Abstract
Objectives:
Small interfering RNAs (siRNAs) represent a promising therapeutic platform addressing unmet clinical needs. Recent advances have improved siRNA design, stability, specificity, and delivery; however, translating preclinical findings into clinical practice remains challenging due to the lack of standardized safety evaluation frameworks. This study conducted a systematic review and meta-analysis of placebo-controlled randomized clinical trials (RCTs) to quantitatively assess the safety of siRNA therapeutics and evaluate the adequacy of current preclinical safety assessments.
Methods:
PubMed, Embase, and Cochrane databases were searched from inception to July 2025. Two reviewers independently performed study selection and data extraction according to predefined criteria. Safety outcomes included death, serious adverse events (SAEs), severe adverse events, and adverse events (AEs) leading to treatment discontinuation. Pooled relative risks (RRs) and 95% confidence intervals (CIs) were calculated using R packages meta and metafor. Methodological quality for AE assessment was evaluated using the McHarm scale. The protocol was registered with PROSPERO (CRD420251120555).
Results:
Of 5,527 records screened, 57 studies (53 reports) evaluating 28 siRNA agents were included. Compared with controls, siRNA treatment did not increase the risk of death (RR 0.77, 95% CI 0.62–0.95) and severe AEs (RR 0.85, 95% CI 0.76–0.95), with no statistical heterogeneity (I2 = 0%). Risks of SAEs (RR 0.94, 95% CI 0.88–1.00) and treatment discontinuation (RR 0.86, 95% CI 0.66–1.13) were not increased.
Conclusion:
Chemical and delivery advancements have effectively enhanced siRNA safety. Nevertheless, ongoing vigilance and comprehensive safety monitoring during early clinical phases remain essential to identify unforeseen toxicities.
Systematic Review Registration:
Identifier CRD420251120555.
Introduction
The management of rare hereditary diseases has largely been confined to symptomatic management rather than curative treatments, resulting in unmet clinical needs for patients (Chaud et al., 2025). To address this limitation, oligonucleotide (OGN)-based therapeutics have emerged as a promising strategy (Levin, 2017). Small interfering RNAs (siRNAs) are a powerful tool for modulating gene expression in a sequence-specific manner (Ranjbar et al., 2023). The mechanism of siRNA involves the binding of its guide strand to the RNA-induced silencing complex (RISC), which triggers RNA interference (RNAi) within the cell to achieve a gene-silencing effect (Chi et al., 2017). The identification of this interference process led to development of agents that target disease-causing genes in rare genetic disorders as well as several common (Ranjbar et al., 2023; Wang et al., 2020). To date, seven siRNA therapeutics have received FDA approval (inclisiran, lumasiran, givosiran, patisiran, vutrisiran, nedosiran, and fitusiran), with several others currently undergoing evaluation for efficacy and safety in clinical trials.
Early siRNA therapeutic agents showed numerous limitations, including low in vivo stability leading to rapid degradation, as well as potential toxicity issues such as off-target effects and immunogenicity (Alshaer et al., 2021). The currently approved siRNA therapies and those in development have incorporated several chemical modifications to overcome these problems (Khvorova and Watts, 2017). Key modifications include a phosphorothioate (PS) backbone and ribose modifications like 2′-O-methyl (2′-OME) and 2′-fluoro (2′-F), which enhance resistance to nuclease degradation and reduce immunogenicity (Judge et al., 2006; Roberts et al., 2020). To improve intracellular delivery efficiency, triantennary N-acetylgalactosamine (GalNAc) conjugation was introduced (Springer and Dowdy). GalNAc-conjugates target the asialoglycoprotein receptor (ASGPR), which is predominantly expressed on hepatocytes, facilitating uptake via receptor-mediated endocytosis (Springer and Dowdy; Nair et al., 2014). This mechanism enables highly selective delivery to the liver and minimizes unnecessary drug distribution to other tissues (Janas et al., 2018). Owing to these advantages, GalNAc has become the preferred drug delivery system for liver-targeting siRNA therapeutics (Anand et al., 2025). In silico analysis has also been used to design sequences that minimize off-target effects (Goyenvalle et al., 2023; Patzel, 2007) and preclinical safety assessments have been performed to predict and mitigate potential toxicity before advancing siRNA agents to clinical trials (Goyenvalle et al., 2023; Andersson, 2022). These toxicology studies, performed in compliance with Good Laboratory Practices (GLP), determines the initial starting dose for human clinical trials (Jeon et al., 2022).
