 2 RNAse III enzyme called Drosha and transported from the nucleus to the cytosol via the exportin-5 protein.Herewith, pre-miRNAs, as well as exogenous, synthetic siRNAs, shRNAs, and bi-shRNAs undergo processing by the RNAse III enzyme Dicer, resulting in mature molecules that will undergo loading into the RNA-induced silencing complex (RISC), serving as the antisense guide for target recognition.Upon binding to a complementary mRNA target, the antisense guide

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7][108] RNAi molecules binding to the target is conditioned by the seed region (nucleotides 2-8) and supplementary region (nucleotides 13-16 of the miRNA's 3 0 region) that recognize mRNA. 109,110However, being a short sequence of nucleobases, seed sequences of RNAi molecules have the potential to bind to the 3 0 UTRs of many different genes, leading to a mixture of on-and off-target effects. 6vertheless, as various types of cancers exhibit abnormal levels of miRNA expression as a consequence of gene alterations, abnormal transcriptional control, dysregulated epigenetic changes, and defects in miRNA biogenesis machinery 111 several miRNA mimics and anti-miRNAs have been synthesized and tested in clinical trials (Table 2) as well as used to better understand their possible impact on cancer development.Besides miRNAs, siRNA stands out as the most promising for future medical applications due to its easy synthesis and efficacy in gene silencing, independent of genome integration.Its therapeutic index can be significantly increased after encapsulation in nanoscale delivery systems.Therefore, promising applications of siRNA have been explored in the management of breast, 112 lung, 113 brain, 114 thyroid, 115 and bladder cancers. 116Nevertheless, the major limitation of siRNA-based therapies is their short lifespan in vivo.
Two strategies have emerged to address this issue: the addition of chemical modifications of the siRNAs and the utilization of shRNA, which can be processed intracellularly into siRNA. 117Multiple chemical modifications of the backbone have been proposed to increase siRNA efficiency in cancer treatments.9][120] Moreover, the ribonuclease resistance has been further enhanced by substituting oxygen from the phosphate backbone with either sulfur, fluorine, or boron (clinical trial NCT04169711, Table 2). 120Notably, to increase the siRNAs' and shRNAs' efficacy as well as their lifespan in vivo for future clinical applications, bi-shRNA has been developed. 121To enhance knockdown potency, bi-shRNA utilizes two different shRNAs: one with mismatched guide and passenger strands for cleavage-independent RISC loading that induces the rapid inhibition of protein synthesis and the other with perfectly matched strands for cleavage-dependent RISC loading that is responsible for a delayed effect via mRNA cleavage and degradation. 122,123Thus, this approach enabled the administration of lower systemic doses and reduced off-target effects in comparison with other RNAi therapeutics. 123

Aptamers
Aptamers are a class of ONs often referred to as "chemical antibodies" in the literature. 124They are short ssDNA or ssRNA molecules, ranging from 20 to 60 nucleotides in length.These molecules adopt three-dimensional structures and demonstrated the capacity to bind with high affinity to target molecules 125 (Table 1; Figure 1) through specific mechanisms dependent on their geometry, electrostatic interactions, van der Waals forces, and hydrogen bond formation. 8,126his improved property of the aptamers is conferred by the in vitro a Special case ASO (instead of focusing on mRNA, its action is directed at the RNA component of the ribonucleoprotein known as telomerase).
