Research progress on non-protein-targeted drugs for cancer therapy

pmc J Exp Clin Cancer Res J Exp Clin Cancer Res 618 jeccr Journal of Experimental & Clinical Cancer Research : CR 0392-9078 1756-9966 BMC PMC10011800 PMC10011800.2 10011800 10012541 36918935 10.1186/s13046-023-02635-y 2635 2 Review Research progress on non-protein-targeted drugs for cancer therapy Zhang Yiwen 1 2 Lu Lu 1 2 Song Feifeng 1 Zou Xiaozhou 1 2 Liu Yujia 1 Zheng Xiaowei 1 Qian Jinjun 3 Gu Chunyan 3 Huang Ping huangping@hmc.edu.cn 1 2 http://orcid.org/0000-0003-0228-5102 Yang Ye 290422@njucm.edu.cn yangye876@sina.com 3 1 Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, 158 Shangtang Road, Hangzhou, 310014 Zhejiang China 2 Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, 158 Shangtang Road, Hangzhou, 310014 China 3 grid.410745.3 0000 0004 1765 1045 School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing, 210023 China 14 3 2023 2023 42 424974 62 5 1 2023 28 2 2023 14 03 2023 14 03 2023 07 01 2025 10011800 10.1186/s13046-023-02635-y 1 14 03 2023 10012541 10.1186/s13046-023-02635-y 2 14 03 2023 © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Non-protein target drugs, especially RNA-based gene therapies for treating hereditary diseases, have been recognized worldwide. As cancer is an insurmountable challenge, no miracle drug is currently available. With the advancements in the field of biopharmaceuticals, research on cancer therapy has gradually focused on non-protein target-targeted drugs, especially RNA therapeutics, including oligonucleotide drugs and mRNA vaccines. This review mainly summarizes the clinical research progress in RNA therapeutics and highlights that appropriate target selection and optimized delivery vehicles are key factors in increasing the effectiveness of cancer treatment in vivo. Keywords Cancer therapy Oligonucleotide drugs mRNA vaccines Target Delivery http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 82173855 pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement no pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY issue-copyright-statement © The Author(s) 2023 Introduction Cancer treatment remains a challenge worldwide. Although overall survival is improved by surgical removal of tumor tissues, chemotherapy, and radiotherapy, recurrence and metastasis of cancers cannot be avoided [ 1 ]. Moreover, chemotherapy has serious adverse effects, such as systemic toxicity and multiple drug resistance, which require the development of novel and effective therapeutic drugs [ 2 ]. Small molecule agents and antibodies that can target intracellular or extracellular proteins in tumor cells have become increasingly popular because of their strong antitumor effects [ 3 , 4 ]. However, they fail to block some transcription factors and oncoproteins, such as RAS [ 5 ], one of the most frequently mutated proteins in cancer. Therefore, non-protein-targeted drugs have emerged to address this dilemma. In particular, RNA-based drugs, which are important components of gene therapy, are the most notable and serve as potential therapeutics that can specifically target and silence any gene target [ 6 ]. The molecular weight of therapeutic RNAs is generally 7–20 kDa, which is much greater than that of small-molecule drugs (< 1 kDa) but less than that of antibodies (> 100 kDa). Full-length mRNA vaccines are also large (> 100 kDa) [ 7 ]. Owing to the development of and improvements in RNA technology, certain synthesized oligonucleotide drugs and macromolecular RNA drugs, such as antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs), and mRNA vaccines (Table 1 ), have been approved for marketing worldwide [ 8 ]. Additionally, an increasing number of oligonucleotide drugs (such as ASOs, siRNAs, and miRNAs) and mRNA drugs are entering clinical trials worldwide [ 9 ]. Table 1 Approved RNA-based drugs in market at present Class Drug name Target Disease Year of approval ASO Nusinersen Exon 7 of SMN2 SMA 2016 Eteplirsen Exon 51 of DMD DMD 2016 Inotersen TTR mRNA FAP 2018 Volanesorsen ApoC3 FCS 2019 Golodirsen Exon 53 of DMD DMD 2019 Vitolarsen Exon 53 of DMD DMD 2020 Casimersen Exon 45 of DMD DMD 2021 siRNA Patisiran TTR mRNA FAP 2018 Givosiran ALAS1 mRNA AHP 2020 Lumasiran HAO1 mRNA PH1 2020 Inclisiran PCSK9 Hypercholesterolaemia 2020 mRNA BNT162b2 Spike protein SARA-CoV-2 2020 mRNA-1273 Spike protein SARA-CoV-2 2020 SMN2 survival pf motor neuron-2, SMA spinal muscular atrophy, TTR transthyretin, FAP familial amyloid polyneuropathy, ApoC3 apolipoprotein C3, FCS Familial chylomicronemia syndrome, DMD duchene muscular dystrophy, ALAS1 aminolevulinate synthase 1, AHP Acute hepatic porphyria, HAO1 Hydroxyacid oxidase 1, PH1 Primary hyperoxaluria type 1, PCSK9 proprotein convertase subtilisin/kexin 9, SARS-CoV-2 syndrome coronavirus 2 RNA-based drugs have played a important role in various diseases, ranging from genetic diseases to viral infections, and clinical studies on RNA-based therapeutics have yielded satisfactory results. Therefore, RNA molecules under development are potential candidates and powerful tools for cancer treatment [ 10 ]. This paper summarizes the research progress of the non-protein target drugs, mainly RNA-based drugs in cancer treatment in recent years, including oligonucleotide drugs (ASOs, siRNA, microRNA), and mRNA vaccines, and puts forward suggestions on the challenges brought by this class of new drugs, and fully exerts their therapeutic potential. This paper summarizes the recent research progress on non-protein-targeted drugs, mainly RNA-based drugs, including oligonucleotide drugs (ASOs, siRNA, and microRNA) and mRNA vaccines, in cancer treatment and enumerates the current challenges faced by researchers studying this new class of drugs. Oligonucleotide therapeutics Approximately 40 years ago, Paul Zamecnik and Mary Stephenson successfully used synthetic oligonucleotides to block the translation of viral RNA [ 11 ]. Currently, owing to the benefits of Watson–Crick base-pairing rules and maturation of RNA technologies, oligonucleotides can be used to treat diseases by binding to specific DNA or RNA sequences or proteins and interfering with target gene expression. Oligonucleotide therapeutics are drugs consisting of 10–50 nucleotides in length, including ASOs, siRNAs, and microRNAs (miRNAs), and can regulate the post-transcriptional level and are expected to target special proteins that are otherwise difficult to target directly [ 12 ]. Hence, oligonucleotide therapeutics are considered the third pillar of drug development, after small-molecule drugs and antibodies [ 13 ]. Drug constructs based on the genomic sequences of target genes are simple to design, and drug candidates only require the identification of the target regions in the RNA associated with the disease process. The key is to design sequences that are highly specific to the target RNA and avoid hybridization with unexpected but homologous “bystander” RNAs. ASOs, siRNAs, and miRNAs are currently the most extensively studied drugs for treating malignant tumors. Here, we provide an overview of recent clinical research progress. Antisense oligonucleotide (ASO) In 1978, Zamecnik and Stephenson used a 13-nucleotide ASO targeting the sequence of the Rous sarcoma virus to inhibit viral replication in vitro. This was the first study to report the therapeutic application of ASOs [ 11 ]. Subsequently, some commercial companies have focused on antisense therapeutics; thus, progress on oligonucleotide chemistry and formulations and the distribution and safety of ASOs have achieved satisfactory results (Table 2 ). Table 2 Antisense oligonucleotides cancer therapeutics in clinal trials Target Drug name Cancer ClinicalTrials. gov Identifier Current status Bcl-2 Oblimersen Solid tumors NCT00543231 Phase I completed Oblimersen Solid tumors NCT00636545 Phase I completed Oblimersen plus carboplatin and paclitaxel Advanced solid tumors NCT00054548 Phase I completed Oblimersen plus etoposide and carboplatin Lung cancer NCT00017251 Phase I completed Olimersen plus paclitaxel Lung cancer NCT00005032 Phase I/II completed Olimersen plus Irinotecan Colorectal cancer NCT00004870 Phase I/II completed Oblimersen CLL NCT00021749 Phase I/II completed Oblimersen plus rituximab and fludarabine CLL NCT00078234 Phase I/II completed Oblimersen plus doxorubicin and docetaxel Metastatic or locally advanced breast cancer NCT00063934 phase I/II terminated Oblimersen plus docetaxel Prostate cancer NCT00085228 phase II completed Oblimersen with interferon alfa mRCC NCT00059813 Phase II completed Oblimersen plus dacarbazine Melanoma NCT00016263 Phase III completed Oblimersen plus dexamethasone Multiple myeloma, plasma cell neoplasm NCT00017602 Phase III completed Oblimersen plus fludarabine and cyclophosphamide CLL NCT00024440 Phase III completed BP1002 Advanced lymphoid malignancies NCT04072458 Phase I recruiting Grb2 BP1001 with or without LDAC AML, CML, ALL, MDS NCT01159028 Phase I completed BP1001-A plus paclitaxel Advanced or recurrent solid tumors NCT04196257 Phase I recruiting BP1001 plus ventoclax and decitabine AML NCT02781883 Phase II recruiting CLU OGX-011 with hormone therapy Prostate cancer NCT00054106 Phase I completed OGX-011 plus docetaxel Metastatic or locally recurrent solid tumors NCT00471432 Phase I completed OGX-011 plus docetaxel Breast cancer NCT00258375 Phase II completed OGX-011 plus docetaxel/prednisone mCRPC NCT01188187 Phase III completed OGX-011 plus docetaxel/prednisone mCRPC NCT01578655 Phase III completed Hsp27 OGX-427 plus docetaxel Neoplasms NCT00487786 Phase I completed OGX-427 CRPC NCT01120470 Phase II completed OGX-427 plus docetaxel Relapsed or refractory metastatic bladder cancer NCT01780545 Phase II completed STAT3 AZD9150 plus Durvalumab Diffuse large B-cell lymphoma NCT02549651 Phase I completed AZD9150 Advanced cancers NCT01563302 Phase I/II completed AZD9150 Advanced or metastatic hepatocellular carcinoma NCT01839604 Phase I/Ib completed AZD9150 Malignant ascites NCT02417753 Phase II Terminated (Could not find these types of patients) Raf-1 LErafAON Advanced cancer NCT00100672 Phase I completed LErafAON Advanced solid tumors NCT00024661 Phase I completed LErafAON plus radiotherapy Neoplasms NCT00024648 Phase I completed Raf-1/Pkc-α ISIS 5132 plus ISIS 3521 Matastatic breast cancer NCT00003236 Phase II completed HIF-1α EZN-2968 Neoplasms, liver metastases NCT01120288 Phase I completed EZN-2968 Advanced solid tumors or lymphoma NCT00466583 Phase I completed EZN-2968 HCC NCT02564614 Phase I completed AZD4785 Advanced solid tumors NCT03101839 Phase I completed AR AZD5312 Advanced solid tumors with AR pathway as a potential factor NCT02144051 Phase I completed c-myb c-myb AS ODN Hematologic malignancies NCT00780052 Phase I completed R2 component of mRNA GTI-2040 plus capecitabine mRCC NCT00056173 Phase I/II completed XIAP AEG35156 plus paclitaxel Advanced breast cancer NCT00558545 phase I/II terminated (Avastin approved for first-in-line treatment) AEG35156 plus gemcitabine Advanced pancreatic cancer NCT00557596 Phase I/II terminated TGF-β2 TASO-001 Solid tumor NCT04862767 Phase I recruiting Akt-1 WGI-0301 Advanced solid tumors NCT05267899 Phase I recruiting FOXP3 AZD8701 plus durvalumab Advanced solid tumors NCT04504669 Phase I recruiting Bcl-2 B-cell lymphoma 2, Grb-2 growth factor receptor-bound protein-2, CLU clusterin, Hsp27 Heat shock protein 27, STAT3 signal transduction and transcriptional activator 3, PKC-α protein kinase C-alpha, HIF-1 hypoxia-inducible factor-1, AR androgen receptor, XIAP X-linked inhibitor of apoptosis, TGF-β2 transforming growth factor beta 2, FOXP3 forkhead box P3, CLL chronic lymphocytic leukemia, mRCC metastatic renal cell cancer, AML acute myeloid leukemia, CML chronic myelogenous leukemia, ALL acute lymphoblastic leukemia, MDS myelodysplastic syndrome, mCRPC metastatic castrate resistant prostate cancer, CRPC castrate resistant prostate cancer, HCC hepatocellular carcinoma ASOs are chemically synthesized oligonucleotides, typically 1–30 nucleotides in length, that bind to RNA following Watson–Crick base-pairing rules. The length of the ASOs allows them to bind uniquely to only one target RNA. Although the first two marketed ASO medications, Fomivirsen and Mipomersen [ 14 ], have been discontinued, there are still seven approved ASO drugs for medical use in the market [ 15 , 16 ], mainly for treating diseases, such as Duchenne muscular dystrophy (DMD) [ 17 ], spinal muscular atrophy, familial amyloid polyneuropathy [ 18 , 19 ], and familial chylomicronemia syndrome. Proteins of the B-cell lymphoma 2 (Bcl-2) family play a role in the regulation of apoptosis and confer resistance to traditional cytotoxic chemotherapy and monoclonal antibodies, making Bcl-2 an attractive target for therapeutic intervention in cancers. Oblimersen sodium (Genasense™, G3139) is an antisense oligonucleotide that hybridizes to the first six codons of the open reading frame of the Bcl-2 mRNA, resulting in Bcl-2 mRNA degradation and induction of apoptosis [ 20 ]. There have been many clinical trials on oblimersen, combined with chemotherapy drugs, such as carboplatin [ 21 , 22 ], paclitaxel [ 23 ], docetaxel [ 24 – 27 ], and irinotecan [ 28 ], for treating solid tumors. In a phase I/II trial, the combination of oblimersen and the prodrug irinotecan was well tolerated in patients with metastatic colorectal cancer; one patient experienced a partial response, and another 10 patients had stable disease lasting for 2.5–10 months ( NCT00004870 ) [ 28 ]. Safety data from clinical trials further support the clinical development of oblimersen in combination with cytotoxic agents. BP1001 is a liposome-incorporated antisense oligodeoxynucleotide designed to inhibit the expression of growth factor receptor-bound protein-2 (Grb-2), an essential oncoprotein