Despite these advancements, translating preclinical findings to the clinical setting has been challenging because of the absence of a gold-standard guideline or regulation for the safety assessment of siRNA therapeutics (Ranjbar et al., 2023). While the FDA released a draft guidance on preclinical safety studies for oligonucleotide drugs in November 2024 (USFaD, 2024), whether this guidance adequately addresses the unique characteristics of siRNA therapies has not been determined. This suggests that safety issues not identified in the preclinical phase could potentially be observed as adverse events in clinical trials. Determining whether reported safety issues are attributable to siRNA therapeutics can be challenging, as the targeted diseases are often intractable, with high baseline mortality and hospitalization rates. Therefore, to properly evaluate the safety profile of these agents, comparative studies against a placebo or treatment-as-usual (TAU) with a randomized-controlled design, rather than single-arm studies, are necessary. While individual randomized controlled trials (RCTs) reporting the efficacy and safety of siRNA therapeutics have been published, a systematic review and meta-analysis synthesizing this safety data is currently lacking. Thus, we conducted a systematic review and meta-analysis of published placebo-controlled RCTs to quantitatively assess the safety of siRNA therapeutics that have entered clinical development. Through this analysis, we evaluated the adequacy of the current preclinical safety framework for siRNA-based drugs.
Methods
Study registration
The protocol for this systematic review was registered with the International Prospective Register of Systematic Reviews (PROSPERO) (CRD420251120555).
Search strategy and study selection
We conducted a systematic search and critical review of the literature published from inception through July 2025 in MEDLINE, Embase, and Cochrane databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (http://www.prisma-statement.org/) (Supplementary Materials S1, S2). Eligibility criteria were as follows: (Chaud et al., 2025) RCTs that used siRNA drugs as the intervention; and (Levin, 2017) studies that used placebo or treatment-as-usual group as comparison. Exclusion criteria were as follows: 1) reviews, commentaries, or editorials; 2) studies published in abstract or poster presentations; 3) open-label single arm studies; 4) Phase 1 trials with healthy volunteers only; 5) pharmacokinetics or pharmacodynamics studies; and 6) in vitro or in vivo studies. Study selection and data extraction were conducted independently by two reviewers (CA, HJ) following these pre-specified criteria. The two investigators independently screened all titles and abstracts to extract relevant studies that met the inclusion and exclusion criteria and then independently assessed a full text review for the relevant studies. Disagreements were resolved by consensus involving a third reviewer (HWY).
Data extraction
Two investigators extracted data using customized data extraction forms. The following data were extracted: year of publication, drug name, indication for use, disease category, number of participants, follow-up period, delivery mechanism, chemical modification, trial phase, and development status. In the disease category, an indication was classified as “rare” if it had a low prevalence (affecting fewer than 200,000 Americans in the US) and no curative treatment was available. An indication was classified as “common” if the prevalence rate was high (affecting more than 200,000 Americans in the US) or standard treatment option was available.
Safety outcomes, including death, serious adverse events (SAEs), severe adverse events, and adverse events (AEs) leading to treatment discontinuation, were extracted from each study. For the intervention group, data on the number of participants who received at least one dose of siRNA and the corresponding number of events for each safety outcome were collected. For the control group, data on the total number of participants randomized and the number of events for each safety outcome were also obtained.
Quality assessment
Methodological quality of AE assessment was evaluated using questions from the McHarm quality assessment scale for AEs. Reports were evaluated as to whether studies used precise, pre-defined criteria for classifying AEs (e.g., “death,” “serious adverse event”), specified personnel and methods involved in harms collection, and provided quantitative data for both the total number of adverse events and the number of participants affected. For assessing mode of harms collection, active collection was defined as proactive solicitation of adverse event data by an investigator or qualified designee using methods such as checklists or interviews. Passive collection was defined as unsolicited, spontaneous reports of adverse events initiated by study participants. Each item was scored as a “yes,” “no,” or “unable to determine” (Chou et al., 2010). Assessment was based on the main publication, and the trial protocol was also referenced when available. Each item was rated “yes” if specified in the publication or protocol or “no” if not specified. Items for which a definitive assessment could not be made because of an inaccessible protocol were marked as “unable to determine.” The McHarm score for each study was calculated as the total number of items rated “yes.” The McHarm score ranges from 0 to 15, with higher scores indicating a higher quality of harm reporting. Quality assessments were independently conducted by two authors (CA and HJ). Disagreements were resolved by discussion with a third author (HWY).
Statistical analyses
We estimated the pooled effect using either Mantel-Haenszel method for the fixed effect model or DerSimonian-Laird method for the random-effect model. We presented forest plots in the safety outcomes including death, SAEs, severe adverse events, and AEs leading to treatment discontinuation as relative risks (RRs) and 95% confidence intervals (CIs) for the intervention group compared with the control group. For studies reporting zero events in one or both arms, a standard continuity correction of 0.5 was added to all cells to enable the calculation of relative risks for rare events. An RR less than 1 was interpreted as indicating fewer occurrences of death, SAEs, severe adverse events, and AE leading to treatment discontinuation in the intervention group compared with the control group. Statistical heterogeneity across the studies was assessed using the I2 statistics and defined as low (25%–50%), moderate (50%–75%), or high (>75%). Subgroup analyses were conducted to explore whether the risk of death varied by disease category, delivery mechanism, study phase, and product development status. Disease categories were classified as either rare or common. For the delivery mechanism, because of the predominance of studies using GalNAc conjugation, subgroups were defined as GalNAc-based versus non-GalNAc delivery. The study phase was categorized as Phase 1, Phase 2, or Phase 3. Product development status was classified into commercially available products (marketed) versus those still in development or discontinued/terminated. For each subgroup, the number of included trials and the number of participants in both the intervention and control groups were represented. For each subgroup, the effect estimates for the risk of death in the intervention group compared with the control group were presented as RRs with corresponding 95% CIs. Publication bias was assessed using funnel plots, and asymmetry in the plots was examined to evaluate the presence of potential publication bias. All analyzes used the R “meta” and “metafor” packages.
Results
Literature search
The initial database search identified 5,527 articles. After removing duplicates and screening titles and abstracts, 258 articles underwent a full-text review for eligibility. During the full-text review, 94 articles were excluded for being an ineligible article type (e.g., abstracts only) and 53 were excluded for being trial protocols or containing only trial results. An additional 27 reports were excluded for not having a randomized controlled trial design, and 8 were excluded for enrolling only healthy volunteers. Through this process, 47 reports were initially selected. A subsequent manual search of the reference lists of included articles and a search of clinicaltrials.gov for registered siRNA trials identified an additional 6 reports. Finally, a total of 53 reports were included Nissen et al., 2023; Nissen et al., 2025; O’Donoghue et al., 2022; Gane E. J. et al., 2023; Benitez-Del-Castillo et al., 2016; Nissen et al., 2024a; Nissen et al., 2024b; Bakris et al., 2024; Desai et al., 2023; Rosenson et al., 2024; Desai et al., 2025; Koren et al., 2022; Nissen et al., 2022; Gottlieb et al., 2016; Gane E. et al., 2023; Ray et al., 2025a; Ray et al., 2025b; Fabbrini et al., 2024; Zamora et al., 2011; Suzuki et al., 2017; Yuen et al., 2020; Hou et al., 2024, Raal et al., 2023; Raal et al., 2020; Ray et al., 2017; Ray et al., 2020; Huo et al., 2024; Yamashita et al., 2024; Watts et al., 2025; Luo et al., 2023; Ballantyne et al., 2024; Gaudet et al., 2024; Wiegman et al., 2025; Koren et al., 2024; Taub et al., 2025; Nissen et al., 2024a; Nissen et al., 2024b; Bakris et al., 2024; Desai et al., 2023; Rosenson et al., 2024; Desai et al., 2025; Koren et al., 2022; Nissen et al., 2022; Gottlieb et al., 2016; Gane E. et al., 2023; Ray et al., 2025a; Ray et al., 2025b; Fabbrini et al., 2024; Zamora et al., 2011; Suzuki et al., 2017; Yuen et al., 2020; Hou et al., 2024; Koren et al., 2024; Taub et al., 2025. Among this group, 4 reports each presented 2 separate studies (Ray et al., 2020; Benitez-Del-Castillo et al., 2016; Fabbrini et al., 2024; Yuen et al., 2020); thus, 57 studies from 53 reports covering 28 different siRNA agents were included in the analysis (Figure 1).
FIGURE 1
Study characteristics
The included RCTs were published from 2011 onwards, with 33 studies (58%) published in 2023 or later. Out of 28 distinct siRNA therapeutic agents, 12 (43%) were developed exclusively for rare diseases (Adams et al., 2018; Coelho et al., 2013; Frishberg et al., 2021; Garrelfs et al., 2021; Balwani et al., 2020; Maurer et al., 2023; Sardh et al., 2019; Turner et al., 2018; Barratt et al., 2024; Clark et al., 2024; Baum et al., 2023; Goldfarb et al., 2023; Fontana et al., 2025; Judge et al., 2020; Srivast et al., 2023; Young et al., 2023; Doi et al., 2023), 14 (50%) for common diseases (Thielmann et al., 2021; Nissen et al., 2023; Nissen et al., 2025; O'Donoghue et al., 2022; Gane E. J. et al., 2023; Benitez-Del-Castillo et al., 2016; Nissen et al., 2024a; Nissen et al., 2024b; Bakris et al., 2024; Desai et al., 2023; Rosenson et al., 2024; Desai et al., 2025; Koren et al., 2022; Nissen et al., 2022; Gottlieb et al., 2016; Gane E. et al., 2023; Ray et al., 2025a; Ray et al., 2025b; Fabbrini et al., 2024; Zamora et al., 2011; Suzuki et al., 2017; Yuen et al., 2020; Hou et al., 2024), and 2 (7%) for indications spanning both categories (Raal et al., 2023; Raal et al., 2020; Ray et al., 2017; Ray et al., 2020; Huo et al., 2024; Yamashita et al., 2024; Watts et al., 2025; Luo et al., 2023; Ballantyne et al., 2024; Gaudet et al., 2024; Wiegman et al., 2025; Koren et al., 2024; Taub et al., 2025). Of the 57 studies, 37 focused on common diseases (Raal et al., 2020; Ray et al., 2017; Ray et al., 2020; Thielmann et al., 2021; Huo et al., 2024; Yamashita et al., 2024; Luo et al., 2023; Ballantyne et al., 2024; Gaudet et al., 2024; Nissen et al., 2023; Nissen et al., 2025; O'Donoghue et al., 2022; Gane E. J. et al., 2023; Benitez-Del-Castillo et al., 2016; Nissen et al., 2024a; Nissen et al., 2024b; Bakris et al., 2024; Desai et al., 2023; Rosenson et al., 2024; Desai et al., 2025; Koren et al., 2022; Nissen et al., 2022; Gottlieb et al., 2016; Gane E. et al., 2023; Ray et al., 2025a; Ray et al., 2025b; Fabbrini et al., 2024; Zamora et al., 2011; Suzuki et al., 2017; Yuen et al., 2020; Hou et al., 2024; Koren et al., 2024; Taub et al., 2025), and hyperlipidemia was the most frequently investigated condition (21 studies) (Raal et al., 2020; Ray et al., 2017; Ray et al., 2020; Huo et al., 2024; Yamashita et al., 2024; Luo et al., 2023; Ballantyne et al., 2024; Gaudet et al., 2024; Nissen et al., 2023; Nissen et al., 2025; O'Donoghue et al., 2022; Nissen et al., 2024a; Nissen et al., 2024b; Rosenson et al., 2024; Koren et al., 2022; Nissen et al., 2022; Ray et al., 2025a; Ray et al., 2025b; Koren et al., 2024; Taub et al., 2025). The distribution by clinical trial phase was as follows: 16 Phase 1 (28%) (Coelho et al., 2013; Sardh et al., 2019; Luo et al., 2023; Turner et al., 2018; Nissen et al., 2023; Goldfarb et al., 2023; Gane E. J. et al., 2023; Nissen et al., 2024a; Desai et al., 2023; Koren et al., 2022; Nissen et al., 2022; Ray et al., 2025a; Fabbrini et al., 2024; Suzuki et al., 2017; Doi et al., 2023), 24 Phase 2 (42%) (Frishberg et al., 2021; Ray et al., 2017; Thielmann et al., 2021; Yamashita et al., 2024; Ballantyne et al., 2024; Gaudet et al., 2024; Barratt et al., 2024; Clark et al., 2024; Nissen et al., 2025; Baum et al., 2023; O'Donoghue et al., 2022; Benitez-Del-Castillo et al., 2016; Nissen et al., 2024b; Bakris et al., 2024; Rosenson et al., 2024; Desai et al., 2025; Gottlieb et al., 2016; Gane E. et al., 2023; Ray et al., 2025b; Zamora et al., 2011; Yuen et al., 2020; Hou et al., 2024), and 17 Phase 3 (30%) (Adams et al., 2018; Garrelfs et al., 2021; Balwani et al., 2020; Maurer et al., 2023; Raal et al., 2023; Raal et al., 2020; Ray et al., 2020; Huo et al., 2024; Watts et al., 2025; Fontana et al., 2025; Judge et al., 2020; Wiegman et al., 2025; Srivast et al., 2023; Young et al., 2023; Koren et al., 2024; Taub et al., 2025) studies. A total of 10 studies involved discontinuation of drug development or early termination of clinical trial (Thielmann et al., 2021; Turner et al., 2018; Gane E. J. et al., 2023; Judge et al., 2020; Gottlieb et al., 2016; Zamora et al., 2011; Yuen et al., 2020; Hou et al., 2024); in 4 of these cases, discontinuation was because of safety concerns related to the siRNA agent (Turner et al., 2018; Judge et al., 2020; Yuen et al., 2020). Regarding chemical modifications, 6 of the 7 FDA-approved therapeutics used GalNAc conjugation along with 2′-OMe, 2′-F, and PS backbone modifications (Frishberg et al., 2021; Garrelfs et al., 2021; Balwani et al., 2020; Raal et al., 2023; Raal et al., 2020; Ray et al., 2017; Ray et al., 2020; Sardh et al., 2019; Huo et al., 2024; Yamashita et al., 2024; Luo et al., 2023; Baum et al., 2023; Goldfarb et al., 2023; Fontana et al., 2025; Wiegman et al., 2025; Srivast et al., 2023; Young et al., 2023; Koren et al., 2024; Taub et al., 2025). Therapeutic agents currently in development also feature these modifications, with 4 agents incorporating other variations (Benitez-Del-Castillo et al., 2016; Gane E. et al., 2023; Suzuki et al., 2017; Doi et al., 2023). The number of participants in the 57 RCTs ranged from 6 to 1,617, and the follow-up duration ranged from 10 days (0.3 months) to a maximum of 36 months (Table 1).
TABLE 1
| First author, year [References] | Drug name | Indication | Disease category | Sample size (Tx; co) | Follow up (mo) | Delivery mechanism | Chemical modification | Trial phase | Development status[trial number] |
|---|---|---|---|---|---|---|---|---|---|
| Adams et al. (2018) | Patisiran | hARRT amyloidosis | Rare | 148; 77 | 18 | LNP | 2′-OMe | 3 | Approved (ONPATTRO) [NCT01960348] |
| Coelho et al. (2013) | ALN-TTR01 | hARRT amyloidosis | Rare | 24; 8 | 1 | LNP | 2′-OMe | 1 | Discontinued [NCT01148953] |
| Frishberg et al. (2021) | Lumasiran | PH1 | Rare | 17; 3 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Approved (OXLUMO) [NCT02706886] |
| Garrelfs et al. (2021) | Lumasiran | PH1 | Rare | 26; 13 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (OXLUMO) [NCT03681184] |
| Balwani et al. (2020) | Givosiran | Acute hepatic porphyria | Rare | 48; 46 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (GIVLAARI) [NCT03338816] |
| Maurer et al. (2023) | Patisiran | hARRT amyloidosis | Rare | 181; 179 | 12 | LNP | 2′-OMe | 3 | Approved (ONPATTRO) [NCT03997383] |
| Raal et al. (2023) | Inclisiran | HoFH | Rare | 37; 19 | 5 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT03851705] |
| Raal et al. (2020) | Inclisiran | HeFH or ASCVD | Common | 242; 240 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT03397121] |
| Ray et al. (2017) | Inclisiran | HeFH or ASCVD | Common | 370; 127 | 7 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Approved (LEQVIO) [NCT02597127] |
| Ray et al. (2020)* | Inclisiran | HeFH or ASCVD | Common | 781; 780 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT03399370] |
| Ray et al. (2020)* | Inclisiran | HeFH or ASCVD | Common | 810; 807 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT03400800] |
| Sardh et al. (2019) | Givosiran | Acute hepatic porphyria | Rare | 33; 10 | 3 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Approved (GIVLAARI) [NCT02452372] |
| Thielmann et al. (2021) | Teprasiran | AKI | Common | 165; 176 | 3 | Unmodified | 2′-OMe | 2 | Terminated [NCT02610283] |
| Huo et al. (2024) | Inclisiran | HeFH or ASCVD | Common | 171; 174 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT04765657] |
| Yamashita et al. (2024) | Inclisiran | HeFH or ASCVD | Common | 255; 57 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Approved (LEQVIO) [NCT04666298] |
| Watts et al. (2025) | Plozasiran | FCS or symptomatic persistent chylomicronemia | Rare | 50; 25 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Ongoing [NCT05089084] |
| Luo et al. (2023) | Inclisiran | Elevated LDL-C | Common | 30; 10 | 3 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Approved (LEQVIO) [NCT04774003] |
| Ballantyne et al. (2024) | Plozasiran | Mixed hyperlipidemia | Common | 266; 87 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT04998201] |
| Gaudet et al. (2024) | Plozasiran | Severe hypertriglyceridemia | Common | 166; 60 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT04720534] |
| Turner et al. (2018) | ARC-AAT | Alpha-1 antitrypsin deficiency | Rare | 7; 4 | 1 | ARC-EX1 | 2′-OMe, 2′-F, PS | 1 | Terminated [NCT02363946] |
| Barratt et al. (2024) | Cemdisiran | IgA nephropathy | Rare | 22; 9 | 9 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT03841448] |
| Clark et al. (2024) | Fazirsiran | Alpha-1 antitrypsin deficiency | Rare | 26; 14 | 22 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT03945292] |
| Nissen et al. (2023) | Lepodisiran | Lipoprotein disorder | Common | 36; 12 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT04914546] |
| Nissen et al. (2025) | Lepodisiran | Lipoprotein disorder | Common | 251; 69 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT05565742] |
| Baum et al. (2023) | Nedosiran | PH1 or PH2 | Rare | 23; 12 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Approved (Rivfloza) [NCT03847909] |
| Goldfarb et al. (2023) | Nedosiran | PH3 | Rare | 4; 2 | 3 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Approved (Rivfloza) [NCT04555486] |
| O'Donoghue et al. (2022) | Olpasiran | ASCVD | Common | 227; 54 | 18 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT04270760] |
| Gane E. J. et al. (2023) | RG6346 | Chronic HBV infection | Common | 18; 9 | 4 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Discontinued [NCT03772249] |
| Benitez-Del-Castillo et al. (2016)† | SYL1001 | Dry eye disease | Common | 40; 20 | 0.6 | Unmodified | Unmodified | 2 | Ongoing [NCT01776658] |
| Benitez-Del-Castillo et al. (2016)† | SYL1001 | Dry eye disease | Common | 44; 24 | 0.6 | Unmodified | Unmodified | 2 | Ongoing [NCT02455999] |
| Fontana et al. (2025) | Vutrisiran | ATTR-CM | Rare | 326; 328 | 36 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (AMVUTTRA) [NCT04153149] |
| Nissen et al. (2024a) | Zerlasiran | ASCVD | Common | 27; 9 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT04606602] |
| Nissen et al. (2024b) | Zerlasiran | ASCVD | Common | 131; 47 | 15 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT05537571] |
| Bakris et al. (2024) | Zilebesiran | Hypertension | Common | 302; 75 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT04936035] |
| Desai et al. (2023) | Zilebesiran | Hypertension | Common | 64; 32 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT03934307] |
| Rosenson et al. (2024) | Zodasiran | Mixed hyperlipidemia | Common | 153; 51 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT04832971] |
| Judge et al. (2020) | Revusiran | hATTR-CM | Rare | 140; 66 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Discontinued [NCT02319005] |
| Desai et al. (2025) | Zilebesiran | Hypertension | