Current Advances in RNA Therapeutics for Human Diseases.

1. Introduction

For years it was believed that DNA was transcribed into RNA and that this RNA (messenger RNA) was then translated into a protein; however, this all became more complicated when RNA interference (RNAi) was discovered [ 1 , 2 ]. RNAi is a conserved biological process in which there is a repression of gene expression caused by small RNAs (i.e., microRNAs (miRNAs) and synthetic small interfering RNAs (siRNAs)) interacting with protein complexes, such as the RNA-induced silencing complex (RISC) [ 3 , 4 ]. Once bound, the small RNAs can then bind to their respective target mRNA in a sequence-specific manner to either stop translation or target the mRNA for degradation [ 5 ]. This discovery led to a huge increase in research focused on treatments for diseases that would exploit RNAi instead of traditional treatments focused on utilizing small molecules and proteins.

Most drugs currently on the market are either small molecules or proteins. Small molecule-based drugs are commonly competitive inhibitors of their target proteins, while protein-based drugs are commonly used to bind to target proteins, replace non-functional target proteins, or supplement for an inadequate amount of a target protein [ 6 , 7 , 8 ]. A serious issue with protein-based drugs is that most proteins are too large to enter their target cells and therefore are only effective when their target molecule is extracellular or excreted [ 9 ]. While small molecule- and protein-based drugs have been found to be effective in many cases, there is still a plethora of diseases that are unable to be treated using either small molecules or proteins. For example, many diabetic patients develop insulin resistance, and supplementing additional insulin is no longer effective in lowering their blood glucose levels. However, RNA-based treatments may have therapeutic potential for diseases, such as diabetes, cancer, and Huntington’s disease [ 2 , 10 , 11 , 12 , 13 ]. RNA therapies may provide better treatment options to target the pathophysiological mechanisms of these disorders, which may lead to better outcomes for patients. Additionally, many RNA therapies have already been approved by the United States Food and Drug Administration (FDA), with more therapies in various phases of clinical trials demonstrating the validity of such RNA therapies for various diseases. This review will cover the mechanisms of RNA therapy design, current FDA-approved RNA therapies, RNA therapies in clinical trials, and RNAs with clinical potential for treating patients suffering from various conditions.

2. RNA Therapeutics

RNAs as therapeutic agents have been vastly studied over the past few decades, as they are more cost-effective and easier to develop than traditional small molecule- or protein-based therapeutics [ 2 ]. As of now, there are five different categories of RNA therapeutics ( Figure 1 ): (1) messenger RNAs (mRNAs) that encode for proteins, (2) antisense oligonucleotides (ASOs) that are small (~15–25 nucleotides) single-stranded RNAs (or DNAs, but for the purposes of this review, we focus only on RNA ASOs) that can either promote or repress their targets expression, (3) small interfering RNAs (siRNAs) that are similar to ASOs in size; however, they are double-stranded and primarily cause translational repression of their target protein, (4) microRNAs (miRNAs) that are small RNAs that can either inhibit protein synthesis when they bind to an mRNA target (miRNA mimics) or free up mRNA by binding to the miRNA that represses the translation of that particular mRNA (miRNA inhibitors), and (5) aptamers that are short single-stranded nucleic acids that form secondary and tertiary structures and interact with a specific enzyme or molecule and therefore can promote or inhibit many different molecular pathways. Within these five categories, there are a handful of already US FDA-approved medications ( Table 1 ), many of which are in clinical trials ( Table 2 ), and an even more that have been found to have possible therapeutic potential but are not yet in clinical trials. While RNA therapeutics seem to have immense potential, there are many hurdles that need to be surmounted when they are developed clinically to make them effective potential treatment options.

Before RNAs were used therapeutically, there were many challenges that had to be overcome in order to make them feasible treatment options for human diseases. For example, nucleic acids are negatively charged and do not passively cross the hydrophobic lipid barrier of the cell. Further, exogenous RNAs are degraded rapidly by RNases once they are injected into the host. Finally, some exogenous RNAs cause an immune response that hampers the translation of the target protein or causes the development of a toxic cell environment. Luckily, scientists over the past couple of decades have substantially overcome these barriers with the use of many unique delivery methods, such as nanoparticles that protect the RNA and enable cell-specific delivery of the therapeutic agent.

2.1. mRNA Therapeutics and Functional Implications

mRNA is coding RNA that is transcribed using genomic DNA as a template and serves to encode proteins [ 68 ]. mRNAs are typically around 2 kb in length and characteristically contain a 5′ cap, 5′ UTR, coding region, 3′ UTR, and poly(A) tail ( Figure 1 A) [ 69 ]. mRNAs are excellent candidates for the treatment of diseases with a known genetic component. Traditionally, mRNAs have been used for replacement therapy when diseases are caused by a lack of expression of a particular protein [ 70 ]. Additionally, CRISPR–Cas-based mRNA therapies can be used to repair DNA mutations that cause non-functional downstream products [ 71 ]. In 1990, Wolff et al., were one of the first groups to induce expression of a protein in vivo through the injection of a synthetic mRNA that encoded beta-galactosidase, chloramphenicol, or luciferase into the skeletal muscle of mice [ 72 ]. Further, Jirikowski et al., used mRNA to treat diabetes insipidus in Brattleboro rats that have reduced levels of vasopressin [ 73 ]. Synthetic or in vitro exogenous vasopressin mRNA was able to temporarily reverse diabetes insipidus when injected in these rats. These initial reports implicated mRNAs as possible therapeutic options for treating/preventing human diseases.

mRNA Vaccines

mRNAs that encode either adjuvants or antigens have also been proposed as possible vaccine candidates that may be able to prevent many diseases. In 1993, Martinon et al., reported that there was induction of anti-influenza cytotoxic T lymphocytes following immunization with liposome-complexed mRNA-encoding influenza virus nucleoproteins in a murine model [ 74 ]. This was the first report highlighting the possible use of mRNAs as vaccines to prevent many infectious diseases. While this study was conducted decades ago, mRNA vaccines have become more common in recent years, especially since the COVID-19 pandemic. Further, this pandemic has also highlighted the speediness of the development of mRNA vaccines when compared to many of the traditional vaccine technologies. The first vaccines to receive emergency use authorization from the FDA were mRNA vaccines.

Currently, there are two mRNA vaccines that are FDA approved and one that is in clinical trials. The first mRNA vaccine approved by the FDA was BNT162b2 [ 31 ]. This vaccine was produced in collaboration between Pfizer and BioNTech to create immunogenicity and antibody response to SARS-CoV-2, which causes COVID-19. This vaccine candidate was clinically tested in Germany and the US and was found to significantly decrease the risk of contracting COVID-19. BNT162b2 encodes for the full-length membrane-anchored spike (S) protein that includes two minor mutations to increase conformational stability. The second FDA-approved mRNA vaccine was mRNA-1273, which encodes the perfusion stabilized S protein of SARS-CoV-2 as well as the S1–S2 cleavage site [ 32 ]. Similar to BNT162b2, the mRNA-1273 vaccine was manufactured by Moderna to prevent the contraction of COVID-19. Both FDA-approved mRNA COVID-19 vaccines include 1-methyl-pseudouridine to hamper innate immune sensing, while also increasing the translational ability of the mRNA. Additionally, they both are encapsulated by lipid nanoparticles. Further, both vaccines have been shown to provide significant immunity against SARS-CoV-2 infection, while also maintaining high safety standards. The exceptional speed at which both vaccines were developed should be noted. To date, no other vaccine has been developed with such swiftness while also maintaining efficacy. This highlights the clinical advantage of using RNA-based vaccines in treating deadly diseases.

Along with the two FDA-approved mRNA vaccines, there are two additional vaccine candidates. The first is CVnCoV, which is currently in Phase III clinical trials [ 52 ]. This vaccine candidate is produced by CureVac AG and is a chemically unmodified mRNA that encodes the full-length S protein of SARS-CoV-2 and utilizes the RNActive mRNA vaccine platform [ 52 ]. CVnCoV induces a robust immune response and provides immunity against SARS-CoV-2 infection. The second vaccine candidate is CV7202 and is in Phase I clinical trials for rabies prevention [ 75 ]. This vaccine is composed of rabies virus glycoprotein mRNA to induce a rabies neutralizing antibody response. Additionally, there are many mRNA vaccine candidates that are currently in pre-clinical trials or that have pre-clinical potential [ 74 , 76 , 77 , 78 , 79 , 80 , 81 ].

To date, there are several mRNA drug candidates in clinical trials for various human diseases: (1) AZD8601 is a vascular endothelial growth factor A (VEGF-A) drug candidate manufactured by AstraZeneca for ischemic heart disease [ 53 ]. VEGF-A has been promising in preclinical trials for increased vessel collateralization [ 82 ]. This study suggested that VEGF-A might be a good drug candidate for treating ischemic heart disease. However, studies have found that treatment with VEGF-A DNA or viral vectors was safe but not effective in treating ischemic heart disease [ 83 , 84 ]. Therefore, it was hypothesized that VEGF-A mRNA drug candidates may be a more efficacious way of treating this disease. AZD8601 is currently being evaluated in Phase II clinical trials. (2) MRT5005 is manufactured by Translate Bio and is currently in Phase I/II clinical trials for the treatment of cystic fibrosis (CF) lung disease [ 54 ]. CF is caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene. This mutation leads to the buildup of mucous in many organs, especially the lungs [ 85 ]. MRT5005 encodes for CFTR and can be delivered through nebulization. (3) mRNA-3704 is in Phase I/II clinical trials for the treatment of methylmalonic aciduria and is manufactured by Moderna [ 55 , 56 ]. Methylmalonic aciduria is a life-threatening genetic disorder in which there is an inability to break down certain proteins and fats, causing a buildup of methylmalonic acid [ 86 ]. This disorder is caused by a plethora of mutations in the methylmalonyl-CoA mutase gene. mRNA-3704 encodes for a fully functional methylmalonyl-CoA mutase enabling the breakdown of proteins and fats that were previously unable to be broken down [ 55 , 56 ]. (4) BNT111, manufactured by BioNTech SE, is currently in dose-escalation Phase I clinical trials for the treatment of advanced melanoma [ 57 ]. This drug targets NY-ESO-1, MAGEA3, tyrosinase, and TPTE, which are tumor-associated antigens predominantly found in melanoma. This therefore should cause an immune response leading to the destruction of tumor cells. There is also a plethora of other mRNA drug candidates in the preclinical pipeline [ 87 , 88 , 89 , 90 , 91 ].

2.2. ASO Therapeutics and Functional Implications

ASOs are synthetic small single-stranded nucleic acid sequences composed of RNA, DNA, or RNA-DNA heteroduplexes that are typically 8–50 bp long [ 92 ]. The study of ASOs began in the late 1970s when it was found that synthesized oligonucleotides were able to inhibit Rous sarcoma virus replication [ 93 ]. This was achieved through viral protein translation inhibition due to the binding of the synthesized complementary oligonucleotide sequence to the viral 35S mRNA. This study paved the way for ASO-based therapeutics.

Since their discovery, there has been a plethora of other mechanisms of action for ASOs. While there are many different ASOs, they all belong to one of two major categories: RNase H dependent ASOs and RNase H independent/steric block ASOs [ 94 ]. RNase H is an endogenous enzyme that catalyzes the degradation of RNA that is part of an RNA–DNA heteroduplex. Therefore, ASOs composed of DNA or both RNA and DNA (gapmers) typically belong to the RNase H dependent category. Once they bind their complementary target mRNA strand, RNase H recognizes the DNA–RNA heteroduplex and catalyzes the degradation of the mRNA. This leads to downregulation of the target mRNA, which can be a useful tool in therapeutic approaches for the treatment of diseases caused by overexpression of certain genes [ 95 ], such as homozygous familial hypercholesterolemia [ 15 ].