in cancer cell signaling [ 29 ]. In a phase I clinical study ( NCT01159028 ), BP1001 was well tolerated both as monotherapy and in combination with low-dose ara-C (LDAC) [ 30 ]. As a therapeutic target, clusterin is overexpressed in many cancers, inhibiting cell death pathways and modulating pro-survival and transcriptional networks [ 31 ]. OGX-011 (custirsen) is a second-generation antisense clusterin inhibitor. To determine the clinical activity of OGX-011, a randomized phase II study, in combination with docetaxel/prednisone, was conducted in patients with metastatic castration-resistant prostate cancer. Treatment with OGX-011 and docetaxel was well tolerated and associated with improved survival, as OGX-011 enhanced the tumor-killing ability of docetaxel by increasing the sensitivity of tumor cells to the drug [ 32 ]. OGX-011 may also be a new treatment strategy for patients with castration-resistant prostate cancer (CRPC) [ 33 ]. Heat shock protein 27 (Hsp27) is a stress-induced multifunctional chaperone that promotes cancer development through its proliferative and antiapoptotic functions. Hsp27 causes therapeutic resistance in prostate and other cancers, and its targeted inhibition sensitizes cancer cells to hormones and chemotherapy. OGX-427 (Apatoren) is a 2′-methoxyethyl-modified ASO that inhibits Hsp27 expression. Hsp27 participates in endoplasmic reticulum (ER) homeostasis, and the knockdown of Hsp27 using OGX-427 induces ER stress [ 34 ]. In a phase I clinical trial, the safety profile of OGX-427 in patients with advanced cancer showed that OGX-427 was tolerated at the highest dose (1000 mg) ( NCT00487786 ) [ 35 ]. The signal transduction and transcriptional activator 3 (STAT3) is an attractive target for many cancers. However, translating the utility of its inhibition from bench to bedside is challenging. AZD9150 (Danvatirsen, ISIS 481464), a generation 2.5 ASO, is a specific inhibitor of STAT3. Compared with generation 2.0 and previous ASOs, generation 2.5 ASOs have a higher affinity and greater intrinsic potency owing to an 8′–10′ phosphorothioate-modified deoxynucleotide “gap” flanked on either end, with 2–3 cEt nucleotides [ 36 ]. AZD9150 specifically inhibits STAT3 and induces apoptosis in various leukemia cell lines [ 37 ]. AZD9150 showed a good efficacy and safety profile in patients with heavily pretreated lymphoma and solid tumors who have undergone extensive pretreatment [ 38 ]. AZD9150 also decreased tumorigenicity and increased the chemosensitivity of neuroblastoma cells by inhibiting endogenous STAT3 and STAT3 target genes [ 39 ]. The STAT3 transcription network is an important driver of the suppressive tumor microenvironment, thus preventing checkpoint-blockade activity. In two phase I clinical studies ( NCT01563302 and NCT01839604 ), AZD9150 monotherapy induced an immune-mediated antitumor response, suggesting that AZD9150, in combination with checkpoint-inhibitor therapy, is expected to enhance antitumor immunity [ 40 ]. LErafAON is a novel formulation of liposome-entrapped ASO targeting the Raf proto-oncogene, which encodes a factor known to play a critical role in regulating cancer cell proliferation, survival, and differentiation [ 41 ]. The preparation of LErafAON showed high liposome entrapment efficiency and stability at room temperature [ 42 ]. A phase I clinical trial evaluating its tolerability and recommended dose, in combination with radiation therapy ( NCT00024648 ), was conducted [ 43 ]. Pharmacokinetic analysis revealed the persistence of detectable circulating rafAON at 24 h in 7 of 10 patients in the highest two-dose cohorts. Thus, liposomal formulations may promote better intratumoral AON delivery and inhibit degradation in viv o. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays key roles in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. High levels of HIF-1 are associated with poor prognosis and treatment resistance in patients with solid tumors; thus, HIF-1 is an attractive target for cancer therapy. EZN-2968 (also known as RO7070179) is a third-generation ASO that specifically targets HIF-1α, a subunit of HIF-1. EZN-2968 hybridizes with HIF-1α mRNA and blocks HIF-1α protein expression in preclinical models [ 44 ]. EZN-2968 was well tolerated at the described dosage and schedule, and most toxicities reported were class 1 or 2, with no accidental toxicity [ 45 ] (Fig. 1 ). Fig. 1 The schematic diagram of antisense oligonucleotides clinically designed for tumor targets, such as Bcl-2, Grb2, CLU, Hsp27, STAT3, c-Raf, HIF-1α, AR, and XIAP (bold font) Small interfering RNA (siRNA) Since the discovery of RNA interference (RNAi) and its subsequent application in gene knockdown in mammalian cells, siRNA therapeutics has made remarkable progress and have become promising tools against various diseases [ 46 , 47 ]. As a type of noncoding double-stranded RNA (dsRNA) molecule, siRNAs are only 18–25 base pairs in length, with or without two overhanging phosphorylated bases at the 3′ end of each strand [ 48 , 49 ]. As the name suggests, siRNA interferes with the expression of specific genes with complementary nucleotide sequences through mRNA degradation after transcription. Generally, siRNAs can bind to a protein complex called the RNA-induced silencing complex (RISC) in the cytoplasm. Upon binding to RISC, the guide strand is directed to the target mRNA, and the phosphodiester bond at the mRNA nucleotides 10 and 11 paired with the antisense strand is cleaved [ 50 ]. To date, four siRNA drugs (patisiran, givosiran, lumasiran, and inclisiran) have been approved for marketing to treat diseases such as TTR, acute hepatic porphyria, primary hyperoxaluria type 1, and hypercholesterolemia [ 51 ]. Patisiran [ 52 ], an siRNA drug for treating polyneuropathy in adults caused by hereditary transthyretin amyloidosis, was the first United States Food and Drug Administration (FDA)- and European Medicines Agency-approved RNAi-based therapy.siRNA drugs have potential advantages in cancer treatment compared with traditional drugs. First, as a useful therapeutic tool, siRNA can knock down genes that directly or indirectly cause abnormal proliferation of cancer cells. Thus, it is possible to treat gene-based cancers. Second, with extensive siRNA libraries available, targets for selective and specific drug development can be rapidly identified and optimized, and such target identification helps elucidate the role of specific genes in tumorigenesis. Third, the synthesis and manufacturing costs of siRNA drugs are relatively low compared to those of their antibody rivals [ 53 ]. Furthermore, optimized siRNA drugs can provide convenient dosing regimens, such as inclisiran, for biannual treatment. These advantages strongly support the notion that siRNA is among the most critical therapeutic tools for the treatment of cancers, and many siRNA drugs have been tested in clinical trials (Table 3 ). Table 3 siRNA cancer therapeutics in clinal trials Target Drug name Cancer ClinicalTrials. gov Identifier Current status RRM2 CALAA-01 Solid tumor NCT00689065 Phase I terminated PKN3 Atu027 Advanced solid cancer NCT00938574 Phase I completed Atu027 Advanced or metastatic pancreatic cancer NCT01808638 Phase Ib/IIa completed KRAS siG12D LODER Pancreatic cancer NCT01188785 Phase I completed siG12D LODER Pancreatic Cancer NCT01676259 Phase II recruiting NBF-006 NSCLC, pancreatic cancer, CRC NCT03819387 Phase I recruiting KrasG12D mutation KRAS G12D siRNA Pancreatic cancer NCT03608631 Phase I recruiting PLK1 TKM-080301 CRC NCT01437007 Phase I completed TKM-080301 HCC NCT02191878 Phase I/II completed EphA2 siRNA-EphA2 DOPC Advanced or recurrent solid tumors NCT01591356 Phase I recruiting MYC DCR-MYC Solid tumors, multiple myeloma, lymphoma NCT02110563 Phase I terminated DCR-MYC HCC NCT02314052 Phase Ib/II terminated Bcl2L12 NU-0129 GBM NCT03020017 Early Phase I completed TLR9/STAT3 CpG-STAT3 siRNA CAS3/SS3 B-cell non-hodgkin lymphoma NCT04995536 Phase I recruiting TGF-β1/COX-2 STP705 Squamous cell carcinoma NCT04844983 Phase II recruiting RRM2 M2 subunit of ribonucleotide reductase, PKN3 protein kinase N3, KARS kirsten rat sarcoma, PLK1 polo-like kinase-1, COX-2 cyclooxygenase-2, NSCLC non-small cell lung cancer, CRC colorectal cancer, HCC hepatocellular carcinoma, GBM glioblastoma CALAA-01, a polymer-based nanoparticle containing siRNA targeting the M2 subunit of ribonucleotide reductase (RRM2), was the first experimental RNAi-based drug screened against solid tumors by Calando Pharmaceuticals in 2008 [ 54 ]. Phase I clinical trials showed that CALAA-01 was quickly eliminated in the blood after intravenous administration and the clearance is associated with body weight [ 55 ]. Another siRNA drug, Atu027, is encapsulated inside a lipid nanoparticle (LNP) to target the protein kinase N3 ( PKN3 ), an essential gene for cancer growth and metastasis [ 56 , 57 ]. Clinical trial results showed that Atu027 serves a new treatment strategy for solid tumors and has good safety and activity profile in patients with advanced or metastatic pancreatic adenocarcinoma when combined with the standard chemotherapeutic gemcitabine ( NCT00938574 ) [ 58 ]. Since the Kirsten rat sarcoma (KRAS) protein binds very closely to nucleotides, which makes it nearly impossible to identify competing nucleotide analogs, the KRAS protein has been considered undruggable for many years. Khvalevsky et al. developed a local prolonged siRNA delivery system, siG12D LODER, against mutated KRAS. This siRNA drug provides an alternative approach for controlling KRAS expression in pancreatic cancer[ 59 ]. A phase I study showed the tolerability, safety, and efficacy of siG12D LODE in patients diagnosed with pancreatic cancer and reported no obvious toxicity. Currently, siG12D LODER is undergoing phase II clinical trials [ 60 ]. TKM-080301 is an LNP formulation containing the siRNA-targeting polo-like kinase-1 ( PLK1 ) gene. PLK1 is overexpressed in hepatocellular carcinoma (HCC), and inhibition of PLK1 activity can rapidly induce mitotic arrest and apoptosis in cancer cells. TKM-080301 improved the overall survival of patients with advanced HCC [ 61 , 62 ]. SiRNA-EphA2-DOPC is an siRNA drug encapsulated in neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes, which targets the ephrin type-A receptor 2 [ 63 ]. EphA2 is overexpressed in several cancer cells. Preclinical results revealed that siRNA-EphA2 DOPC had no dose-dependent adverse effects in primates, and phase I clinical trials of siRNA-EphA2 DOPC are currently under recruitment [ 64 ]. The MYC oncogene family, which consists of C-MYC , MYCN , and MYCL , whose products regulate the transcription of at least 15% of the entire genome and participate in the growth of many solid tumor malignancies [ 65 ]. The siRNA drug DCR-MYC, designed by Dicerna Pharma, is a novel synthetic dsRNA in a stable lipid particle suspension that targets MYC in HCC, solid tumors, and multiple myeloma [ 66 ]. Phase I studies showed that DCR-MYC regulates tumor size in patients with solid tumors ( NCT02110563 ). Glioblastoma (GBM) is one of the most difficult cancers to treat because of the blood–brain and blood-tumor barriers. NU-0129, based on the spherical nucleic acid platform, is an siRNA drug designed to target the GBM oncogene Bcl2Like12 ( Bcl2L12 ), which can cross the blood–brain barrier and may be a new precision medicine approach for GBM treatment. In an early phase I trial, Bcl2L12 protein levels in tumor tissues were reduced after intravenous administration of NU-0129 [ 67 ]. MicroRNA (miRNA) miRNA, a type of small noncoding RNAs encoded by endogenous genes approximately 19–25 nucleotides in size, participates in the regulation of post-transcriptional gene expression [ 68 ]. miRNA biogenesis occurs in the nucleus, where gene transcription is strictly regulated. Normally, once miRNAs bind to RISC to form miRISC, the relative gene expression is fine-tuned by blocking translation or cleaving the mRNA via RISC-based mechanisms, similar to those used by siRNA [ 69 ]. Thus, RSIC assembly is a key process in performing miRNA functions. Although siRNA and miRNA are both noncoding RNAs with similar roles in gene silencing and regulation, siRNA is perfectly complementary to a single gene at a specific location, whereas one miRNA has multiple targets and can regulate the expression of hundreds or thousands of genes through imperfect base pairing; a gene can be regulated by several different miRNAs [ 70 ]. Thus, the clinical applications and therapeutic potential of these two are different (Fig.  2 ). Fig. 2 The different regulatory mechanisms of siRNA and miRNA In 2002, miRNAs were first suggested to participate in cancer progression owing to the deletion and low expression of miR-15 and miR-16 clusters in chronic lymphocytic leukemia [ 71 ]. Over the past two decades, the association between miRNAs and various cancers has been extensively studied. miRNAs play a non-negligible role in cancer regulation, and several miRNA-based therapies are underway for different cancers. There are two strategies for miRNA-based therapeutics: miRNA mimics and miRNA inhibitors, depending on whether miRNA should be replaced or downregulated to manipulate the amount of mRNA target in the cell [ 72 ]. miRNA mimics are synthetic double-stranded oligonucleotides that can overexpress the corresponding endogenous miRNA sequence and mimic the function of the target miRNA, resulting in the downregulation of cancer cells proliferation, thereby promoting mRNA inhibition. Owing to the tumor-suppressor role of miRNAs, miRNA mimics could potentially serve as therapeutic agents for cancer management [ 73 ]. Unlike miRNA mimics, miRNA inhibitors, also known as anti-miRs, are designed as complementary single-stranded RNA analogs based on the generation of ASOs to target endogenous miRNAs. Anti-miRNAs can specifically block the upregulated expression of miRNAs associated with cancer development [ 74 , 75 ]. As a potential tumor-suppressive miRNA, miR-34a is lacking in stem cells and advanced tumors. MRX34 is an LNP that can bind to miR-34a mimics[ 76 ]. MRX34 could enhance the effect of radiation therapy by inhibiting DNA repair in a non-small cell lung cancer (NSCLC) mouse model [ 77 ]. As a therapeutic target, miR-155 is a well-studied miRNA in many hematological malignancies and is mainly associated with poor prognosis in lymphoma and leukemia [ 78 ]. Cobomarsen (MRG-106), an inhibitor of miR-155, is currently undergoing clinical trials and can suppress the downstream targets or survival pathways of miR-155, including JAK/STAT, MAPK/ERK, and PI3K/AKT in vitro [ 79 ]. Remlarsen (MRG-201) was designed to mimic the activity of miR-29 and is currently being studied to determine whether it can limit the formation of fibrous scar tissues in keloids. Huang et al. found that high expression of miR-29 could regulate the STAT3 signaling pathway to inhibit the proliferation, invasion, and metastasis of uterine leiomyoma in vitro; thus, miR-29 might be a new target for treating uterine leiomyoma [ 80 ] Several miRNA drugs have also undergone preclinical trials. For example, the miR-122 mimic could improve the sensitivity of breast cancer cells to chemotherapy drugs, such as alpelisib and trametinib, and reduce the emergence of drug resistance [ 81 ]. When the miR-151a mimic was transfected into a drug-resistant glioblastoma cell line, the cells showed miR-151a-induced enhancement of chemosensitivity to temozolomide by modulation of XRCC4-mediated DNA repair [ 82 ]. The expression level of miR-634 in gastric cancer was significantly lower than that in normal adjacent tissues, and the proliferation, migration, and invasion abilities of gastric cancer cell lines were inhibited upon transfection of the miR-634 mimic [ 83 ]. Both siRNAs and miRNAs are meaningful gene-silencing tools, and four siRNA drug candidates have been approved for marketing. However, many miRNA drugs were mostly terminated owing to safety issues, and no drug candidates have entered phase III clinical trials. Consequently, it is difficult to identify miRNAs that regulate specific genes, as they can lead to unexpected side effects. Addressing the specificity of miRNA drugs can advance the application of miRNAs in clinical settings (Table 4 ). Table 4 miRNA cancer therapeutics in clinal trials Target Drug name Cancer ClinicalTrials. gov Identifier Current status miR-16 TargomiRs MPM, NSCLC NCT02369198 Phase I completed miR-34a MRX34 Primary liver cancer, solid tumors, hematologic malignancies NCT01829971 Phase I terminated (Five immune related serious adverse events) miR-155 Cobomarsen Lymphoma, leukemia NCT02580552 Phase I completed miR-29 Remlarsen Keloid NCT03601052 Phase II completed MPM malignant pleural mesothelioma, NSCLC non-small cell lung cancer Messenger RNA (mRNA) vaccine mRNA, known as messenger RNA, is a single-stranded RNA complementary to the antisense DNA. It carries genetic information and directs protein synthesis in the cytoplasm [ 84 ]. As an intermediary of the central dogma of molecular biology, mRNA plays a vital role in protein production. Since Wolf et al. first successfully introduced in vitro transcription (IVT) mRNA in animals in 1900 [ 85 ], mRNA-based therapeutics, such as mRNA vaccines, have made significant progress in preventing infectious diseases and tumor immunotherapy over the past decade. In particular, because of the relatively low risk of insertion mutagenesis and lack of need to enter the nucleus for functionality, mRNA vaccines have become a hotspot in the prevention and treatment of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 [ 86 ]. On August 23, 2021, tozinameran (Comirnaty, BNT162b2), developed by Pfizer-BioNTech, became the first mRNA vaccine officially approved for commercialization by the FDA to prevent COVID-19 among individuals aged ≥ 16 years old [ 87 , 88 ]. Subsequently, the mRNA vaccine elasomeran (Spikevax, mRNA-1273), developed by Moderna, was approved for marketing [ 89 ]. These two mRNA vaccines have promoted the development of mRNA-based therapy and served as a blueprint for mRNA vaccines in cancer treatment. Compared to other vaccines, mRNA vaccines have many advantages, such as good safety, high efficacy, shorter development cycle, and lower cost [ 90 ]. First, mRNA can be directly translated into proteins in the cytoplasm, whereas plasmid DNA and viral vectors are at risk of mutations caused by gene insertion or infection. Second, cells do not need to be involved in producing mRNA vaccines using IVT mRNA technology, thus avoiding contamination by proteins or viruses; mRNA vaccines can therefore be rapidly and economically mass-produced. In addition, based on current research data, patients showed good tolerance to mRNA vaccines, allowing repeated inoculation of mRNA vaccines. With the development of mRNA vaccines, mRNA cancer vaccines have gradually become a research focus over the last five years (Table  5 ). Since cancer progression is correlated with immune response, mRNA cancer vaccines also show significant advantages in cancer immunotherapy. Through artificial design, mRNA cancer vaccines can deliver and express cancer antigens and activate innate immunity [ 91 , 92 ]. Moreover, with the help of IVT mRNA technology, mRNA cancer vaccines can be used to advance personalized tumor immunotherapy. Therefore, mRNA cancer vaccines have great potential for use in antitumor therapy.mRNA cancer vaccines work by using related delivery vectors and adjuvants to deliver mRNA fragments encoding tumor antigen proteins or immunomodulatory molecules directly targeting cells. Once the tumor antigen is recognized by human immune cells, the body triggers an antitumor immune response [ 93 ]. mRNA cancer vaccines can be divided into two categories: mRNA direct cancer vaccines and mRNA dendritic cell (DC) vaccines. Using granulocyte–macrophage colony-stimulating factor (GM-CSF) as an adjuvant, mRNA direct cancer vaccines induce tumor-specific T-cell responses for tumor rejection by encoding cancer antigens, such as tumor-associated antigens (TAAs) and tumor-specific antigens. In contrast, mRNA DC vaccines obtain mRNA using IVT technology. After transfection into DCs, mRNA is translated into antigens in the cytoplasm to activate DCs, and activated DCs can present TAAs and stimulate the immune system response against tumors. Currently, there is sufficient promising preclinical evidence and many ongoing clinical trials on mRNA vaccines for cancer treatment [ 94 ] (Fig. 3 ). Table 5 mRNA vaccine cancer therapeutics in clinal trials Intervention Cancer ClinicalTrials. gov Identifier Current status DC vaccine Breast cancer, malignant melanoma NCT00978913 Phase I completed DC vaccine AML NCT01734304 Phase I/II completed DC vaccine Melanoma NCT00940004 Phase I/II completed DC vaccine with mRNA from tumor stem cells GBM NCT00846456 Phase I/II completed mDC vaccine/ pDC vaccine mCRPC NCT02692976 Phase II completed DC vaccine plus cisplatin Melanoma NCT02285413 Phase II completed DC vaccine plus docetaxel mCRPC NCT01446731 Phase II completed DC vaccine AML NCT05000801 Recruiting DC vaccine plus temozolomide GBM NCT02649582 Phase I/II recruiting DC vaccine plus temozolomide High grade glioma, diffuse intrinsic pontine glioma NCT04911621 Phase I/II recruiting DC vaccine AML NCT01686334 Phase II recruiting DC vaccine plus radiotherapy and IFN-α Malignant melanoma NCT01973322 Phase II recruiting RNA-loaded DC vaccine plus basiliximab Malignant neoplasms brain NCT00626483 Phase I completed TriMix-DC Melanoma NCT01066390 Phase I completed TriMix-DC plus ipilimumab Melanoma NCT01302496 Phase II completed TriMix Breast cancer NCT03788083 Phase I recruiting BTSC mRNA-loaded DCs GBM NCT00890032 Phase I completed CT7, MAGE-A3, and WT1 mRNA-electroporated LCs Multiple myeloma NCT01995708 Phase I completed CEA-loaded DC vaccine CRC NCT00228189 Phase I/II completed MiHA-loaded PD-L-silenced DC Hematological malignancies NCT02528682 Phase I/II completed mRNA transfected DC Androgen resistant metastatic prostate cancer NCT01278914 Phase I/II completed GRNVAC1 AML NCT00510133 Phase II completed mRNA transfected DC plus docetaxel Prostate cancer NCT01446731 Phase II completed Human CMV pp65-LAMP mRNA-pulsed autologous DCs GBM NCT02366728 Phase II completed Human CMV pp65-LAMP mRNA-pulsed autologous DCs with or without varlilumab GBM NCT03688178 Phase II recruiting pp65-shLAMP DC with GM-CSF/ pp65-flLAMP DC with GM-CSF GBM NCT02465268 Phase II recruiting Autologous DCs loaded with autologous tumor RNA Uveal melanoma NCT01983748 Phase III recruiting CV9103 HRPC NCT00831467 Phase I/II completed CV9103 HRPC NCT00906243 Phase I/II Terminated (Study closed after completion of Phase I) CV9104 Prostate Cancer NCT01817738 Phase I/II terminated (Follow up period after primary analysis was prematurely stopped because more mature data will not impact the study outcome) CV9104 Prostate Cancer NCT02140138 Phase II terminated (Recruitment was terminated after enrolment of 35 instead of 36 evaluable patients for administrative reasons.) CV9201 NSCLC NCT00923312 Phase I/II completed CV9202 and local radiation NSCLC NCT01915524 Phase I terminated (Slow recruitment in stratum 3: enrolled only 2 instead of 8 pts. within predicted time) CV9202 plus durvalumab and tremelimumab NSCLC NCT03164772 Phase I/II completed mRNA-5671/V941 with or without pembrolizumab KRAS mutant advanced or metastatic NSCLC, CRC or pancreatic adenocarcinoma NCT03948763 Phase I completed mRNA-2416 plus durvalumab Advanced malignancies NCT03323398 Phase I/II terminated (This study was halted prematurely because the efficacy endpoints were not met for either treatment arm.) mRNA-4157 plus Pembrolizumab Melanoma NCT03897881 Phase II active, not recruiting mRNA-4157 plus pembrolizumab Solid tumors NCT03313778 Phase I recruiting mRNA-2752 plus durvalumab Advanced or metastatic solid tumor malignancies or lymphoma NCT03739931 Phase I recruiting mRNA-4359 plus pembrolizumab Advanced solid tumors NCT05533697 Phase I/II recruiting mRNA RNA loaded lipid particles GBM NCT04573140 Phase I recruiting OTX-2002 HCC and other solid tumor types known for association with the MYC oncogene NCT05497453 Phase I/II recruiting BNT141 plus nab-paclitaxel and gemcitabine Advanced unresectable or metastatic CLDN18.2-positive solid tumors NCT04683939 Phase I/II recruiting BNT113 HPV16 + head and neck cancer NCT03418480 Phase I/II recruiting BNT113 plus pembrolizumab Unresectable head and neck SCC NCT04534205 Phase II recruiting BNT111 Melanoma NCT02410733 Phase I active, not recruiting BNT111 plus cemiplimab Unresectable Stage III or IV melanoma NCT04526899 Phase II recruiting Stabilized tumor-mRNA plus GM-CSF Malignant melanoma NCT00204607 Phase I/II completed mRNA coding for melanoma associated antigens plus GM-CSF Malignant melanoma NCT00204516 Phase I/II completed Personalized cellular vaccine Brain cancer NCT02808416 Phase I completed Neoantigen tumor vaccine with or without PD-1/L1 Advanced gastric cancer, esophageal cancer, and liver cancer NCT05192460 Recruiting Neoantigen mRNA personalised cancer SW1115C3 Advanced malignant solid tumors NCT05198752 Phase I recruiting RO7198457 with or without atezolizumab Advanced or metastatic tumors NCT03289962 Phase I active, not recruiting RO7198457 plus atezolizumab and mFOLFIRINOX Pancreatic cancer NCT04161755 Phase I active, not recruiting RO7198457 plus pembrolizumab Advanced melanoma NCT03815058 Phase II active, not recruiting RO7198457 Stage II and stage III colorectal cancer NCT04486378 Phase II recruiting HB-201 HPV 16 + confirmed oropharynx cancer, cervical cancer NCT04630353 Early Phase 1 recruiting GRANITE (GRT-C901/GRT-R902) Colon cancer NCT05456165 Phase II recruiting DC dendritic cell, mDC myeloid dendritic cells, pDC plasmacytoid dendritic cells, LCs langerhans cells, GM-CSF granulocyte–macrophage colony-stimulating factor, CEA carcinoembryonic antigen, mCRPC metastatic castration-resistant prostate cancer, AML acute myeloid leukemia, GBM glioblastoma, mCRPC metastatic castration-resistant prostate cancer, CRC Colorectal cancer, AML acute myelogenous leukemia, HRPC hormonal refractory prostate cancer, NSCLC non-small cell lung cancer, HCC hepatocellular carcinoma, CLDN18.2 Claudin 18.2, SCC squamous cell carcinoma Fig. 3 The schematic diagram of mRNA direct cancer vaccine and mRNA dendritic cell (DC) vaccine excitation of immune cells to kill tumor cell TriMixDC is an autologous monocyte-derived DC electroporated with mRNA encoding a mixture of three immune-modulating molecules, including active TLR-4, CD40 ligand, and CD70 [ 95 ], which can stimulate T cells. TriMixDC-MEL, obtained by co-electroporation of TriMixDC with an mRNA encoding melanoma-associated antigens, showed favorable safety, strong immunogenicity. It produced a durable tumor response in 4 of 15 patients with advanced melanoma after intravenous and intradermal combined administration ( NCT01066390 ) [ 96 ]. When combined with the immune-checkpoint blocker ipilimumab to overcome immune tolerance, the median progression-free survival and overall survival rates improved in patients with advanced melanoma treated with TriMixDC-MEL, and robust CD8 + T-cell responses were detected ( NCT01302496 ) [ 97 ]. CV9103 and CV9104 are both mRNA-based vaccines based on RNActive® technology. CV9103 encodes four specific antigens present in cancer cells: prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and six-transmembrane epithelial antigen of the prostate (STEAP). The follow-up vaccine CV9104 encodes six antigens, i.e., PSA, PSMA, PSCA, STEAP, mucin 1, and prostatic acid phosphatase (PAP), which are overexpressed in prostate cancer cells compared to those in healthy tissues [ 98 ]. These antigens are appropriate targets for intervention and can induce adaptive immunity in humans. In a phase I/II clinical trial, CV9103 displayed safety and activated immunogenicity in patients with advanced prostate cancer, and one patient showed a confirmed PSA response [ 99 ]. However, the subsequent trial with CV9104 for prostate cancer was terminated because there was no significant improvement in overall survival compared to that in patients treated with placebo [ 100 ]. CV9201 is another mRNA vaccine based on RNActive®, which encodes five NSCLC antigens, including melanoma antigen family C1 (MAGE-C1), MAGE family C2, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), trophoblast glycoprotein (5T4), and survivin. CV9201 showed an acceptable tolerability profile and evidence of immune activation in a phase I/IIa dose-escalation experiment ( NCT00923312 ) [ 101 ]. CV9202 is also a self-adjuvanted mRNA vaccine encoding six NSCLC-associated antigens, namely NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1, which induce targeted immune responses. A phase Ib clinical trial demonstrated that treatment with CV9202 combined with radiotherapy in 26 patients with stage IV NSCLC was well tolerated, and antigen-specific immune responses were detected in 84% of patients ( NCT01915524 ) [ 102 ]. Further clinical trials on CV9202 evaluating its safety and preliminary efficacy, combined with the immune checkpoint inhibitors durvalumab (anti-PD-L1) or remelimumab (anti-CTLA-4), have been conducted ( NCT03164772 ) [ 103 ]. BNT111 is an intravenously administered tetravalent liposomal RNA vaccine encoding four TAAs: NY-ESO-1, melanoma-associated antigen A3 (MAGE-A3), tyrosinase, and transmembrane phosphatase with tensin homology. These antigens show restricted normal tissue expression, high immunogenicity, and high prevalence in melanoma. When entering the body, BNT111 is taken up by antigen-presenting cells (APCs), translocated to the cytoplasm, and translated into four tumor-associated proteins, ultimately triggering antigen-specific CD8 + and CD4 + T cell responses. A first-in-human dose-escalation phase I clinical study showed that BNT111 exhibited good safety and induced durable objective immune responses in patients with advanced melanoma ( NCT02410733 ) [ 104 ]. An open-label, randomized, multicenter phase II trial is currently ongoing to evaluate the safety, tolerability, and efficacy of BNT111, in combination with cemiplimab, in patients with unresectable stage III or IV melanoma with anti-PD-1-refractory or relapse after anti-PD-1 therapy ( NCT04526899 ). Autogene cevumeran, also called RO7198457, consists of RNA-Lipoplex (RNA-LPX) and is an individualized neoantigen-specific therapy (iNeST) that can potentially stimulate and expand neoantigen-specific CD4 + and CD8 + T cells, leading to antitumor responses. Currently, four clinical trials are underway or under recruitment. One is a first-human phase I study designed to evaluate the safety, tolerability, immune response, and pharmacokinetics of RO7198457 as a single agent or in combination with the anti-PD-L1 antibody atezolizumab in participants with locally advanced or metastatic tumors ( NCT03289962 ). A randomized phase II study of RO7198457 in combination with pembrolizumab was conducted in patients with previously untreated advanced melanoma ( NCT03815058 ). Challenges Despite considerable progress in RNA-based therapeutics, two major challenges remain for clinical application: selecting the best drug target from a large number of possible targets and optimizing the delivery of RNA drugs to individual tumors [ 105 ]. The choice of targets and delivery routes can enhance drug efficacy while minimizing side effects in normal tissues and increasing drug safety. Target Cancer is caused by a variety of complex factors, including genetic lesions. Many small-molecule therapeutics directly target key genetic genes for cancer treatment. In RNA-based drug development, we should seriously consider potential genetic targets and concentrate on those that are difficult to target using small molecules. For example, the MYC oncogene family is frequently deregulated in most human cancers and is associated with poor prognosis and unfavorable patient survival [ 65 ]. One of the potential ways to treat cancer is to inhibit MYC expression; however, owing to the disorderly structure of the MYC protein, there is currently no small-molecule inhibitor with good activity and high selectivity that directly targets MYC  [ 106 ]. KRAS is among the most common oncogenes in solid tumors. However, few KRAS -targeted drugs are currently available. Currently, only Lumakras (Sotorasib, Amgen), approved by the FDA on May 28, 2021, is used to treat patients with a proto-oncogene KRAS G12C-mutated NSCLC, the first targeted drug approved for KRAS mutations [ 107 ]. Therefore, these oncogenes can be preferred targets against which oligonucleotide drugs can be developed. Cancer is a multifactorial disease that involves multiple genes. Thus, targeting only one associated gene may be insufficient. Combination therapies that simultaneously target multiple affected genes can be a viable approach in the future. Oligonucleotide therapeutics are particularly amenable to combination therapy because the same drug modality can be applied to target multiple cancer drivers [ 12 ]. Although neoantigens have shown great potential in cancer immunotherapies, identifying suitable cancer neoantigens that can be targeted by mRNA vaccines remains a challenge. Alternative splicing occurs widely in tumors and has been proven to contribute to the generation of candidate neoantigens [ 108 ]. However, abnormal alternative splicing occurs in many tumors, which may lead to the translation of abnormal transcripts into tumor-specific proteins. High-throughput technologies enable systematic characterization of alternative splicing and may identify alternative splicing-derived cancer neoantigens from RNA-seq data. It is also possible to design personalized mRNA vaccines based on alternative splicing-derived cancer neoantigens [ 109 ]. Delivery Currently, delivery is among the greatest barriers to the widespread application of RNA-based therapeutics. In particular, safe, efficient, and targeted delivery of oligonucleotide drugs and mRNA vaccines remains a major challenge [ 16 , 110 ] (Table 6 ). First, naked and unmodified RNAs are poorly stable, easily degraded by multiple circulating ribonucleases (RNases) and hydrolases, and rapidly cleared by renal clearance upon systemic injection. Second, as a hydrophilic negatively charged macromolecule, oligonucleotide drugs have limited ability to penetrate cell membranes, making it difficult to enter the cytoplasm or nucleus. In addition, ASO and siRNA sequences may have off-target effects, leading to non-specific gene knockdown and activation of the innate immune system via Toll-like receptors. Thus, optimized RNA drug delivery systems can protect RNA structures from degradation, increase targeting capacity, and reduce toxic side effects. Table 6 Currently developed delivery platforms in RNA therapeutics Delivery platform Classification Pros Cons Viral vectors Adenovirus, adeno-associated virus, lentivirus High transfection efficiency Immunogenicity, high cost, toxicity Lipid-based delivery system Micelles, liposomes, lipid nanoparticles Easy to production, lack of immunogenicity, biodegradability Difficult to large-scale Polymer-based nanoparticles Cationic polymers, dendrimers Small size, low immunogenicity and toxicity Poor biodegradability Inorganic nanoparticles Gold nanoparticles, silica nanoparticles, carbon nanotubes Easy functionalization, good biocompatibility, high load capacity, mass production Limited transfection efficiency, lack of clinical trials With the development of feasible technologies that improve the druggability of RNA molecules, various viral and non-viral delivery systems have emerged. Currently, there are three key viral vectors for gene therapy: adenovirus (AdV), adeno-associated virus (AAV), and lentivirus [ 111 ]. Over the past two decades, they have achieved preclinical and clinical successes. AAV was first identified in laboratory AdV preparations in the mid-1960s [ 112 ]. Recombinant AAV is also a leading platform for in vivo delivery of gene therapies [ 113 ]. However, viral vectors pose toxicity issues and are unsafe for humans owing to their inflammatory and immunogenic effects, which limit their clinical translation [ 114 ]. Compared with viral vectors, non-viral vectors have a wider range of application, and they have overcome some issues, including high cost, immunogenicity, and toxictity [ 115 ]. Therefore, relatively safe non-viral vectors, such as lipid-based delivery systems, polymer-based nanoparticles, and inorganic nanoparticles, are rapidly evolving [ 116 ]. Lipid-based delivery systems, such as micelles, liposomes, and LNP, can be easily synthesized through chemical reactions [ 117 , 118 ]. The efficiency of delivering RNA therapy to the liver is greatly improved by distinct chemical structures and more reasonable lipid molecular design. LNPs are one of the most widely used non-viral delivery systems for oligonucleotide drugs and mRNA vaccines, and their advantages include ease of production, biodegradability, protection of the embedded RNA from RNase degradation and renal clearance, promotion of cellular uptake, and endosomal escape [ 119 , 120 ]. Recently, LNP has received global attention as an important component of mRNA vaccines, playing a key role in effectively protecting and transporting mRNA into cells. Polymers are the second largest class of nucleic acid-delivery vehicles after lipids. Cationic polymers form stable complexes with anionic nucleic acids, providing a versatile, scalable, and easily adaptable platform for efficient nucleic acid delivery while minimizing the immune response and cytotoxicity [ 121 ]. The efficiency of RNA delivery into cells can be altered by adjusting polymer polarity, degradation, and molecular weight. Dendrimers are another type of polymer that deliver RNA [ 122 ]. These macromolecules are centered on a core molecule and synthesize highly branched polymers via repetitive growth reactions. Modifying the dendrimer structure can protect nucleotides from enzymatic degradation. With the development of nanomaterials, inorganic nanocarriers provide a unique platform for the effective delivery of nucleic acid drugs to tumor cells due to their high stability, good biocompatibility, low immunogenicity, and mass production, such as gold nanoparticles (AuNPs) [ 123 , 124 ], silica nanoparticles [ 125 ], and carbon nanotubes. AuNPs are [ 126 ] a classical inorganic nanocarrier with good chemical stability and biocompatibility [ 127 ]. Nucleic acid chains are covalently attached to the AuNP core via mercaptan groups. The abovementioned NU-0129 is a siRNA drug designed based on AuNPs to target the oncogene Bcl2L12 in GBM treatment. Silica is another type of biodegradable, safe, and stable carrier nanomaterial. Mesoporous silica nanoparticles (MSNs) have attracted great interest for their easy functionalization, biocompatibility, high specific surface area, and biodegradability [ 128 ]. MSNs can effectively deliver drugs to cells and easily escape from endosomes, thereby enhancing anti-tumor effects [ 129 ]. Bertucci et al. co-delivered anti-miR-221 PNA and temozolomide to induce drug-resistant glioma cell apoptosis by using MSNs [ 130 ]. Viral vectors are more effective but more immunogenic than non-viral delivery systems. Non-viral gene vectors are generally versatile, simple, cost-effective, and potentially safer alternatives but may lack adequate clinical efficacy. Therefore, when selecting a delivery vehicle for an RNA drug, it is necessary to consider many aspects and select the most suitable one to maximize efficacy and minimize side effects. Conclusion RNAs can be used both as a target and a drug. The successful development of various new oligonucleotide drugs and mRNA COVID-19 vaccines has resulted in an increasing number of RNA-based drugs that show great promise for clinical translation. RNA therapy offers an innovative approach to new drugs for cancer treatment, with several important advantages, including high specificity for the target, modular development by replacing RNA sequences, predictability in terms of pharmacokinetics and pharmacodynamics, and relative safety. However, some challenges are associated with this therapy, including the selection of suitable targets, innovation, and optimization of delivery systems. Although these non-protein-targeted drugs have certain limitations, the market potential of RNA therapeutics in the treatment of tumors and other diseases cannot be ignored, along with the continuous breakthrough of core technologies, such as chemical modification and delivery systems. The successful commercialization of oligonucleotide drugs and mRNA vaccines has promoted a wave of nucleic acid drug research and development, and large-scale production and economic benefits have now become the main focus point. Non-protein-targeted drugs can overcome the limitations of the druggability of small molecule and antibody drugs and are thus expected to become the third major drug type. With a deeper understanding of the multiple types and functions of RNA, the ability to generate modified RNAs with higher stability and drug activity, and nanotechnology-based vectors capable of targeted delivery of these RNAs into cells, the development of targeted RNA therapeutic options with multiple specificities is expected to change the landscape of cancer treatment in humans.

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