www.moleculartherapy.orgReview procedure of selecting these structures, known as the systematic evolution of ligands by exponential enrichment (SELEX). 127Owing to their high specificity, aptamers hold promise as agents against various targets in cancer therapy (Table 2), including extracellular ligands and cell surface proteins. 124Furthermore, unlike antibodies, aptamers are characterized by minimal immunogenicity, low molecular weight, stable structure, plasticity of chemical groups, and efficient and low-cost chemical synthesis. 128,129vertheless, aptamers present several challenges that need to be addressed, such as stability and high renal clearance.Various chemical modifications and technological advances are being explored to address these issues. 130][132] Moreover, the half-life and thermal stability of a linear aptamer can be enhanced by circularization using either chemical or enzymatic ligation processes.The chemical ligation method offers a more adaptable approach as diverse linking strategies can be employed.Nevertheless, this approach requires complex organic synthesis and may lead to the generation of harmful byproducts.Enzymatic circularization enables the utilization of natural nucleotides, thereby avoiding the toxicity associated with chemical alteration.It represents a quick and easy way to modify aptamers to enhance their stability and broaden their range of applications while exerting minimal influence on their folding and functionality.The most frequently utilized enzymatic ligation strategy involves the application of T4 ligase and CircLigase. 133,134However, enzymatic reactions have a relatively low circularization yield due to their low selectivity for intramolecular circularization over intermolecular ligation. 135An alternative approach to circularizing ssDNA molecules through ligase-mediated ligation involves utilizing Twister ribozymes to flank the RNA of interest leading to cleavage followed by subsequent ligation of both ends by endogenous RNA ligase RtcB. 136Multimerizing individual aptamers or conjugating them with bulky moieties increases their size, thereby overcoming rapid renal filtration and prolonging circulation time. 1379][140] To enhance the binding affinity, base modifications with naphthyl, triptamino, isobutyl, and benzyl groups can be employed. 141,142For example, 5-(N-benzylcarboxyamide)-2-deoxyuridine modification of the AS1411 aptamer selectively increased its target affinity to cancer cells (clinical studies NCT00881244 and NCT00512083, Table 2).

DNAzymes
DNAzymes are specific short (15-40 nucleotides) ssDNA sequences with catalytic activity. 143Scientists have used in vitro selection strategies to identify DNAzymes capable of catalyzing RNA cleavage, RNA and DNA ligation, and covalent modifications of nucleic acid substrates. 144Similar to SELEX, a nucleotide library is incubated with the substrate of interest to select the optimal DNAzymes with the required activity, affinity, and specificity.RNA-cleaving DNAzymes are the most studied DNAzymes in cancer research due to their gene silencing potential.In the presence of specific metal ions such as Mg 2+ , Pb 2+ , Mn 2+ , Cu 2+ , and Na + , DNAzymes can cleave the target mRNA (Table 1; Figure 1) by catalyzing the hydrolysis of the phosphodiester bond. 145Despite promising results in vitro, further research on DNAzymes as gene silencing agents revealed that their in vivo efficacy is limited by reduced catalytic activity caused by the poor availability of metal ions under physiological conditions. 146,147ence, only one clinical trial has been completed for DNAzymes in oncology thus far (Table 2).

TFDs
TFDs are small dsDNA fragments designed to mimic the precise binding site of a target transcription factor involved in cancer development (Table 1; Figure 1). 148,149After cell internalization, TFDs can effectively disrupt the abnormal expression of multiple disease-associated genes by selectively binding to specific transcription factors responsible for regulating the expression of these genes. 150Most of the clinical investigations have focused on TFDs that target nuclear factor kappa-light-chain-enhancer of activated B cells and signal transducer and activator of transcription 3, two transcription factors implicated in carcinogenesis, tumor progression, and drug resistance in many types of cancers (Table 2). 151,152RNA First described as an "adaptive immune system" in bacteria and archaea to safeguard against viruses, 153 the CRISPR-Cas system proved to be an efficient tool in cancer drug development due to its ability to precisely cleave and target multiple genomic regions associated with cellular malignant transformation. 154Thus, this technology facilitates the correction of genomic errors, regulation of gene expression, and cost-effective manipulation of genes in cells (Table 1; Figure 1). 155e CRISPR-Cas system consists of two main components, an sgRNA molecule and the Cas9 nuclease, forming together a complex that can cleave specific DNA sites. 156The primary function of the sgRNA is to guide Cas9 endonuclease to a specific location within the genome, where it induces a double-stranded break in the DNA.The inherent DNA repair processes subsequently maintain genomic stability through two distinct pathways: an error-prone non-homologous end-joining method, which can result in deletions and insertions (indels), and a less-frequent homology-directed repair mechanism, which mends the DNA damage by incorporating external repair templates to the damaged area. 157The sgRNA is composed of two segments: a constant sequence that creates a scaffold by several stem-loops for binding the Cas9 nuclease and an adaptable 5 0 end segment of 20 nucleotides that is altered to match the target DNA sequence, allowing customization to various targets. 156 alternative CRISPR-Cas technique employs a modified, catalytically inactive Cas9 enzyme, known as nuclease-dead Cas9 (dCas9) to either activate or repress targeted genes. 157dCas9 is an RNAguided DNA binding protein generated by the inactivation of its

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two catalytic domains that are fused to transcription modulating domains. 158Then, gene-specific sgRNAs guide the dCas9-transcription modulating domain complex to effector domains of specific DNA sequences to either repress (CRISPRi) or activate (CRISPRa) the transcription of target genes.While CRISPRi possesses the capability to inhibit transcription by either directly obstructing RNA polymerase activity or employing effector domains (Krüppel-associated box domains), CRISPRa solely utilizes effector domains to induce transcriptional activation. 159Nuclease-dead Cas9 has the potential to advance research on the biological functions of various genes in cancer. 160

NECESSITY FOR TUMOR-TARGETED DELIVERY OF ONs
Cancer therapies based on naked ONs have significant limitations regarding their therapeutic index, which depends on their ability to overcome different biological barriers associated with specific administration routes in the human body (Figure 2).Thus, upon systemic administration, naked ONs encounter extracellular obstacles such as non-specific biodistribution, elimination through the reticuloendothelial system (RES), nuclease degradation in the serum, renal clearance, and specific organ barriers including endothelial and bloodbrain barrier (BBB) or mucus barrier in the intestine (Figure 2). 161,162al administration of ONs might be a very convenient route for systemic delivery due to the ease of administration, different dosage possibilities, and no constraints regarding sterility, size, and charge of the formulation.However, this route is not currently used in the clinic as there are important barriers that hinder ON accumulation at the target tissue, such as degradation determined by low pH and gastrointestinal enzymes, mucus barrier, low permeability of the intestinal mucosa due to the tight junctions, macrophage clearance, and intestinal peristalsis. 163Different parenteral routes have been used for the administration of ONs in cancer therapy according to their pharmacokinetics as well as the targeted tissues.Among systemic delivery, subcutaneous and intravenous (i.v.) routes are the most used for ON administration in the clinic due to their rapid systemic distribution, especially i.v.injection.However, the half-life and bioavailability of the i.v.administered ONs could also be increased when their binding to plasma proteins is strong. 164,165Although systemic delivery favors the ONs accumulation at the site of action, a significant amount also reaches highly vascularized organs with fenestrated endothelia, such as the kidney, liver, and spleen, showing hepatotoxicity-and/or nephrotoxicity-associated risks. 162,166,167Moreover, ONs are small molecules (about 3-6 nm) that undergo kidney ultrafiltration, being rapidly cleared from the organism. 168Besides kidney clearance, the RES mononuclear phagocytes, including Kupffer cells and splenic macrophages, play a significant role in degrading naked ONs from the bloodstream. 169 addition to systemic administration, local administration routes of ONs to tumors have gained increased interest due to several advantages such as bypass of various organ anatomical barriers, increased accumulation and retention time at the target site resulting in efficient uptake, fewer off-target effects, and reduced toxicity.Several local administration methods can be used to target ONs in different organs: intratumoral injection (direct injection or by using other platforms such as hydrogels), intrathecal injection, intraperitoneal injection, nose-to-brain administration, direct intravitreal injection, inhalation or intratracheal administration, etc. [170][171][172] Although not currently used in the clinic, intratumor injection of ONs allows the achievement of high drug concentration in situ while using a lower dose, but it can be technically difficult in human patients. 173,174Moreover, the extracellular matrix is a dense barrier within tumors that significantly limits ON diffusion to deep tumor sites due to their macromolecular nature. 175Unlike healthy tissues, in tumors, the lymphatic drainage is hampered, with a leaky vasculature, leading to elevated interstitial fluid pressure that increases proportional to the distance from the www.moleculartherapy.orgReview vessel, hindering the homogeneous distribution of ONs throughout the tumor. 176In addition, tumor microenvironment conditions, such as low pH resulting from acid metabolite accumulation, can also contribute to ON delivery failure. 177trathecal and intravitreal injections are invasive and induce significant inflammation at the injection site.Similar to blood vessel endothelium, the BBB prevents ONs as well as other drugs from entering the brain parenchyma since endothelial cells are interconnected by tight junctions that provide a barrier function with a higher electrical resistance than peripheral capillaries, which prevents extravasation of molecules larger than 400 Da. 178,179Intraperitoneal administration of ON therapeutics is used to treat the tumors located in the peritoneal cavity or scattered throughout the peritoneum where systemic drug delivery is not successful.The main advantages of intraperitoneal administration of ONs are prolonged retention and the capability to administer large volumes of drug suspension. 180sides extracellular barriers associated with different delivery routes significant obstacles given by the cellular structures of the target sites must be overcome to fulfill ONs' pharmacological effects (Figure 2).Thus, intracellular challenges that most ONs encounter start with cellular uptake.Because of their negative charge, ONs are restricted from passing through the negatively charged cellular membrane due to electrostatic repulsion. 181In addition, altered membrane lipid structure and elevated cholesterol concentration make the tumor cell membrane less permeable. 182Upon internalization, successfully delivered ONs are often taken up into endosomes where they can be degraded by compartment-specific enzymes, impairing their escape to the cytoplasm. 183,184Within the cytoplasm, an important obstacle is the potential off-target effects caused by binding to unintended targets with similar sequences to the target RNA and proteins. 11,185[188] TARGETED DELIVERY OF ONs TO TUMORS Tumor-targeted delivery of ONs might efficiently counteract the abovementioned limitations and significantly improve their bioavailability and therapeutic efficacy, avoiding side effects on healthy cells.Therefore, the incorporation of ONs in viral and non-viral delivery systems ensures protection from endonuclease degradation and enhances cellular uptake, inducing their endosomal escape. 189The present section provides an overview of the main advantages and disadvantages of these potential delivery systems (Figure 3).Since the first successful gene therapy in humans in 1992, when a retroviral recombinant virus was used to deliver adenosine deaminase gene to T cells, several viral vector-based therapies have been developed for the treatment of different genetic diseases and cancer. 190iral vectors represent efficient delivery vehicles as they can transduce human cells and transfer genetic material, allowing for shortand long-term gene expression. 191 overcome cancer-related challenges, several viral vectors including adenoviruses (Advs), lentiviruses, and adeno-associated viruses (AAVs) have been the most used (Figure 3). 192Advs are icosahedral viruses characterized by a dsDNA structure and a diameter ranging from 90 to 100 nm.These viruses not only possess the capability to transport ONs but also activate the complement system and Tolllike receptors through their capsid and nucleic acid components, boosting intratumoral immune responses. 193AAVs, measuring 25 nm, are relevant in clinical trials due to lack of human pathogenicity, non-toxicity, and tissue tropism. 194,195Advs or AAVs can provide almost 100% transduction efficiency without integrating into the host genome, making them suitable for transient gene knockdown. 196entiviruses are enveloped, spherical retroviruses measuring approximately 100 nm in diameter with exposed glycoprotein that defines its tropism. 195,197One of the advantages of lentiviruses is the ability to perform a stable gene integration with a lower transduction efficiency but for a longer-term gene knockdown. 196 oncology, viral vectors are mainly used as gene delivery platforms in virus-based cancer vaccines, chimeric antigen receptor T cell as well as targeted oncolytic therapies. 198ONs such as siRNA and ASO became increasingly important in cancer treatment due to advances in their chemical modifications that were translated into increased stability.A great number of delivery methods to targeted cells have been developed, but their safety and efficacy need to be improved.Several preclinical studies used virus-based vectors to deliver different types of ONs to cancer cells. 199,200For example, a retroviral vector expressing siRNA targeting the mutant Kirsten rat sarcoma viral oncogene homolog (KRASV12) allele was shown to efficiently induce knockdown of the gene in the pancreatic cancer CAPAN-1 cells.In addition, the retroviral vector fully inhibited the tumorigenic capacity of the same cell line in vivo. 201Similarly, a recombinant adenovirus was used to transfer siRNA specific for survivin in different cancer cell lines and efficiently induced caspase-mediated apoptosis. 202though the safety of the viral vectors used as delivery platforms has experienced significant improvements and they have several advantages over other delivery systems, viral vectors are not currently used in clinic to deliver ONs.Their use was limited mainly due to potential mutation risks, inflammation, and immunogenicity concerns.
Non-viral vectors are considered safer and more efficient in delivering ONs to different targets. 189her ONs, such as aptamers, were conjugated to a chemically modified AAV to target it to MCF-7 breast cancer cells, A549 lung carci-noma epithelial cells, and HeLa cells.Aptamer-conjugated vectors showed a 3-to 9-fold increase in transduction compared with nonconjugated vectors.In addition, in vivo studies showed that the DNA aptamer-virus conjugate showed no off-target effects. 203n-viral delivery systems Non-viral delivery systems for ONs have developed as a promising alternative to viral vectors by offering a safer and more efficient approach to delivering ONs to target cells.Moreover, non-viral systems have emerged to counteract the limitations of therapies based on ONs, such as lack of specificity for tumor tissue, physico-chemical properties (size, charge), as well as interaction with several biological barriers of the human body, including nuclease degradation, immune system, and quick clearance. 204These systems consist of various nanostructures, including polymer and lipid-based carriers, conjugates with cell-penetrating peptides and antibodies, and inorganic carriers (Figure 3). 189,2053][214][215] Furthermore, delivery systems based on positively charged polymers (polyethyleneimine [PEI], chitosan, carbosilane, polyamidoamine, polypropylenimine) possess high encapsulation efficiency, enabling high ON concentrations accumulation in the tumor microenvironment.However, the significant toxicity and immunogenicity of the cationic polymers have limited their clinical use.1][232][233][234][235][236][237] Notably, the development of a dual-targeting drug delivery system named Pep-21, combining a PD-L1-binding peptide with anti-miR-21 inhibitor, demonstrated the efficient binding to tumor cells and macrophages inducing decrease of miR-21 levels, tumor cell migration, and a macrophage polarization toward M1-phenotype and finally suppression of B16 melanoma progression. 236Furthermore, synthetic chimeric biomolecules such as antibody-ON conjugates (AOCs) take advantage of both the targeting capabilities of antibodies and the functional specificity provided by ON components.Several AOCs proved to be efficient in triggering receptor-mediated endocytosis upon binding to membrane receptor antigens.Nevertheless, the therapeutic efficacy of AOCs is dependent on several factors, including membrane antigen density, receptor turnover rate, and antibody-antigen affinity. 2318][249] Moreover, to improve targeting and selectivity for specific cells, the lipid bilayer can also be functionalized with ligands. 189n addition, different lipid types (cationic, anionic, neutral, and ionizable) from liposome composition make these carriers versatile platforms for ON delivery to tumors.Thus, cationic liposomes, often used for siRNA delivery due to their large cargo capacities, face challenges such as serum clearance and off-target effects that can be counteracted by strategies such as PEG conjugation. 250,251In a clinical trial, cationic liposomes carrying anti-vascular endothelial growth factor (VEGF) and anti-kinesin spindle protein (KSP) siRNA, known as ALN-VSP, showed an increased uptake in tumor cells and significant downregulation of VEGF and KSP levels when administered to patients with multiple types of cancer (Table 2). 51Moreover, liposomes made of ionizable lipids are able to induce destabilization of the endosomal membrane and efficient ON delivery into the cytosol. 252- 255With this regard, Liu et al. developed a novel method to treat glioma cells by using hypoxia-responsive ionizable liposome-carrying anti-PLK1 siRNA that enhanced cellular uptake of siRNA, inducing a significant decrease in glioma cell proliferation both in vitro and in vivo. 256Nevertheless, latest studies suggested EVs as a better alternative for liposomes due to certain advantages over lipid nanoparticles. 2579][260][261][262][263] Furthermore, a previous study demonstrated that the therapy based on fibroblast exosomes to transport anti-KRAS siRNAs eliminated metastatic pancreatic cancer in mice.Exosomes exhibited prolonged circulation time due to CD47-mediated immune evasion. 264Notably, these exosomes are also the subject of a phase I active clinical trial (NCT03608631) that focuses on the safety and optimal dosage of mesenchymal stromal cell-derived exosomes carrying anti-KrasG12D siRNA (iExosomes) in pancreatic cancer (Table 2).
Another strategy to deliver ONs to tumor tissues investigated the use of inorganic delivery systems (Figure 3) that offer advantages over lipid-based carriers due to their versatile functionalities and ease of synthesis with controllable size and surface characteristics. 2657][268] Previous studies reported that AuNP conjugation with miRNA induced efficient knockdown in MM.1S multiple myeloma cells 269 and AuNPs covalently linked to aptamer AS1411 improved the effects of imiquimod on HeLa cells and HEC-1-A human endometrial carcinoma cells. 2701][282][283] Thus, Chen et al. showed that HeLa cancer cell membrane-coated NPs could efficiently deliver a nanocore-loaded with doxorubicin and anti-PD-L1 siRNA, leading to suppression of PD-L1 and a stronger antitumor effect. 284

Clinical trials
Since 1998, when the first ON-based therapy (fomivirsen) was approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis, several other ON-based therapies were approved by the FDA as well as the European Union's European Medicines Agency (EMA) for the treatment of different diseases, other than cancer. 285,286In 2023, both the FDA and EMA approved two novel ON therapies for the treatment of geographic atrophy secondary to age-related macular degeneration (Iveric Bio) and amyotrophic lateral sclerosis (tofersen).Also, nedosiran (treatment of primary hyperoxaluria type 1) and eplontersen (treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults) were approved in 2023 but only by the FDA. 287We can see that the FDA generally approved new drugs in a shorter time compared with the EMA. 285Both the FDA and EMA have developed programs to allow faster approval of medicines with potential major public health interests.Therefore, in 2018, the FDA has initiated the Breakthrough Therapy and Fast Track designation programs, while the EMA introduced the PRIority Medicines designation plan.Nevertheless, there are several differences between the two agencies, including organization, advanced therapies classification, and clinical trial supervision.For example, the FDA has a broader classification of the gene therapy products compared with EMA. 288spite this remarkable progress, the main limit to the widespread usage of ON therapeutics is their poor accumulation at the target tissue as a consequence of their enhanced clearance. 289Therefore, half of the ON-based therapeutics approved by the FDA have the liver as the target organ: inclisiran, lumasiran, givosiran, volanesorsen, patisiran, inotersen, defibrotide, mipomersen, and nedosiran. 290Yet, various strategies have been used to overcome this limitation.For example, Molecular Therapy: Nucleic Acids Vol.35 September 2024 15 www.moleculartherapy.orgReview the first ON-based therapeutic, fomivirsen, is locally administered by intravitreal injection and accumulates mainly in the retina and iris with minimal systemic exposure.Nusinersen, a splice-switching ON approved for the treatment of spinal muscular atrophy, is also locally administered by intrathecal injection, resulting in a good biodistribution.The most recently FDA-approved ON-based drug for the treatment of amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 gene, tofersen, is also administered by the intrathecal route.On the other hand, ON drugs used for the treatment of Duchenne muscular dystrophy are systemically administered by i.v.injection, and it has been shown that they achieve the highest concentration in the kidneys. 285Another important characteristic of these successful ON therapies that can be exploited for the development of novel ON-based therapeutics for cancer treatment is the chemical modification of the nucleotides needed to increase their stability.For example, both the first and second generation of modifications such as PS, 2 0 -fluoro RNA, and 2 0 -OMe RNA were used in the development of several FDA-approved ON therapeutics: fomivirsen, pagaptanib, mipomersen, nusinersen, inotersen, givosiran, volanesorsen, lumasiran, and inclisiran.All the four ONs approved for the treatment of Duchenne muscular dystrophy have a phosphorodiamidate morpholino backbone.To further increase their chemical stability, accumulation at the target tissue and biodistribution, and to optimize the pharmacological effect, some of the FDA-approved ONs were delivered by different systems such as N-acetyl-galactosamine for givosiran, lumasiran, inclisiran, and vutrisiran, or lipid nanoparticles for patisiran. 291The latest siRNAbased therapeutic approved in 2023, nedosiran, uses Dicerna's GalXC proprietary delivery platform consisting of N-acetylgalactosamine sugars attached to the extended region of a dicer substrate siRNA molecule with a unique tetraloop configuration that ensures high stability and targeting to hepatocytes. 292 mentioned earlier, no ON-based therapy has been approved for cancer treatment by the end of 2023.However, there is an important number of ON-based therapies in clinical trials.Table 2 presents an overview of clinical studies investigating the potential of ONs as a promising therapeutic approach for various types of cancer treatment.These studies have explored different types of ONs, primarily focusing on chemically modified ASOs.In addition, miRNA and siRNA have been shown to be effective in several clinical studies.Among the most frequently targeted gene products presented in the table is B cell lymphoma 2 (BCL-2).These targets are being investigated for selective inhibition to suppress cancer cell growth, enhance apoptosis, and reduce tumor proliferation.Table 2 also provides information on the different phases of the clinical studies (phase I, II, and III) based on the specific target and delivery system being utilized.The phase of each study indicates its progress in the research process, ranging from early-stage exploratory trials (phase I) to larger-scale evaluations of efficacy and safety (phase III).A considerable number of clinical studies have reached the advanced stage of phase III, with many of them targeting BCL-2, clusterin, and protein kinase C-alpha.In contrast, a significant fraction of studies remain in the early stages of phase I, with only a limited number in phase II or I/II.Notably, all phase III studies employ ASO as the chosen ON platform.Other ONs, such as siRNA and miRNA, are predominantly found in the first, second, or firstto-second stage of development.As ongoing research advances, even though chemically modified ASOs remain at the forefront of investigational cancer therapies, miRNA and siRNA demonstrate promising potential for personalized cancer therapy.

CONCLUSIONS AND FUTURE PERSPECTIVES
In the past few years, the research and development of ONs for cancer therapy have been fueled by the successful approval of several ON-based drugs for different non-cancer diseases as well as by the large-scale administration of mRNA-based COVID-19 vaccines.The development of ASOs, RNAi molecules, aptamers, DNAzymes, and TFDs has expanded the range of targets, especially for difficult or previously undruggable targets, making these ONs a promising class of biotherapeutics for a new era of anti-cancer therapies.However, the clinical use of ON-based therapeutics has been hindered by their high susceptibility to nuclease degradation, rapid blood clearance, immunogenicity, lack of inherent targeting mechanisms, low capability to cross physical and biological barriers, poor cellular uptake, and limited endosomal escape.
Numerous preclinical studies have been conducted to address the intracellular and extracellular challenges of naked ONs and to enhance their pharmacodynamic and pharmacokinetic properties for increased therapeutic efficacy against tumoral cells.Thus, experimental studies have demonstrated that chemical modifications, conjugation with different molecules, and utilization of nanoscale carriers can be employed to improve the delivery of ON-based therapeutics.However, further research is still required before the clinical translation of ONs can be fully realized.

Figure 1 .
Figure 1.Standard schematic representation of ON activity as cancer therapeutics Antisense ONs (ASOs) can physically obstruct or impede the splicing process.sgRNA guides the Cas9 endonuclease toward creating a double-stranded break at a designated position within the genome, favoring deletions and insertions.Transcription factor decoys bind to transcription factors of targeted DNA during the initial stage, blocking their activity.ASOs, gapmers, small interfering RNAs (siRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), bifunctional short hairpin RNAs (bi-shRNAs), and DNAzymes act to target pre-mRNAs/mRNAs to downregulate or block the production of proteins.Aptamers directly inhibit proteins involved in pathogenesis.Created with BioRender.com.

Figure 2 .
Figure 2. Extracellular and intracellular challenges of in vivo delivery of ONsSystemic administration of naked ON drugs often results in inadequate targeting of the specific tissue or cells, leading to non-specific distribution.Only a small ON fraction evades macrophage uptake, nuclease degradation, renal clearance, and serum protein adsorption.In addition, physiological barriers such as endothelium, cell membranes, nuclear membranes, or extracellular matrix (ECM) impede ONs from reaching their therapeutic target.Following cellular uptake, ONs must bypass endosomal entrapment to reach the target.A large proportion of the originally administered ON drugs does not effectively reach its final target.Created with BioRender.com.

Figure
Figure 3. Viral and non-viral delivery systems for ONs in cancerAdenovirus, adeno-associated virus, and lentivirus are the most used viral delivery systems.Lipids, polymers, antibodies, and peptides are used for covalent conjugation to ONs for passive and active targeting.Polymer nanoparticles, dendrimers, lipid-based nano-carriers (liposomes, stable nucleic acid lipid particles, and extracellular vesicles), and inorganic nanoparticles (mesoporous silica and membrane-core nanoparticles) are the main non-viral vectors used to encapsulate ONs.Polyplexes, dendrimers, lipoplexes, and gold and iron oxide nanoparticles bind negatively charged ONs through electrostatic interactions.Created with BioRender.com.

Table 2 .
Completed, active, or recruiting clinical trials based on ONs therapy for cancer treatment