RNase H independent/steric block ASOs are typically composed of only RNA and act by binding directly to the target pre-mRNA or mature mRNA to cause inhibition of mRNA translation ( Figure 1 B), alternative splicing, promotion of mRNA translation, or alternative polyadenylation. When the ASO binds to the pre-mRNA at a splice recognition site, it will cause alternative splicing of the target RNA. This approach can be extremely useful for the treatment of disorders that are caused by mutations that can be avoided by selective alternative splicing [ 96 ]. In general, this type of ASO can also lead to increased expression of the target protein [ 97 ]; however, they can also be used to cause exon skipping in order to block translation of the target mRNA [ 98 ]. This approach is also known as splice corruption. ASOs have also been known to bind regions of the mRNA, such as the translation initiation codon, to inhibit translation of the mRNA [ 99 ]. Further, ASOs that bind regulatory regions upstream of the open reading frame (ORF) have been shown to promote target translation [ 100 ]. This is caused by the blocking of regulatory regions typically responsible for translational suppression. ASOs have also been shown to cause alternative polyadenylation by blocking certain polyadenylation sites [ 101 ]. This commonly leads to shorter transcripts that contain less destabilized segments, typically leading to increased stability of the mRNA. The many molecular mechanisms of ASOs have enabled their use for many different therapeutic approaches to treat human diseases.

2.2.1. FDA-Approved ASOs

The first FDA-approved ASO was fomivirsen, which is also known as Vitravene. Formivirsen was developed to treat patients with cytomegalovirus (CMV) retinitis, a serious viral eye infection [ 102 ]. This ASO therapeutic is composed of 21 phosphorothioate oligodeoxynucleotides that target CMV immediate-early-2 mRNA, which is essential for viral replication [ 103 ]. Clinical trials have shown that injections of this drug into the vitreous humor delayed disease progression when compared to untreated controls. Successful clinical trials led to fomivirsen obtaining FDA approval in 1999 [ 102 ]. However, disease progression was still inevitable. This, along with the discovery of anti-retroviral therapies, made this medication less pertinent, and it was eventually taken off the market in 2006.

Mipomersen was approved by the FDA in 2013 for the treatment of homozygous familial hypercholesterolemia [ 104 ]. This genetic disorder is characterized by increased levels of low-density lipoprotein (LDL) cholesterol due to decreased liver uptake of plasma LDL [ 105 ]. This condition leads to premature cardiovascular disease even when treated with previously established lipid-lowering therapies, such as statin therapy [ 106 ]. However, treatment with mipomersen, a gapmer 20 oligonucleotides long that targets and reduces expression of the ApoB mRNA, led to significantly reduced LDL levels [ 15 ]. ApoB is an essential protein for the clearance of LDL and aids in the production of low-density lipoprotein (VLDL), which is a precursor of LDL [ 106 ]. Therefore, reduction of ApoB likely leads to the reduced plasma levels of LDL following mipomersen treatment.

In 2016, nusinersen was approved by the FDA for the treatment of spinal muscular atrophy (SMA) [ 107 ]. This disorder is caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene. This leads to inadequate SMN protein expression, causing weakness and atrophy of skeletal and respiratory muscles [ 108 ]. Nusinersen modulates splicing of SMN2 , which varies only from SMN1 in that it undergoes alternative splicing and excludes exon 7 [ 16 ]. This exclusion results in a truncated protein that only has 5% to 10% functionality. However, nusinersen regulates alternative splicing such that exon 7 is included, resulting in fully functional SMN leading to improved motor function in patients with SMA.

Eteplirsen was approved by the FDA in 2016 for the treatment of Duchenne muscular dystrophy (DMD) [ 109 ]. Mutations in the DMD gene encoding the dystrophin protein leads to the development of DMD [ 110 ]. The most common mutation resulting in DMD is located in exon 51; therefore, eteplirsen (a 30-mer ASO) targets exon 51 of the DMD gene, causing this exon to be excluded during alternative splicing [ 17 ]. This prevents frame shift mutations that lead to the production of non-functional dystrophin. The resulting dystrophin protein is slightly shorter than its wild-type counterpart but maintains its functionality [ 111 ].

Inotersen received FDA approval in 2018 for the treatment of familial amyloid polyneuropathy. This disorder is caused by autosomal dominant mutations in the transthyretin ( TTR ) gene. These mutations disrupt the TTR tetramer leading to aggregation of TTR monomers into amyloid deposits throughout the body [ 112 ]. In order to combat the buildup of TTR, inotersen targets the 3′ UTR of the TTR mRNA, preventing the production of TTR, thus inhibiting disease progression [ 113 ]. Clinical trials demonstrated the efficacy and safety of inotersen in the reduction of circulating TTR levels [ 18 ].

Another ASO that has been FDA approved for the treatment of DMD is golodirsen. This drug behaves similarly to eteplirsen, in that it enables exon skipping; however, it leads to the exclusion of exon 53 instead of exon 51 [ 114 ]. This results in functional dystrophin and improves symptomology of patients with DMD caused by mutations that are acquiescent to exon 53 skipping [ 19 ].

Milasen was developed in less than a year to treat Mila Makovec’s CLN7 gene mutation leading to the development of Batten disease. This is a rare disease caused by one of at least 13 known mutations to the CLN gene that affects the cell’s ability to remove waste, such as excess proteins and lipids [ 115 ]. This disease is eventually fatal if left untreated. After genomic sequencing of Mila Makovec’s unique CLN7 gene mutation, it was clear that this form of Batten disease was caused by improper exon splicing and the resulting premature translational termination [ 20 ]. Milasen targets a specific CLN7 splice site, restoring proper splicing and function of CLN7. It eventually received FDA approval in 2018.

The FDA approved the use of casimersen in 2021 for the treatment of DMD caused by a mutation in the DMD gene that is amenable to exon 45 skipping [ 21 ]. Again, the mechanism of action of this drug is similar to that of golodirsen and eteplirsen.

2.2.2. ASOs in Clinical Trials

ISS 1018 is in Phase II clinical trials for its synergistic effect with rituximab for the treatment of Non-Hodgkin’s lymphoma (NHL) [ 33 ]. NHL is a common cancer that begins in the lymphatic system and eventually spreads to other organs [ 116 ]. Rituximab has been extremely successful in treating many forms of NHL; however, there are still some forms that do not see any improvement following rituximab treatment. ISS 1018 was thought to have a beneficial effect when given to patients simultaneously with rituximab treatment. This drug can illicit immunostimulatory effects by signaling through the Toll-like receptor 9 and leads to proliferation and immunoglobulin production by B cells and the induction of tumor necrosis factor α (TNF-α), interleukin-12 (IL-12), interferon-α (IFN-α), and IFN-β by plasmacytoid dendritic cells [ 33 ]. The production of these cytokines triggers a powerful response in various other immune cell types that are not targeted directly by ISS 1018. Further, ISS 1018 can cause the maturation of dendritic cells that in turn cause an initiation of NK-cell and T-cell responses to tumor antigens [ 117 , 118 ]. To date, clinical trials have shown promise for 1018 ISS when used in conjunction with rituximab for the treatment of NHL [ 34 ].

Apatorsen (OGX-427) is in Phase I/II clinical trials for the treatment of castration-resistant prostate cancer (CRPC) or other metastatic cancers that have been demonstrated to express Hsp27, such as ovarian, breast, bladder, and non-small-cell lung cancers [ 35 ]. Apatorsen works by inhibiting the expression of Hsp27 by binding to and blocking translation of its mRNA [ 35 ]. Studies have shown effectiveness and tolerance of apatorsen in high doses in clinical trials thus far, with many patients showing decreased expression of cancer markers [ 35 ].

Phase II clinical trials of cenersen (EL625) have been conducted to test the efficacy of the drug for the treatment of acute myeloid leukemia [ 36 ]. Cenersen targets mutated p53, which is a proto-oncogene and leads to degradation of the p53 mRNA. This allows for cancer cells to respond to DNA damaging agents that once were not sensitive to such agents. Thus far, clinical trials have shown that patients that had not responded to standard chemotherapy or had relapsed shortly after standard chemotherapy had significantly better clinical outcomes following additional treatment with cenersen [ 36 ].

ARRx (AZD5312) has undergone Phase I/II testing for the treatment of CRPC [ 37 ]. ARRx was designed to target the androgen receptor (AR), which plays an important role in CRPC disease development and progression [ 119 ]. Preclinical trials have demonstrated the efficacy of this drug when used in conjunction with the pan-AKT inhibitor, AZD5363, for treating CRPC in a murine model [ 37 ].

Custirsen (OGX-011) is in Phase I/II clinical trials for treatment of advanced non-small-cell lung cancer that has previously been left untreated [ 38 ]. Custirsen targets the mRNA clusterin, which encodes for a chaperone protein that enables cell survival and causes resistance to various treatments [ 39 ]. Custirsen was shown to have minimal toxicity while significantly reducing the expression of clusterin in primary prostate tumors [ 38 ].

2.3. siRNA Therapeutics and Functional Implications

siRNAs are double-stranded RNAs that are relatively small (~21–25 nucleotides) and function to silence gene expression ( Figure 1 C) [ 120 ]. They occur naturally or can be chemically synthesized. Naturally occurring siRNAs originate from endogenous or viral precursor siRNAs. These precursors are roughly around 100 nucleotides long and are cleaved by Dicer into their mature siRNA structures [ 121 ]. Dicer leaves a 3′ overhang of two nucleotides that allows the siRNA to interact with the RISC complex, where it will initiate gene silencing [ 122 ]. Once it is bound, the RISC protein argonaute 2 (AGO) carries out cleavage of the sense strand [ 123 ]. This allows for the antisense strand to bind its target mRNA. Once the target RNA is bound to the antisense strand, its phosphodiester backbone is cleaved by AGO2. This leads to sequence-specific knockdown of the target mRNA and therefore causes gene silencing. Synthetic or naturally occurring siRNAs can therefore be used to knockdown the expression of a single protein coding gene. The use of the double-stranded siRNA to reduce expression of a target gene was first utilized in 1998 to target hlh-1, unc-54, unc-22, and fem1 in Caenorhabditis elegans ( C. elegans ) [ 1 ]. This groundbreaking work by Fire et al., eventually led to a Nobel prize award. Most importantly, they found that double-stranded RNAs (siRNAs) are more effective in downregulating their target mRNA than their single-stranded counterparts (ASOs) [ 1 ]. This highlights the advantage of siRNA technology over ASOs for the treatment of most human diseases.

2.3.1. FDA-Approved siRNAs

The first siRNA therapeutic approved for use by the FDA was Patisiran [ 124 ]. It was approved in 2018 for the treatment of polyneuropathy caused by hereditary transthyretin-mediated (hATTR) amyloidosis [ 24 ]. hATTR amyloidosis is a genetic disorder that causes the buildup of abnormal TTR, which generally causes polyneuropathy when the build-up occurs in the peripheral nervous system [ 125 ]. Patisiran is an siRNA drug that targets the mutated TTR mRNA leading to mRNA degradation and decreased TTR protein expression [ 24 ]. This has been shown to greatly reduce TTR deposition in patients with polyneuropathy caused by hATTR amyloidosis [ 24 ].

Givosiran was the second FDA-approved siRNA therapeutic and is used to treat acute hepatic porphyria [ 126 ]. This disorder is caused by a plethora of deficiencies in enzymes involved in heme production and leads to a toxic buildup of porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) [ 127 ]. Givosiran targets the mRNA of ALA synthase 1 in the liver and reduces the levels of disease-causing neurotoxic intermediates aminolevulinic acid and porphobilinogen [ 25 ].

Lumasiran was approved by the FDA for the treatment of primary hyperoxaluria type 1 (PH1) in 2020 [ 128 ]. Various mutations in the enzyme alanine-glyoxylate aminotransferase causes increased oxalate concentrations and calcium oxalate crystal formation leading to the development of PH1 [ 26 ]. Lumasiran targets the mRNA that encodes glycolate oxidase, leading to the depletion of the substrate for oxalate synthesis and sufficiently reducing oxalate concentrations [ 128 ].

In December 2021, inclisiran was approved by the FDA for the treatment of atherosclerotic cardiovascular disease (ASCVD) or heterozygous familial hypercholesterolemia (HeFH) ( https://www.fda.gov/drugs/news-events-human-drugs/fda-approves-add-therapy-lower-cholesterol-among-certain-high-risk-adults (accessed on 30 December 2021)). These conditions are characterized by high LDL-C levels. Inclisiran works to lower LDL-C levels by targeting the mRNA encoding for proprotein convertase subtilsin/kexin type 9 (PCSK9), which is involved in lipid metabolism and the regulation of cholesterol levels [ 27 ]. Inclisiran has been shown to reduce LDL-C levels in patients that were unable to reduce these levels with statins alone [ 27 ]. Additionally, inclisiran has demonstrated increased efficacy in lowering LDL-C levels when administered in conjunction with statins in patients that statins alone have been partially effective in lowering LDL-C levels [ 27 ].

2.3.2. siRNAs in Clinical Trials

TKM-080301 is in Phase I/II clinical trials for the treatment of hepatocellular carcinoma (HCC) [ 40 ]. HCC is typically characterized by the overexpression of Polo-like kinase 1 (PLK1) [ 129 ]. Therefore, targeting PLK1 may have beneficial effects for the treatment of HCC. To date, clinical trials have shown limited antitumor effects of TKM-080301 in patients with HCC [ 40 ].

Atu027 is in Phase I/II clinical trials for the treatment of advanced solid tumors and pancreatic adenocarcinoma [ 42 , 43 ]. It is designed to target the mRNA encoding protein kinase N3 (PKN3) in order to reduce the metastatic activity of tumors. Clinical trials have demonstrated the safety and efficacy of Atu027 in preventing adverse outcomes in patients with metastatic cancer [ 43 ].

siG12D LODER is in Phase I/IIa clinical trials for the treatment of pancreatic tumors in combination with chemotherapy [ 44 ]. It is a biodegradable implant containing a siRNA that targets the mRNA of the mutated KRAS oncogene, which can be surgically embedded in pancreatic tumors [ 45 ]. Mutated KRAS has been implicated in the development of most pancreatic cancers and is correlated with a worse prognosis for the patient [ 130 ]. Clinical studies have shown the potential efficacy of siG12D LODER in preventing tumor progression [ 44 ].

ARO-HIF2 is in Phase I clinical trials for the treatment of clear cell renal cell carcinoma ( NCT04169711 ). This form of carcinoma is the most diagnosed form of kidney cancer [ 131 ]. In addition, it is associated with the inactivation of von Hippel–Lindau tumor-suppressor protein (pVHL) propelled by hypoxia-inducible factor 2 (HIF2) transcription factor deregulation [ 132 ]. Therefore, ARO-HIF2 aims to target the mRNA of HIF2 to inhibit tumor growth.

APN401 is currently in Phase I clinical trials to test its efficacy in treating patients with either metastatic or recurrent colorectal cancer, pancreatic cancer, or other solid tumors that are not surgically accessible [ 47 ]. It works by targeting casitas-B-lineage lymphoma protein-b (Cbl-b) that has been shown to limit lymphocyte activation [ 133 ]. Preclinical studies using murine tumor models demonstrated that Cbl-b inhibition enhances natural killer cell and T cell-mediated antitumor activity [ 133 , 134 ].

Vutrisiran (ALN-TTRSC02) is in Phase III clinical trials for the treatment of transthyretin (ATTR)-mediated amyloidosis with ( NCT04153149 ) or without ( NCT03759379 ) cardiomyopathy. ATTR-mediated amyloidosis is a condition caused by a buildup of TTR either caused by mutations in the TTR gene [ 48 ]. By targeting the TTR mRNA, vutrisiran is able to reduce TTR protein expression, leading to better outcomes in patients with ATTR-mediated amyloidosis [ 49 ].

2.4. miRNA Therapeutics and Functional Implications

miRNAs are non-coding RNAs that consist of ~20 nucleotides that are highly conserved between eukaryotic species. miRNAs were discovered in C. elegans by Ambros in 1993 [ 135 ]. That same year, Ruvkun found the first miRNA target genes [ 136 ]. The discovery that miRNAs can be used to downregulate target genes paved the way for miRNA therapeutics. miRNAs have excellent therapeutic potential due to their extraordinary targeting capability. For example, one miRNA can target anywhere from ten to hundreds of genes. Additionally, they tend to target multiple genes within the same pathway. miRNAs are naturally occurring molecules endogenous to our cells. Therefore, there is less chance for immunogenic response with miRNA therapeutics than their other synthetic RNA counterparts. Further, miRNA inhibitors and mimics can be used to restore or inhibit protein synthesis, respectively ( Figure 1 D).

Inhibition of protein synthesis or mRNA degradation is achieved through the miRNA associating with a variety of AGO proteins and modulating gene expression through the activity of the RISC complex [ 137 ]. When the miRNA is associated with AGO and the RISC complex is formed, the miRNA guides AGO to its target mRNA [ 138 ]. The seed sequence of the miRNA binds to the mRNA and causes either translational repression or mRNA degradation [ 139 ]. This leads to reduced target protein expression and therefore plays a key role in post-transcriptional gene regulation. This pathway can be enhanced by the supplementation of a miRNA mimic that is identical in sequence to the endogenous miRNA duplex. Restoring miRNA levels will lead to the repression of mRNAs that are overexpressed in certain conditions.

In contrast, restoration of protein synthesis is achieved by administering a miRNA inhibitor, a single-stranded miRNA that is complementary to a target miRNA. Once the miRNA inhibitor binds to the miRNA, it prevents the miRNA from associating with AGO. Therefore, miRNA inhibitors block the mRNA targeting ability of miRNAs and restore protein synthesis. This approach can be used to treat disorders caused by overexpression of a miRNA that leads to downregulation of certain disease preventing mRNAs.

miRNAs in Clinical Trials

While there are currently no miRNAs that are FDA approved, there are many in clinical development. For example, miravirsen has completed Phase II clinical trials for the treatment of Hepatitis (Hep) C [ 58 ]. Miravirsen is a miR-122 inhibitor that sequesters miR-122, which has been implicated in the promotion of the Hep C virus (HCV) life cycle [ 140 ]. Clinical trials to date have shown a significant reduction of HCV viral load in patients treated with miravirsen [ 58 ].

RG-012, also known as lademirsen, is in Phase II clinical trials for treatment of Alport syndrome ( NCT02855268 ). This condition is caused by various mutations in the genes encoding collagen IV and leads to kidney disease as well as to both ocular and hearing deficiencies [ 141 ]. This syndrome is associated with increased levels of miR-21; therefore, it was hypothesized that a miR-21 inhibitor may work in treating this condition. Pre-clinical trials have demonstrated that a miR-21 inhibitor is extremely successful in preventing the onset of Alport syndrome, thus highlighting the therapeutic potential for this drug [ 59 ].

Cobomarsen is currently in clinical development for the treatment of various leukemias and lymphomas, such as adult T-cell leukemia/lymphoma (ATLL), chronic lymphocytic leukemia (CLL), the mycosis fungoides (MF) subtype of cutaneous T-cell lymphoma (CTCL), and the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) [ 142 ]. It works by targeting miR-155, which is associated with inflammation and the development of various leukemias and lymphomas [ 60 , 142 , 143 , 144 ]. Phase II clinical trials were initiated following successful Phase I trials ( NCT02580552 ); however, they were terminated early due to business reasons unassociated with safety or efficacy ( NCT03713320 ).

MRG-110 has completed Phase I clinical trials to test the safety efficacy of the miR-92a inhibitor in healthy patients ( NCT03603431 ). Treatment with this inhibitor was found to significantly reduce miR-92a expression in these individuals when compared to patients treated with a placebo [ 145 ]. Additionally, increased expression of miR-92a has been associated with poor wound healing [ 61 ]. Further, miR-92a inhibition has been shown to improve wound healing in vivo in preclinical models [ 146 ]. Therefore, MRG-110 may be effective in treating impaired wound healing in conditions such as diabetes.

RG-125 (AZD4076) completed Phase I/IIa clinical trials for the treatment of Type 2 Diabetes (T2D) and Non-Alcoholic Fatty Liver Disease (NAFLD) ( NCT02826525 ). This drug inhibits miR-103/107, in which overexpression of these miRNAs has been shown to correlate with the development of T2D and NAFLD [ 147 ]. Further, preclinical studies have demonstrated that a miR-103/107 inhibitor can improve insulin sensitivity in obese mice [ 62 ]. This along with successful Phase I/IIa clinical trials emphasizes the therapeutic potential of miR-103/107 inhibitors for the treatment of T2D and NAFLD.

RGLS4326 completed Phase 1b clinical trials for the treatment of autosomal dominant polycystic kidney disease (ADPKD) ( NCT04536688 ). This disease is caused by mutations in PKD1 and PKD2 , resulting in decreased expression of PC1 and PC2 [ 148 ]. miR-17 has been shown to bind and downregulate PKD1 and PKD2 gene expression, while miR-17 inhibitors have been shown to restore PKD1 and PKD2 expression [ 63 ]. Finally, inhibition of miR-17 by RGLS4326 in humans was also shown to significantly increase PC1 and PC2 levels in patients with ADPKD ( NCT04536688 ).

CDR132L has completed Phase I clinical trials for the treatment of heart failure of ischemic origin ( NCT04045405 ). Hypertrophy of cardiomyocytes has been shown to be caused by overexpression of miR-212/132 family, leading to heart failure [ 149 ]. Further, preclinical studies have shown that inhibiting miR-132 can improve heart function in animal models of heart failure [ 150 , 151 ]. Finally, initial clinical studies have shown exceptional efficacy of CDR132L for treating patients with heart failure ( NCT04045405 ).

TargomiRs has been studied as either a second- or third-line treatment for recurrent malignant pleural mesothelioma and non-small-cell lung cancer. Downregulation of miR-16 has been implicated in the development of many types of cancer, such as chronic lymphocytic leukemia and non-small-cell lung cancer [ 152 , 153 ]. Additionally, miR-16 mimics act to increase miR-16 levels in order to target and downregulate multiple oncogenes and lead to tumor regression [ 154 ]. TargomiRs, a miR-16 mimic, has shown substantial preclinical efficacy in the treatment of many types of cancer [ 155 , 156 ]. Finally, initial clinical trials have shown that TargomiRs has antitumor effects in patients with malignant pleural mesothelioma [ 65 ].

Remlarsen has completed Phase II clinical trials for the treatment of keloid scars. Keloid scars are caused by a fibroproliferative disorder that causes excess production of extracellular matrix proteins and collagen [ 157 ]. miR-29 has been shown to negatively regulate multiple genes involved in the fibrotic response [ 158 , 159 ] and therefore reduces fibrosis [ 160 ]. Further, remlarsen, a miR-29 mimic, has shown a significant reduction in collagen expression and fibrosis in skin wounds [ 161 ]. Therefore, remlarsen may have therapeutic potential in treating keloid scars as well as scleroderma.

There have been some miRNA drug candidates that have demonstrated severe adverse effects. For example, MRX34 is a miR-34 mimic that showed success in preclinical trials for the treatment of cancer [ 162 , 163 , 164 ]. Expression of miR-34 has been shown to be significantly reduced in many different types of cancer [ 165 , 166 , 167 ]. Further, miR-34 targets many different oncogenes and therefore can theoretically hamper tumor progression [ 168 , 169 , 170 ]. While early Phase I clinical trials were successful in reducing the miR-34a target oncogenes in a dose-dependent manner [ 67 , 171 ], they were eventually halted after several patients had severe adverse reactions to treatment [ 171 ].

Finally, there are multiple miRNA therapeutics that have demonstrated substantial preclinical efficacy for various disorders. For example, miR-10b-5p is in preclinical development for the treatment of diabetes and associated gut motility disorders [ 13 ]. Additionally, a miR-101-3p inhibitor in combination with chemotherapeutic agents has been shown to effectively reduce CRC cell proliferation [ 172 ]. Further, miR-221 is another possible target for many different types of cancer, as its expression is increased in glioblastoma, osteosarcoma, CRC, etc. [ 173 , 174 , 175 ]. The therapeutic potential of miRNAs is limitless and will likely lead to improved treatment options for many different diseases, particularly ones with multiple underlying pathophysiological mechanisms.

2.5. Aptamer Therapeutics and Functional Implications

Aptamers are single-stranded RNA, DNA, or RNA-DNA hybrids that have often been classified as chemical antibodies. They often are around 20–100 base pairs long and fold into specific tertiary structures that allow them to specifically bind to their respective targets ( Figure 1 E) [ 176 ]. Aptamers can be designed to target carbohydrates, peptides, proteins, and various other molecules, making them an attractive therapeutic option for various diseases.

Aptamers are generated using the systematic evolution of ligands by exponential enrichment (SELEX) method. This procedure uses a randomized library that contains ~4 # of nucleotides individual sequences that can be tested simultaneously [ 177 ]. While aptamers have been synthesized containing 8–228 nucleotides, most are around 20 nucleotides long [ 178 ]. This large library allows for trillions of sequences to be tested to find ones that are able to bind the target molecule. These sequences then continue on to further rounds of selection, thus increasing the population of aptamers that are able to bind the target with high affinity. Eventually, there are specific sequences that dominate the population of library species. This process is extremely fast in comparison to traditional peptide synthesis strategies.

Aptamers can act through three main mechanisms of action: (1) aptamers that are specific to a particular cell type can deliver other therapeutic agents to the target tissue or cells; (2) aptamers can act as an agonist and thus functionally activate their target molecules; (3) aptamers work as antagonists and block the interaction of molecules in pathways associated with disease development [ 176 ].

2.5.1. FDA-Approved Aptamers

Pegaptanib was the first-ever FDA-approved aptamer and is used for the treatment of neovascular age-related macular degeneration. This disease is characterized by retinal degeneration, causing vision loss [ 179 ]. Increased vascular endothelial growth factor (VEGF) has been associated with this condition [ 180 ]. Therefore, anti-VEGF treatment was thought to be an efficient method for treating this disease. Pegaptanib was found to have high affinity for VEGF and caused it to be sequestered, preventing it from binding to its receptor. After successful clinical trials showing that pegaptanib improved or halted vision loss [ 28 ], it was approved for use by the FDA in 2004.

Defibrotide was approved by the FDA in 2020 for the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome [ 29 ]. This can be a life-threatening complication caused by chemotherapy and hematopoietic stem cell transplant (HSCT) conditioning [ 181 ]. Defibrotide has been reported to stabilize endothelial cells via reduced endothelial cell activation [ 182 ]. This protects endothelial cells from further damage and rescues this condition. Both FDA-approved medicines have minimal side effects and highlight the promise of aptamer-based therapeutics.

2.5.2. Aptamers in Clinical Trials

NOX-A12 is an RNA aptamer that is in clinical trials for the treatment of various types of cancers, for example, pancreatic cancer, colorectal cancer, and multiple myeloma [ 50 , 183 , 184 ]. This drug works by neutralizing CXCL12, which leads to an increase in circulating tumor-infiltrating T-cells [ 185 ]. To date, clinical trials have demonstrated the potential of NOX-A12 in the treatment of various types of cancer [ 50 , 183 , 184 ].

NOX-E36 has completed Phase I clinical trials for the treatment of diabetes and albuminuria ( NCT01547897 ). Increased HbA1c and albumin/creatinine ratio (ACR) are hallmarks of these conditions. NOX-E36 has been shown to reduce these levels in patients with diabetes and albuminuria [ 51 ] by binding to CCL2, a pro-inflammatory cytokine, with high affinity [ 186 ]. Therefore, this therapeutic drug may have beneficial effects on the treatment of diabetes and related conditions.

There are other aptamers in clinical trials for various human diseases that are composed of DNA. For example, AS1411 is a DNA aptamer for the treatment of cancers such as acute myeloid leukemia [ 187 ]. However, since the purpose of this review is to highlight the potential of RNA therapeutics, we will not discuss these aptamers in detail.

3. Conclusions and Future Perspectives

RNA molecules are multi-functional and are extremely versatile. State-of-the-art studies have demonstrated considerable promise for the clinical use of RNA therapeutics to treat and prevent human diseases. Further, RNA therapeutics are relatively cheaper, easier, and faster to develop than traditional protein- and small molecule-based drugs. RNA therapeutic approaches vary in how they treat different clinical conditions. For example, siRNAs are highly specific with only one mRNA target; therefore, they are good for the treatment of diseases where pathologies are caused by the alternations of only one single gene. However, miRNAs have the virtue of targeting multiple mRNAs; consequently, they are suitable for the treatment of diseases in which various pathologies and/or alternations of many genes are involved. The current challenges for RNA therapeutics include: (i) Cell specificity—ideally, the best RNA therapeutic molecule would have on-target cell specificity without off-target and undesired on-target effects. (ii) Cell-specific delivery agents—one of the most significant challenges in RNA therapeutics is efficient and stable delivery of the molecule to the cell type of interest and being functionally active to perform their role.

Clinical trials should focus heavily on early study design to prevent possible adverse outcomes such as acute toxicity. Additionally, this early study design should focus on in vivo functional assays rather than in vitro functional assays alone. Further, the most critical step in RNA therapeutics development is to compare the clinical outcomes in their ability to fix the mechanistic parameters. To accomplish this goal, the RNA therapeutic candidates must be rigorously examined, particularly for their immune tolerance, pharmacokinetics, and pharmacodynamics. However, RNA therapeutic agents are likely developed based on the cellular and molecular mechanisms underlying pathologies of the diseases; thus, these molecules are placed in a prime position for future clinical trials. The current knowledge gaps warrant a modern approach to better understand the pathologies at the cellular and molecular level that will enable us to tackle the therapeutic approaches to treat the disease, not only improving the symptomology, but also fixing the exact cause.

While there are currently challenges to RNA therapeutic development, unprecedented interdisciplinary approaches and promising developments in modern science, along with improved early study design for clinical trials, will overcome these obstacles in the foreseeable future. This will provide substantial hope for the clinical utility of RNA therapeutics for different disease conditions and lead to a better quality of life for millions of patients.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author Contributions

H.Z. and R.S. reviewed the literature and drafted the original manuscript. S.R. critically revised the manuscript and provided important intellectual directives. H.Z. and R.S. contributed equally as co-first authors to this study. All authors have read and agreed to the published version of the manuscript.

Funding

This review received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 1998 391 806 811 10.1038/35888 9486653

2. Winkle M. El-Daly S.M. Fabbri M. Calin G.A. Noncoding RNA therapeutics—Challenges and potential solutions Nat. Rev. Drug Discov. 2021 20 629 651 10.1038/s41573-021-00219-z 34145432 PMC8212082

3. Rand T.A. Ginalski K. Grishin N.V. Wang X. Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity Proc. Natl. Acad. Sci. USA 2004 101 14385 14389 10.1073/pnas.0405913101 15452342 PMC521941

4. Singh R. Zogg H. Ro S. Role of microRNAs in Disorders of Gut-Brain Interactions: Clinical Insights and Therapeutic Alternatives J. Pers. Med. 2021 11 1021 10.3390/jpm11101021 34683162 PMC8541612

5. Matranga C. Tomari Y. Shin C. Bartel D.P. Zamore P.D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes Cell 2005 123 607 620 10.1016/j.cell.2005.08.044 16271386

6. Zhong L. Li Y. Xiong L. Wang W. Wu M. Yuan T. Yang W. Tian C. Miao Z. Wang T. Small molecules in targeted cancer therapy: Advances, challenges, and future perspectives Signal Transduct. Target. Ther. 2021 6 201 10.1038/s41392-021-00572-w 34054126 PMC8165101

7. Lu H. Zhou Q. He J. Jiang Z. Peng C. Tong R. Shi J. Recent advances in the development of protein-protein interactions modulators: Mechanisms and clinical trials Signal Transduct. Target. Ther. 2020 5 213 10.1038/s41392-020-00315-3 32968059 PMC7511340

8. Dimitrov D.S. Therapeutic proteins Methods Mol. Biol. 2012 899 1 26 10.1007/978-1-61779-921-1_1 22735943 PMC6988726

9. Bruno B.J. Miller G.D. Lim C.S. Basics and recent advances in peptide and protein drug delivery Ther. Deliv. 2013 4 1443 1467 10.4155/tde.13.104 24228993 PMC3956587

10. Bajan S. Hutvagner G. RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs Cells 2020 9 137 10.3390/cells9010137 PMC7016530 31936122

11. Komatsu H. Innovative Therapeutic Approaches for Huntington’s Disease: From Nucleic Acids to GPCR-Targeting Small Molecules Front. Cell Neurosci. 2021 15 785703 10.3389/fncel.2021.785703 34899193 PMC8662694

12. Hanna J. Hossain G.S. Kocerha J. The Potential for microRNA Therapeutics and Clinical Research Front. Genet. 2019 10 478 10.3389/fgene.2019.00478 31156715 PMC6532434

13. Singh R. Ha S.E. Wei L. Jin B. Zogg H. Poudrier S.M. Jorgensen B.G. Park C. Ronkon C.F. Bartlett A. miR-10b-5p Rescues Diabetes and Gastrointestinal Dysmotility Gastroenterology 2021 160 1662 1678.e18 10.1053/j.gastro.2020.12.062 33421511 PMC8532043

14. Vitravene Study G. A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS Am. J. Ophthalmol. 2002 133 467 474 10.1016/s0002-9394(02)01327-2 11931780

15. Santos R.D. Raal F.J. Donovan J.M. Cromwell W.C. Mipomersen preferentially reduces small low-density lipoprotein particle number in patients with hypercholesterolemia J. Clin. Lipidol. 2015 9 201 209 10.1016/j.jacl.2014.12.008 25911076

16. Mercuri E. Darras B.T. Chiriboga C.A. Day J.W. Campbell C. Connolly A.M. Iannaccone S.T. Kirschner J. Kuntz N.L. Saito K. Nusinersen versus Sham Control in Later-Onset Spinal Muscular Atrophy N. Engl. J. Med. 2018 378 625 635 10.1056/NEJMoa1710504 29443664

17. Mendell J.R. Rodino-Klapac L.R. Sahenk Z. Roush K. Bird L. Lowes L.P. Alfano L. Gomez A.M. Lewis S. Kota J. Eteplirsen for the treatment of Duchenne muscular dystrophy Ann. Neurol. 2013 74 637 647 10.1002/ana.23982 23907995

18. Benson M.D. Waddington-Cruz M. Berk J.L. Polydefkis M. Dyck P.J. Wang A.K. Plante-Bordeneuve V. Barroso F.A. Merlini G. Obici L. Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis N. Engl. J. Med. 2018 379 22 31 10.1056/NEJMoa1716793 29972757 PMC12611561

19. Scaglioni D. Catapano F. Ellis M. Torelli S. Chambers D. Feng L. Beck M. Sewry C. Monforte M. Harriman S. The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy Acta Neuropathol. Commun. 2021 9 7 10.1186/s40478-020-01106-1 33407808 PMC7789286

20. Kim J. Hu C. Moufawad El Achkar C. Black L.E. Douville J. Larson A. Pendergast M.K. Goldkind S.F. Lee E.A. Kuniholm A. Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease N. Engl. J. Med. 2019 381 1644 1652 10.1056/NEJMoa1813279 31597037 PMC6961983

21. Shirley M. Casimersen: First Approval Drugs 2021 81 875 879 10.1007/s40265-021-01512-2 33861387

22. Wagner K.R. Kuntz N.L. Koenig E. East L. Upadhyay S. Han B. Shieh P.B. Safety, tolerability, and pharmacokinetics of casimersen in patients with Duchenne muscular dystrophy amenable to exon 45 skipping: A randomized, double-blind, placebo-controlled, dose-titration trial Muscle Nerve 2021 64 285 292 10.1002/mus.27347 34105177 PMC9290993

23. Clemens P.R. Rao V.K. Connolly A.M. Harper A.D. Mah J.K. Smith E.C. McDonald C.M. Zaidman C.M. Morgenroth L.P. Osaki H. Safety, Tolerability, and Efficacy of Viltolarsen in Boys With Duchenne Muscular Dystrophy Amenable to Exon 53 Skipping: A Phase 2 Randomized Clinical Trial JAMA Neurol. 2020 77 982 991 10.1001/jamaneurol.2020.1264 32453377 PMC7251505

24. Adams D. Gonzalez-Duarte A. O’Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis N. Engl. J. Med. 2018 379 11 21 10.1056/NEJMoa1716153 29972753

25. Balwani M. Sardh E. Ventura P. Peiro P.A. Rees D.C. Stolzel U. Bissell D.M. Bonkovsky H.L. Windyga J. Anderson K.E. Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria N. Engl. J. Med. 2020 382 2289 2301 10.1056/NEJMoa1913147 32521132

26. Liebow A. Li X. Racie T. Hettinger J. Bettencourt B.R. Najafian N. Haslett P. Fitzgerald K. Holmes R.P. Erbe D. An Investigational RNAi Therapeutic Targeting Glycolate Oxidase Reduces Oxalate Production in Models of Primary Hyperoxaluria J. Am. Soc. Nephrol. 2017 28 494 503 10.1681/ASN.2016030338 27432743 PMC5280024

27. Lamb Y.N. Inclisiran: First Approval Drugs 2021 81 389 395 10.1007/s40265-021-01473-6 33620677 PMC7900795

28. Gragoudas E.S. Adamis A.P. Cunningham E.T. Jr. Feinsod M. Guyer D.R. Group V.I.S.i.O.N.C.T. Pegaptanib for neovascular age-related macular degeneration N. Engl. J. Med. 2004 351 2805 2816 10.1056/NEJMoa042760 15625332

29. Richardson P.G. Smith A.R. Triplett B.M. Kernan N.A. Grupp S.A. Antin J.H. Lehmann L. Shore T. Iacobelli M. Miloslavsky M. Defibrotide for Patients with Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome: Interim Results from a Treatment IND Study Biol. Blood Marrow Transplant. 2017 23 997 1004 10.1016/j.bbmt.2017.03.008 28285079

30. Lee T.B. Yang K. Ko H.J. Shim J.R. Choi B.H. Lee J.H. Ryu J.H. Successful defibrotide treatment of a patient with veno-occlusive disease after living-donor liver transplantation: A case report Medicine 2021 100 e26463 10.1097/MD.0000000000026463 34160449 PMC8238349

31. Polack F.P. Thomas S.J. Kitchin N. Absalon J. Gurtman A. Lockhart S. Perez J.L. Perez Marc G. Moreira E.D. Zerbini C. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine N. Engl. J. Med. 2020 383 2603 2615 10.1056/NEJMoa2034577 33301246 PMC7745181

32. Baden L.R. El Sahly H.M. Essink B. Kotloff K. Frey S. Novak R. Diemert D. Spector S.A. Rouphael N. Creech C.B. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine N. Engl. J. Med. 2021 384 403 416 10.1056/NEJMoa2035389 33378609 PMC7787219

33. Friedberg J.W. Kim H. McCauley M. Hessel E.M. Sims P. Fisher D.C. Nadler L.M. Coffman R.L. Freedman A.S. Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: Increased interferon-alpha/beta-inducible gene expression, without significant toxicity Blood 2005 105 489 495 10.1182/blood-2004-06-2156 15358617

34. Friedberg J.W. Kelly J.L. Neuberg D. Peterson D.R. Kutok J.L. Salloum R. Brenn T. Fisher D.C. Ronan E. Dalton V. Phase II study of a TLR-9 agonist (1018 ISS) with rituximab in patients with relapsed or refractory follicular lymphoma Br. J. Haematol. 2009 146 282 291 10.1111/j.1365-2141.2009.07773.x 19519691 PMC2747261

35. Chi K.N. Yu E.Y. Jacobs C. Bazov J. Kollmannsberger C. Higano C.S. Mukherjee S.D. Gleave M.E. Stewart P.S. Hotte S.J. A phase I dose-escalation study of apatorsen (OGX-427), an antisense inhibitor targeting heat shock protein 27 (Hsp27), in patients with castration-resistant prostate cancer and other advanced cancers Ann. Oncol. 2016 27 1116 1122 10.1093/annonc/mdw068 27022067

36. Cortes J. Kantarjian H. Ball E.D. Dipersio J. Kolitz J.E. Fernandez H.F. Goodman M. Borthakur G. Baer M.R. Wetzler M. Phase 2 randomized study of p53 antisense oligonucleotide (cenersen) plus idarubicin with or without cytarabine in refractory and relapsed acute myeloid leukemia Cancer 2012 118 418 427 10.1002/cncr.26292 21717444 PMC4977021

37. De Velasco M.A. Kura Y. Sakai K. Hatanaka Y. Davies B.R. Campbell H. Klein S. Kim Y. MacLeod A.R. Sugimoto K. Targeting castration-resistant prostate cancer with androgen receptor antisense oligonucleotide therapy JCI Insight 2019 4 e122688 10.1172/jci.insight.122688 PMC6777919 31484823

38. Laskin J.J. Nicholas G. Lee C. Gitlitz B. Vincent M. Cormier Y. Stephenson J. Ung Y. Sanborn R. Pressnail B. Phase I/II trial of custirsen (OGX-011), an inhibitor of clusterin, in combination with a gemcitabine and platinum regimen in patients with previously untreated advanced non-small cell lung cancer J. Thorac. Oncol. 2012 7 579 586 10.1097/JTO.0b013e31823f459c 22198426

39. Chi K.N. Eisenhauer E. Fazli L. Jones E.C. Goldenberg S.L. Powers J. Tu D. Gleave M.E. A phase I pharmacokinetic and pharmacodynamic study of OGX-011, a 2′-methoxyethyl antisense oligonucleotide to clusterin, in patients with localized prostate cancer J. Natl. Cancer Inst. 2005 97 1287 1296 10.1093/jnci/dji252 16145049

40. El Dika I. Lim H.Y. Yong W.P. Lin C.C. Yoon J.H. Modiano M. Freilich B. Choi H.J. Chao T.Y. Kelley R.K. An Open-Label, Multicenter, Phase I, Dose Escalation Study with Phase II Expansion Cohort to Determine the Safety, Pharmacokinetics, and Preliminary Antitumor Activity of Intravenous TKM-080301 in Subjects with Advanced Hepatocellular Carcinoma Oncologist 2019 24 747–e218 10.1634/theoncologist.2018-0838 30598500 PMC6656521

41. Steegmaier M. Hoffmann M. Baum A. Lenart P. Petronczki M. Krssak M. Gurtler U. Garin-Chesa P. Lieb S. Quant J. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo Curr. Biol. 2007 17 316 322 10.1016/j.cub.2006.12.037 17291758

42. Schultheis B. Strumberg D. Santel A. Vank C. Gebhardt F. Keil O. Lange C. Giese K. Kaufmann J. Khan M. First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors J. Clin. Oncol. 2014 32 4141 4148 10.1200/JCO.2013.55.0376 25403217

43. Schultheis B. Strumberg D. Kuhlmann J. Wolf M. Link K. Seufferlein T. Kaufmann J. Feist M. Gebhardt F. Khan M. Safety, Efficacy and Pharcacokinetics of Targeted Therapy with The Liposomal RNA Interference Therapeutic Atu027 Combined with Gemcitabine in Patients with Pancreatic Adenocarcinoma. A Randomized Phase Ib/IIa Study Cancers 2020 12 3130 10.3390/cancers12113130 33114652 PMC7693593

44. Golan T. Khvalevsky E.Z. Hubert A. Gabai R.M. Hen N. Segal A. Domb A. Harari G. David E.B. Raskin S. RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients Oncotarget 2015 6 24560 24570 10.18632/oncotarget.4183 26009994 PMC4695206

45. Zorde Khvalevsky E. Gabai R. Rachmut I.H. Horwitz E. Brunschwig Z. Orbach A. Shemi A. Golan T. Domb A.J. Yavin E. Mutant KRAS is a druggable target for pancreatic cancer Proc. Natl. Acad. Sci. USA 2013 110 20723 20728 10.1073/pnas.1314307110 24297898 PMC3870687

46. Cho H. Kaelin W.G. Targeting HIF2 in Clear Cell Renal Cell Carcinoma Cold Spring Harb. Symp. Quant. Biol. 2016 81 113 121 10.1101/sqb.2016.81.030833 27932568

47. Triozzi P. Kooshki M. Alistar A. Bitting R. Neal A. Lametschwandtner G. Loibner H. Phase I clinical trial of adoptive cellular immunotherapy with APN401 in patients with solid tumors J. ImmunoTherapy Cancer 2015 3 175 10.1186/2051-1426-3-S2-P175

48. Sekijima Y. Wiseman R.L. Matteson J. Hammarstrom P. Miller S.R. Sawkar A.R. Balch W.E. Kelly J.W. The biological and chemical basis for tissue-selective amyloid disease Cell 2005 121 73 85 10.1016/j.cell.2005.01.018 15820680

49. Habtemariam B.A. Karsten V. Attarwala H. Goel V. Melch M. Clausen V.A. Garg P. Vaishnaw A.K. Sweetser M.T. Robbie G.J. Single-Dose Pharmacokinetics and Pharmacodynamics of Transthyretin Targeting N -acetylgalactosamine-Small Interfering Ribonucleic Acid Conjugate, Vutrisiran, in Healthy Subjects Clin. Pharmacol. Ther. 2021 109 372 382 10.1002/cpt.1974 32599652

50. Park E.J. Choi J. Lee K.C. Na D.H. Emerging PEGylated non-biologic drugs Expert Opin. Emerg. Drugs 2019 24 107 119 10.1080/14728214.2019.1604684 30957581

51. Menne J. Eulberg D. Beyer D. Baumann M. Saudek F. Valkusz Z. Wiecek A. Haller H. Emapticap Study G. C-C motif-ligand 2 inhibition with emapticap pegol (NOX-E36) in type 2 diabetic patients with albuminuria Nephrol. Dial. Transplant. 2017 32 307 315 10.1093/ndt/gfv459 28186566 PMC5410979

52. Kremsner P.G. Ahuad Guerrero R.A. Arana-Arri E. Aroca Martinez G.J. Bonten M. Chandler R. Corral G. De Block E.J.L. Ecker L. Gabor J.J. Efficacy and safety of the CVnCoV SARS-CoV-2 mRNA vaccine candidate in ten countries in Europe and Latin America (HERALD): A randomised, observer-blinded, placebo-controlled, phase 2b/3 trial Lancet Infect. Dis. 2021 22 329 340 10.1016/S1473-3099(21)00677-0 34826381 PMC8610426

53. Anttila V. Saraste A. Knuuti J. Jaakkola P. Hedman M. Svedlund S. Lagerstrom-Fermer M. Kjaer M. Jeppsson A. Gan L.M. Synthetic mRNA Encoding VEGF-A in Patients Undergoing Coronary Artery Bypass Grafting: Design of a Phase 2a Clinical Trial Mol. Ther. Methods Clin. Dev. 2020 18 464 472 10.1016/j.omtm.2020.05.030 32728595 PMC7369517

54. Yan Z. McCray P.B. Jr. Engelhardt J.F. Advances in gene therapy for cystic fibrosis lung disease Hum. Mol. Genet 2019 28 R88 R94 10.1093/hmg/ddz139 31332440 PMC6796993

55. Chandler R.J. Venditti C.P. Gene Therapy for Methylmalonic Acidemia: Past, Present, and Future Hum. Gene Ther. 2019 30 1236 1244 10.1089/hum.2019.113 31303064 PMC6763959

56. An D. Schneller J.L. Frassetto A. Liang S. Zhu X. Park J.S. Theisen M. Hong S.J. Zhou J. Rajendran R. Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia Cell Rep. 2017 21 3548 3558 10.1016/j.celrep.2017.11.081 29262333 PMC9667413

57. Sahin U. Oehm P. Derhovanessian E. Jabulowsky R.A. Vormehr M. Gold M. Maurus D. Schwarck-Kokarakis D. Kuhn A.N. Omokoko T. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma Nature 2020 585 107 112 10.1038/s41586-020-2537-9 32728218

58. Janssen H.L. Reesink H.W. Lawitz E.J. Zeuzem S. Rodriguez-Torres M. Patel K. van der Meer A.J. Patick A.K. Chen A. Zhou Y. Treatment of HCV infection by targeting microRNA N. Engl. J. Med. 2013 368 1685 1694 10.1056/NEJMoa1209026 23534542

59. Gomez I.G. MacKenna D.A. Johnson B.G. Kaimal V. Roach A.M. Ren S. Nakagawa N. Xin C. Newitt R. Pandya S. Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways J. Clin. Investig. 2015 125 141 156 10.1172/JCI75852 25415439 PMC4382246

60. Bedewy A.M.L. Elmaghraby S.M. Shehata A.A. Kandil N.S. Prognostic Value of miRNA-155 Expression in B-Cell Non-Hodgkin Lymphoma Turk. J. Haematol. 2017 34 207 212 10.4274/tjh.2016.0286 28148469 PMC5544039

61. Bonauer A. Carmona G. Iwasaki M. Mione M. Koyanagi M. Fischer A. Burchfield J. Fox H. Doebele C. Ohtani K. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice Science 2009 324 1710 1713 10.1126/science.1174381 19460962

62. Trajkovski M. Hausser J. Soutschek J. Bhat B. Akin A. Zavolan M. Heim M.H. Stoffel M. MicroRNAs 103 and 107 regulate insulin sensitivity Nature 2011 474 649 653 10.1038/nature10112 21654750

63. Lee E.C. Valencia T. Allerson C. Schairer A. Flaten A. Yheskel M. Kersjes K. Li J. Gatto S. Takhar M. Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease Nat. Commun. 2019 10 4148 10.1038/s41467-019-11918-y 31515477 PMC6742637

64. Taubel J. Hauke W. Rump S. Viereck J. Batkai S. Poetzsch J. Rode L. Weigt H. Genschel C. Lorch U. Novel antisense therapy targeting microRNA-132 in patients with heart failure: Results of a first-in-human Phase 1b randomized, double-blind, placebo-controlled study Eur. Heart J. 2021 42 178 188 10.1093/eurheartj/ehaa898 33245749 PMC7954267

65. Van Zandwijk N. Pavlakis N. Kao S.C. Linton A. Boyer M.J. Clarke S. Huynh Y. Chrzanowska A. Fulham M.J. Bailey D.L. Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: A first-in-man, phase 1, open-label, dose-escalation study Lancet Oncol. 2017 18 1386 1396 10.1016/S1470-2045(17)30621-6 28870611

66. Cushing L. Kuang P.P. Qian J. Shao F. Wu J. Little F. Thannickal V.J. Cardoso W.V. Lu J. miR-29 is a major regulator of genes associated with pulmonary fibrosis Am. J. Respir. Cell Mol. Biol. 2011 45 287 294 10.1165/rcmb.2010-0323OC 20971881 PMC3175558

67. Beg M.S. Brenner A.J. Sachdev J. Borad M. Kang Y.K. Stoudemire J. Smith S. Bader A.G. Kim S. Hong D.S. Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors Investig. New Drugs 2017 35 180 188 10.1007/s10637-016-0407-y 27917453 PMC5893501

68. Mignone F. Gissi C. Liuni S. Pesole G. Untranslated regions of mRNAs Genome Biol. 2002 3 REVIEWS0004 10.1186/gb-2002-3-3-reviews0004 11897027 PMC139023

69. Leppek K. Das R. Barna M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them Nat. Rev. Mol. Cell Biol. 2018 19 158 174 10.1038/nrm.2017.103 29165424 PMC5820134

70. Da Silva Sanchez A. Paunovska K. Cristian A. Dahlman J.E. Treating Cystic Fibrosis with mRNA and CRISPR Hum. Gene Ther. 2020 31 940 955 10.1089/hum.2020.137 32799680 PMC7495921

71. Musunuru K. Chadwick A.C. Mizoguchi T. Garcia S.P. DeNizio J.E. Reiss C.W. Wang K. Iyer S. Dutta C. Clendaniel V. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates Nature 2021 593 429 434 10.1038/s41586-021-03534-y 34012082

72. Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Direct gene transfer into mouse muscle in vivo Science 1990 247 1465 1468 10.1126/science.1690918 1690918

73. Jirikowski G.F. Sanna P.P. Maciejewski-Lenoir D. Bloom F.E. Reversal of diabetes insipidus in Brattleboro rats: Intrahypothalamic injection of vasopressin mRNA Science 1992 255 996 998 10.1126/science.1546298 1546298

74. Martinon F. Krishnan S. Lenzen G. Magne R. Gomard E. Guillet J.G. Levy J.P. Meulien P. Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA Eur. J. Immunol. 1993 23 1719 1722 10.1002/eji.1830230749 8325342

75. Aldrich C. Leroux-Roels I. Huang K.B. Bica M.A. Loeliger E. Schoenborn-Kellenberger O. Walz L. Leroux-Roels G. von Sonnenburg F. Oostvogels L. Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: A phase 1 trial Vaccine 2021 39 1310 1318 10.1016/j.vaccine.2020.12.070 33487468 PMC7825876

76. Petsch B. Schnee M. Vogel A.B. Lange E. Hoffmann B. Voss D. Schlake T. Thess A. Kallen K.J. Stitz L. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection Nat. Biotechnol. 2012 30 1210 1216 10.1038/nbt.2436 23159882

77. Aliprantis A.O. Shaw C.A. Griffin P. Farinola N. Railkar R.A. Cao X. Liu W. Sachs J.R. Swenson C.J. Lee H. A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults Hum. Vaccin Immunother. 2021 17 1248 1261 10.1080/21645515.2020.1829899 33121346 PMC8078688

78. Schnee M. Vogel A.B. Voss D. Petsch B. Baumhof P. Kramps T. Stitz L. An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs PLoS Negl. Trop. Dis. 2016 10 e0004746 10.1371/journal.pntd.0004746 27336830 PMC4918980

79. Medina-Magues L.G. Gergen J. Jasny E. Petsch B. Lopera-Madrid J. Medina-Magues E.S. Salas-Quinchucua C. Osorio J.E. mRNA Vaccine Protects against Zika Virus Vaccines 2021 9 1464 10.3390/vaccines9121464 34960211 PMC8707647

80. John S. Yuzhakov O. Woods A. Deterling J. Hassett K. Shaw C.A. Ciaramella G. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity Vaccine 2018 36 1689 1699 10.1016/j.vaccine.2018.01.029 29456015

81. Webster H. Valencia S. Kumar A. Chan C. Dennis M. Roark H. Woods A. John S. Carfi A. Permar S.R. Pre-existing immunity to cytomegalovirus in macaques influences human CMV vaccine responses in preclinical models Vaccine 2021 39 5358 5367 10.1016/j.vaccine.2021.08.011 34393017 PMC11694353

82. Conklin L.D. McAninch R.E. Schulz D. Kaluza G.L. LeMaire S.A. Coselli J.S. Raizner A.E. Sutton R.E. HIV-based vectors and angiogenesis following rabbit hindlimb ischemia J. Surg. Res. 2005 123 55 66 10.1016/j.jss.2004.06.010 15652951

83. Giacca M. Zacchigna S. VEGF gene therapy: Therapeutic angiogenesis in the clinic and beyond Gene Ther. 2012 19 622 629 10.1038/gt.2012.17 22378343

84. Gaffney M.M. Hynes S.O. Barry F. O’Brien T. Cardiovascular gene therapy: Current status and therapeutic potential Br. J. Pharmacol. 2007 152 175 188 10.1038/sj.bjp.0707315 17558439 PMC1978263

85. Butnariu L.I. Tarca E. Cojocaru E. Rusu C. Moisa S.M. Leon Constantin M.M. Gorduza E.V. Trandafir L.M. Genetic Modifying Factors of Cystic Fibrosis Phenotype: A Challenge for Modern Medicine J. Clin. Med. 2021 10 5821 10.3390/jcm10245821 34945117 PMC8707808

86. Fraser J.L. Venditti C.P. Methylmalonic and propionic acidemias: Clinical management update Curr. Opin. Pediatr. 2016 28 682 693 10.1097/MOP.0000000000000422 27653704 PMC5393914

87. Connolly B. Isaacs C. Cheng L. Asrani K.H. Subramanian R.R. SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency J. Nucleic Acids 2018 2018 8247935 10.1155/2018/8247935 30009048 PMC6020464

88. Cao J. An D. Galduroz M. Zhuo J. Liang S. Eybye M. Frassetto A. Kuroda E. Funahashi A. Santana J. mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency Mol. Ther. 2019 27 1242 1251 10.1016/j.ymthe.2019.04.017 31056400 PMC6612659

89. Porciuncula A. Morgado M. Gupta R. Syrigos K. Meehan R. Zacharek S.J. Frederick J.P. Schalper K.A. Spatial Mapping and Immunomodulatory Role of the OX40/OX40L Pathway in Human Non-Small Cell Lung Cancer Clin. Cancer Res. 2021 27 6174 6183 10.1158/1078-0432.CCR-21-0987 34518312 PMC8595671

90. Hewitt S.L. Bailey D. Zielinski J. Apte A. Musenge F. Karp R. Burke S. Garcon F. Mishra A. Gurumurthy S. Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment Clin. Cancer Res. 2020 26 6284 6298 10.1158/1078-0432.CCR-20-0472 32817076

91. Zhu X. Yin L. Theisen M. Zhuo J. Siddiqui S. Levy B. Presnyak V. Frassetto A. Milton J. Salerno T. Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates Am. J. Hum. Genet 2019 104 625 637 10.1016/j.ajhg.2019.02.003 30879639 PMC6451694

92. Evers M.M. Toonen L.J. van Roon-Mom W.M. Antisense oligonucleotides in therapy for neurodegenerative disorders Adv. Drug Deliv. Rev. 2015 87 90 103 10.1016/j.addr.2015.03.008 25797014

93. Stephenson M.L. Zamecnik P.C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide Proc. Natl. Acad. Sci. USA 1978 75 285 288 10.1073/pnas.75.1.285 75546 PMC411231

94. Roberts T.C. Langer R. Wood M.J.A. Advances in oligonucleotide drug delivery Nat. Rev. Drug Discov. 2020 19 673 694 10.1038/s41573-020-0075-7 32782413 PMC7419031

95. Wu H. Lima W.F. Zhang H. Fan A. Sun H. Crooke S.T. Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs J. Biol. Chem. 2004 279 17181 17189 10.1074/jbc.M311683200 14960586

96. Wan L. Dreyfuss G. Splicing-Correcting Therapy for SMA Cell 2017 170 5 10.1016/j.cell.2017.06.028 28666123

97. Lim K.H. Han Z. Jeon H.Y. Kach J. Jing E. Weyn-Vanhentenryck S. Downs M. Corrionero A. Oh R. Scharner J. Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression Nat. Commun. 2020 11 3501 10.1038/s41467-020-17093-9 32647108 PMC7347940

98. Ward A.J. Norrbom M. Chun S. Bennett C.F. Rigo F. Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides Nucleic Acids Res. 2014 42 5871 5879 10.1093/nar/gku184 24589581 PMC4027159

99. Boiziau C. Kurfurst R. Cazenave C. Roig V. Thuong N.T. Toulme J.J. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism Nucleic Acids Res. 1991 19 1113 1119 10.1093/nar/19.5.1113 1850511 PMC333789

100. Liang X.H. Shen W. Sun H. Migawa M.T. Vickers T.A. Crooke S.T. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames Nat. Biotechnol. 2016 34 875 880 10.1038/nbt.3589 27398791

101. Vickers T.A. Wyatt J.R. Burckin T. Bennett C.F. Freier S.M. Fully modified 2′ MOE oligonucleotides redirect polyadenylation Nucleic Acids Res. 2001 29 1293 1299 10.1093/nar/29.6.1293 11238995 PMC29745

102. Piascik P. Fomiversen sodium approved to treat CMV retinitis J. Am. Pharm. Assoc. 1999 39 84 85 10.1016/S1086-5802(16)30428-4 9990192

103. Jabs D.A. Griffiths P.D. Fomivirsen for the treatment of cytomegalovirus retinitis Am. J. Ophthalmol. 2002 133 552 556 10.1016/S0002-9394(02)01325-9 11931791

104. Won J.I. Zhang J. Tecson K.M. McCullough P.A. Balancing Low-density Lipoprotein Cholesterol Reduction and Hepatotoxicity With Lomitapide Mesylate and Mipomersen in Patients With Homozygous Familial Hypercholesterolemia Rev. Cardiovasc. Med. 2017 18 21 28 28509890 10.3909/ricm0834

105. Cesaro A. Fimiani F. Gragnano F. Moscarella E. Schiavo A. Vergara A. Akioyamen L. D’Erasmo L. Averna M. Arca M. New Frontiers in the Treatment of Homozygous Familial Hypercholesterolemia Heart Fail Clin. 2022 18 177 188 10.1016/j.hfc.2021.07.008 34776078

106. Gouni-Berthold I. Berthold H.K. Mipomersen and lomitapide: Two new drugs for the treatment of homozygous familial hypercholesterolemia Atheroscler. Suppl. 2015 18 28 34 10.1016/j.atherosclerosissup.2015.02.005 25936301

107. Aartsma-Rus A. FDA Approval of Nusinersen for Spinal Muscular Atrophy Makes 2016 the Year of Splice Modulating Oligonucleotides Nucleic Acid. Ther. 2017 27 67 69 10.1089/nat.2017.0665 28346110

108. Burr P. Reddivari A.K.R. Spinal Muscle Atrophy StatPearls Treasure Island, FL, USA 2022 32809522

109. Aartsma-Rus A. Krieg A.M. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga Nucleic Acid Ther. 2017 27 1 3 10.1089/nat.2016.0657 27929755 PMC5312460

110. Chamberlain J.R. Chamberlain J.S. Progress toward Gene Therapy for Duchenne Muscular Dystrophy Mol. Ther. 2017 25 1125 1131 10.1016/j.ymthe.2017.02.019 28416280 PMC5417844

111. De Angelis F.G. Sthandier O. Berarducci B. Toso S. Galluzzi G. Ricci E. Cossu G. Bozzoni I. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells Proc. Natl. Acad. Sci. USA 2002 99 9456 9461 10.1073/pnas.142302299 12077324 PMC123162

112. Sekijima Y. Hereditary Transthyretin Amyloidosis Adam M.P. Ardinger H.H. Pagon R.A. Wallace S.E. Bean L.J.H. Gripp K.W. Mirzaa G.M. Amemiya A. GeneReviews Seattle, WA, USA 1993

113. Mahfouz M. Maruyama R. Yokota T. Inotersen for the Treatment of Hereditary Transthyretin Amyloidosis Methods Mol. Biol. 2020 2176 87 98 10.1007/978-1-0716-0771-8_6 32865784

114. Iftikhar M. Frey J. Shohan M.J. Malek S. Mousa S.A. Current and emerging therapies for Duchenne muscular dystrophy and spinal muscular atrophy Pharmacol. Ther. 2021 220 107719 10.1016/j.pharmthera.2020.107719 33130193

115. Brudvig J.J. Weimer J.M. On the cusp of cures: Breakthroughs in Batten disease research Curr. Opin. Neurobiol. 2021 72 48 54 10.1016/j.conb.2021.08.003 34571324

116. Schmitz-Feuerhake I. Frentzel-Beyme R. Wolff R. Non-Hodgkin lymphomas and ionizing radiation: Case report and review of the literature Ann. Hematol. 2022 101 243 250 10.1007/s00277-021-04729-z 34881390 PMC8742808

117. Fernandez N.C. Lozier A. Flament C. Ricciardi-Castagnoli P. Bellet D. Suter M. Perricaudet M. Tursz T. Maraskovsky E. Zitvogel L. Dendritic cells directly trigger NK cell functions: Cross-talk relevant in innate anti-tumor immune responses in vivo Nat. Med. 1999 5 405 411 10.1038/7403 10202929

118. Banchereau J. Steinman R.M. Dendritic cells and the control of immunity Nature 1998 392 245 252 10.1038/32588 9521319

119. Yamamoto Y. Loriot Y. Beraldi E. Zhang F. Wyatt A.W. Al Nakouzi N. Mo F. Zhou T. Kim Y. Monia B.P. Generation 2.5 antisense oligonucleotides targeting the androgen receptor and its splice variants suppress enzalutamide-resistant prostate cancer cell growth Clin. Cancer Res. 2015 21 1675 1687 10.1158/1078-0432.CCR-14-1108 25634993

120. Fakhr E. Zare F. Teimoori-Toolabi L. Precise and efficient siRNA design: A key point in competent gene silencing Cancer Gene Ther. 2016 23 73 82 10.1038/cgt.2016.4 26987292

121. Song M.S. Rossi J.J. Molecular mechanisms of Dicer: Endonuclease and enzymatic activity Biochem. J. 2017 474 1603 1618 10.1042/BCJ20160759 28473628 PMC5415849

122. Iwakawa H.O. Tomari Y. Life of RISC: Formation, action, and degradation of RNA-induced silencing complex Mol. Cell 2022 82 30 43 10.1016/j.molcel.2021.11.026 34942118

123. Niaz S. The AGO proteins: An overview Biol. Chem. 2018 399 525 547 10.1515/hsz-2017-0329 29447113

124. Hoy S.M. Patisiran: First Global Approval Drugs 2018 78 1625 1631 10.1007/s40265-018-0983-6 30251172

125. Kaku M. Berk J.L. Neuropathy Associated with Systemic Amyloidosis Semin Neurol. 2019 39 578 588 10.1055/s-0039-1688994 31639841

126. De Paula Brandao P.R. Titze-de-Almeida S.S. Titze-de-Almeida R. Leading RNA Interference Therapeutics Part 2: Silencing Delta-Aminolevulinic Acid Synthase 1, with a Focus on Givosiran Mol. Diagn. Ther. 2020 24 61 68 10.1007/s40291-019-00438-6 31792921

127. Syed Y.Y. Givosiran: A Review in Acute Hepatic Porphyria Drugs 2021 81 841 848 10.1007/s40265-021-01511-3 33871817

128. Scott L.J. Keam S.J. Lumasiran: First Approval Drugs 2021 81 277 282 10.1007/s40265-020-01463-0 33405070

129. Andrisani O.M. Studach L. Merle P. Gene signatures in hepatocellular carcinoma (HCC) Semin. Cancer Biol. 2011 21 4 9 10.1016/j.semcancer.2010.09.002 20851183 PMC3030925

130. Buscail L. Bournet B. Cordelier P. Role of oncogenic KRAS in the diagnosis, prognosis and treatment of pancreatic cancer Nat. Rev. Gastroenterol. Hepatol. 2020 17 153 168 10.1038/s41575-019-0245-4 32005945

131. Wolf M.M. Kimryn Rathmell W. Beckermann K.E. Modeling clear cell renal cell carcinoma and therapeutic implications Oncogene 2020 39 3413 3426 10.1038/s41388-020-1234-3 32123314 PMC7194123

132. Krieg M. Haas R. Brauch H. Acker T. Flamme I. Plate K.H. Up-regulation of hypoxia-inducible factors HIF-1alpha and HIF-2alpha under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function Oncogene 2000 19 5435 5443 10.1038/sj.onc.1203938 11114720

133. Bachmaier K. Krawczyk C. Kozieradzki I. Kong Y.Y. Sasaki T. Oliveira-dos-Santos A. Mariathasan S. Bouchard D. Wakeham A. Itie A. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b Nature 2000 403 211 216 10.1038/35003228 10646608

134. Paolino M. Choidas A. Wallner S. Pranjic B. Uribesalgo I. Loeser S. Jamieson A.M. Langdon W.Y. Ikeda F. Fededa J.P. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells Nature 2014 507 508 512 10.1038/nature12998 24553136 PMC6258903

135. Lee R.C. Feinbaum R.L. Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 Cell 1993 75 843 854 10.1016/0092-8674(93)90529-Y 8252621

136. Wightman B. Ha I. Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans Cell 1993 75 855 862 10.1016/0092-8674(93)90530-4 8252622

137. Fabian M.R. Sonenberg N. The mechanics of miRNA-mediated gene silencing: A look under the hood of miRISC Nat. Struct. Mol. Biol. 2012 19 586 593 10.1038/nsmb.2296 22664986

138. Schirle N.T. Sheu-Gruttadauria J. MacRae I.J. Structural basis for microRNA targeting Science 2014 346 608 613 10.1126/science.1258040 25359968 PMC4313529

139. Jonas S. Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing Nat. Rev. Genet 2015 16 421 433 10.1038/nrg3965 26077373

140. Panigrahi M. Thibault P.A. Wilson J.A. miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections J. Virol. 2021 96 JVI-01903 10.1128/JVI.01903-21 PMC8865533 34908444

141. Kashtan C.E. Alport Syndrome: Achieving Early Diagnosis and Treatment Am. J. Kidney Dis. 2021 77 272 279 10.1053/j.ajkd.2020.03.026 32712016

142. Witten L. Slack F.J. miR-155 as a novel clinical target for hematological malignancies Carcinogenesis 2020 41 2 7 10.1093/carcin/bgz183 31711135

143. Seto A.G. Beatty X. Lynch J.M. Hermreck M. Tetzlaff M. Duvic M. Jackson A.L. Cobomarsen, an oligonucleotide inhibitor of miR-155, co-ordinately regulates multiple survival pathways to reduce cellular proliferation and survival in cutaneous T-cell lymphoma Br. J. Haematol. 2018 183 428 444 10.1111/bjh.15547 30125933

144. Anastasiadou E. Seto A.G. Beatty X. Hermreck M. Gilles M.E. Stroopinsky D. Pinter-Brown L.C. Pestano L. Marchese C. Avigan D. Cobomarsen, an Oligonucleotide Inhibitor of miR-155, Slows DLBCL Tumor Cell Growth In Vitro and In Vivo Clin. Cancer Res. 2021 27 1139 1149 10.1158/1078-0432.CCR-20-3139 33208342 PMC8287597

145. Abplanalp W.T. Fischer A. John D. Zeiher A.M. Gosgnach W. Darville H. Montgomery R. Pestano L. Allee G. Paty I. Efficiency and Target Derepression of Anti-miR-92a: Results of a First in Human Study Nucleic Acid Ther. 2020 30 335 345 10.1089/nat.2020.0871 32707001

146. Gallant-Behm C.L. Piper J. Dickinson B.A. Dalby C.M. Pestano L.A. Jackson A.L. A synthetic microRNA-92a inhibitor (MRG-110) accelerates angiogenesis and wound healing in diabetic and nondiabetic wounds Wound Repair Regen. 2018 26 311 323 10.1111/wrr.12660 30118158

147. Rottiers V. Naar A.M. MicroRNAs in metabolism and metabolic disorders Nat. Rev. Mol. Cell Biol. 2012 13 239 250 10.1038/nrm3313 22436747 PMC4021399

148. Kim D.Y. Park J.H. Genetic Mechanisms of ADPKD Adv. Exp. Med. Biol. 2016 933 13 22 10.1007/978-981-10-2041-4_2 27730431

149. Ucar A. Gupta S.K. Fiedler J. Erikci E. Kardasinski M. Batkai S. Dangwal S. Kumarswamy R. Bang C. Holzmann A. The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy Nat. Commun. 2012 3 1078 10.1038/ncomms2090 23011132 PMC3657998

150. Foinquinos A. Batkai S. Genschel C. Viereck J. Rump S. Gyongyosi M. Traxler D. Riesenhuber M. Spannbauer A. Lukovic D. Preclinical development of a miR-132 inhibitor for heart failure treatment Nat. Commun. 2020 11 633 10.1038/s41467-020-14349-2 32005803 PMC6994493

151. Batkai S. Genschel C. Viereck J. Rump S. Bar C. Borchert T. Traxler D. Riesenhuber M. Spannbauer A. Lukovic D. CDR132L improves systolic and diastolic function in a large animal model of chronic heart failure Eur. Heart J. 2021 42 192 201 10.1093/eurheartj/ehaa791 33089304 PMC7813625

152. Calin G.A. Dumitru C.D. Shimizu M. Bichi R. Zupo S. Noch E. Aldler H. Rattan S. Keating M. Rai K. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia Proc. Natl. Acad. Sci. USA 2002 99 15524 15529 10.1073/pnas.242606799 12434020 PMC137750

153. Bandi N. Zbinden S. Gugger M. Arnold M. Kocher V. Hasan L. Kappeler A. Brunner T. Vassella E. miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer Cancer Res. 2009 69 5553 5559 10.1158/0008-5472.CAN-08-4277 19549910

154. Bonci D. Coppola V. Musumeci M. Addario A. Giuffrida R. Memeo L. D’Urso L. Pagliuca A. Biffoni M. Labbaye C. The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities Nat. Med. 2008 14 1271 1277 10.1038/nm.1880 18931683

155. Reid G. Pel M.E. Kirschner M.B. Cheng Y.Y. Mugridge N. Weiss J. Williams M. Wright C. Edelman J.J. Vallely M.P. Restoring expression of miR-16: A novel approach to therapy for malignant pleural mesothelioma Ann. Oncol. 2013 24 3128 3135 10.1093/annonc/mdt412 24148817

156. Chava S. Reynolds C.P. Pathania A.S. Gorantla S. Poluektova L.Y. Coulter D.W. Gupta S.C. Pandey M.K. Challagundla K.B. miR-15a-5p, miR-15b-5p, and miR-16-5p inhibit tumor progression by directly targeting MYCN in neuroblastoma Mol. Oncol. 2020 14 180 196 10.1002/1878-0261.12588 31637848 PMC6944109

157. Grabowski G. Pacana M.J. Chen E. Keloid and Hypertrophic Scar Formation, Prevention, and Management: Standard Review of Abnormal Scarring in Orthopaedic Surgery J. Am. Acad. Orthop. Surg. 2020 28 e408 e414 10.5435/JAAOS-D-19-00690 32109921

158. Cheng J. Wang Y. Wang D. Wu Y. Identification of collagen 1 as a post-transcriptional target of miR-29b in skin fibroblasts: Therapeutic implication for scar reduction Am. J. Med. Sci. 2013 346 98 103 10.1097/MAJ.0b013e318267680d 23221517

159. Zhu Y. Li Z. Wang Y. Li L. Wang D. Zhang W. Liu L. Jiang H. Yang J. Cheng J. Overexpression of miR-29b reduces collagen biosynthesis by inhibiting heat shock protein 47 during skin wound healing Transl. Res. 2016 178 38 53.e36 10.1016/j.trsl.2016.07.001 27477081

160. Montgomery R.L. Yu G. Latimer P.A. Stack C. Robinson K. Dalby C.M. Kaminski N. van Rooij E. MicroRNA mimicry blocks pulmonary fibrosis EMBO Mol. Med. 2014 6 1347 1356 10.15252/emmm.201303604 25239947 PMC4287936

161. Gallant-Behm C.L. Piper J. Lynch J.M. Seto A.G. Hong S.J. Mustoe T.A. Maari C. Pestano L.A. Dalby C.M. Jackson A.L. A MicroRNA-29 Mimic (Remlarsen) Represses Extracellular Matrix Expression and Fibroplasia in the Skin J. Investig. Dermatol. 2019 139 1073 1081 10.1016/j.jid.2018.11.007 30472058

162. Liu C. Kelnar K. Liu B. Chen X. Calhoun-Davis T. Li H. Patrawala L. Yan H. Jeter C. Honorio S. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44 Nat. Med. 2011 17 211 215 10.1038/nm.2284 21240262 PMC3076220

163. Jiang L. Hermeking H. miR-34a and miR-34b/c Suppress Intestinal Tumorigenesis Cancer Res. 2017 77 2746 2758 10.1158/0008-5472.CAN-16-2183 28363996

164. Ji Q. Hao X. Zhang M. Tang W. Yang M. Li L. Xiang D. Desano J.T. Bommer G.T. Fan D. MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells PLoS ONE 2009 4 e6816 10.1371/journal.pone.0006816 19714243 PMC2729376

165. Sun D. Wu Y. Zhang S. Han Y. Shen J. Zheng W. Wei L. Liu Y. Ren L. Gu Z. Distinct roles of miR-34 family members on suppression of lung squamous cell carcinoma Biomed. Pharmacother. 2021 142 111967 10.1016/j.biopha.2021.111967 34467896

166. Schmid G. Notaro S. Reimer D. Abdel-Azim S. Duggan-Peer M. Holly J. Fiegl H. Rossler J. Wiedemair A. Concin N. Expression and promotor hypermethylation of miR-34a in the various histological subtypes of ovarian cancer BMC Cancer 2016 16 102 10.1186/s12885-016-2135-2 26879132 PMC4754861

167. Zou Y. Huang Y. Yang J. Wu J. Luo C. miR-34a is downregulated in human osteosarcoma stem-like cells and promotes invasion, tumorigenic ability and self-renewal capacity Mol. Med. Rep. 2017 15 1631 1637 10.3892/mmr.2017.6187 28260055 PMC5364984

168. Raver-Shapira N. Marciano E. Meiri E. Spector Y. Rosenfeld N. Moskovits N. Bentwich Z. Oren M. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis Mol. Cell 2007 26 731 743 10.1016/j.molcel.2007.05.017 17540598

169. Chen C. Zhao S. Karnad A. Freeman J.W. The biology and role of CD44 in cancer progression: Therapeutic implications J. Hematol. Oncol. 2018 11 64 10.1186/s13045-018-0605-5 29747682 PMC5946470

170. Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. Menssen A. Meister G. Hermeking H. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest Cell Cycle 2007 6 1586 1593 10.4161/cc.6.13.4436 17554199

171. Siu L. Brody J. Gupta S. Marabelle A. Jimeno A. Munster P. Grilley-Olson J. Rook A.H. Hollebecque A. Wong R.K.S. Safety and clinical activity of intratumoral MEDI9197 alone and in combination with durvalumab and/or palliative radiation therapy in patients with advanced solid tumors J. Immunother. Cancer 2020 8 e001095 10.1136/jitc-2020-001095 33037117 PMC7549442

172. Tao L. Xu C. Shen W. Tan J. Li L. Fan M. Sun D. Lai Y. Cheng H. HIPK3 Inhibition by Exosomal hsa-miR-101-3p Is Related to Metabolic Reprogramming in Colorectal Cancer Front. Oncol. 2021 11 758336 10.3389/fonc.2021.758336 35096570 PMC8792385

173. Chang L. Yin L. Zhang D. Wang C. Li G. Tan C. Zhang X. Su J. MicroRNA-221 Promotes Tumor Progression by Targeting HHIP in Human Glioblastoma Transl. Cancer Res. 2021 10 1073 1081 10.21037/tcr-21-99 35116434 PMC8799047

174. Zhao H. Yan P. Wang J. Zhang Y. Zhang M. Wang Z. Fu Q. Liang W. Clinical Significance of Tumor miR-21, miR-221, miR-143, and miR-106a as Biomarkers in Patients with Osteosarcoma Int. J. Biol. Markers 2019 2 184 193 10.1177/1724600819843537 31084400

175. Iida M. Hazama S. Tsunedomi R. Tanaka H. Takenouchi H. Kanekiyo S. Tokumitsu Y. Tomochika S. Tokuhisa Y. Sakamoto K. Overexpression of miR-221 and miR-222 in the Cancer Stroma Is Associated with Malignant Potential in Colorectal Cancer Oncol. Rep. 2018 40 1621 1631 10.3892/or.2018.6575 30015977

176. Zhou J. Rossi J. Aptamers as targeted therapeutics: Current potential and challenges Nat. Rev. Drug Discov. 2017 16 181 202 10.1038/nrd.2016.199 27807347 PMC5700751

177. Tuerk C. Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase Science 1990 249 505 510 10.1126/science.2200121 2200121

178. Li-Ping Z. Ge Y. Xiao-Min Z. Feng Q. Development of Aptamer Screening against Proteins and Its Applications Chin. J. Anal. Chem. 2020 48 560 572 10.1016/S1872-2040(20)60012-3

179. Ricci F. Bandello F. Navarra P. Staurenghi G. Stumpp M. Zarbin M. Neovascular Age-Related Macular Degeneration: Therapeutic Management and New-Upcoming Approaches Int. J. Mol. Sci. 2020 21 8242 10.3390/ijms21218242 PMC7662479 33153227

180. Jager R.D. Mieler W.F. Miller J.W. Age-related macular degeneration N. Engl. J. Med. 2008 358 2606 2617 10.1056/NEJMra0801537 18550876

181. Kernan N.A. Grupp S. Smith A.R. Arai S. Triplett B. Antin J.H. Lehmann L. Shore T. Ho V.T. Bunin N. Final results from a defibrotide treatment-IND study for patients with hepatic veno-occlusive disease/sinusoidal obstruction syndrome Br. J. Haematol. 2018 181 816 827 10.1111/bjh.15267 29767845 PMC6032999

182. Richardson P.G. Riches M.L. Kernan N.A. Brochstein J.A. Mineishi S. Termuhlen A.M. Arai S. Grupp S.A. Guinan E.C. Martin P.L. Phase 3 trial of defibrotide for the treatment of severe veno-occlusive disease and multi-organ failure Blood 2016 127 1656 1665 10.1182/blood-2015-10-676924 26825712 PMC4817309

183. Steurer M. Montillo M. Scarfo L. Mauro F.R. Andel J. Wildner S. Trentin L. Janssens A. Burgstaller S. Fromming A. Olaptesed pegol (NOX-A12) with bendamustine and rituximab: A phase IIa study in patients with relapsed/refractory chronic lymphocytic leukemia Haematologica 2019 104 2053 2060 10.3324/haematol.2018.205930 31097627 PMC6886437

184. Suarez-Carmona M. Williams A. Schreiber J. Hohmann N. Pruefer U. Krauss J. Jager D. Fromming A. Beyer D. Eulberg D. Combined inhibition of CXCL12 and PD-1 in MSS colorectal and pancreatic cancer: Modulation of the microenvironment and clinical effects J. Immunother. Cancer 2021 9 e002505 10.1136/jitc-2021-002505 34607895 PMC8491418

185. Zboralski D. Hoehlig K. Eulberg D. Fromming A. Vater A. Increasing Tumor-Infiltrating T Cells through Inhibition of CXCL12 with NOX-A12 Synergizes with PD-1 Blockade Cancer Immunol. Res. 2017 5 950 956 10.1158/2326-6066.CIR-16-0303 28963140

186. Oberthur D. Achenbach J. Gabdulkhakov A. Buchner K. Maasch C. Falke S. Rehders D. Klussmann S. Betzel C. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2 Nat. Commun. 2015 6 6923 10.1038/ncomms7923 25901662 PMC4423205

187. Mongelard F. Bouvet P. AS-1411, a guanosine-rich oligonucleotide aptamer targeting nucleolin for the potential treatment of cancer, including acute myeloid leukemia Curr. Opin. Mol. Ther. 2010 12 107 114 20140822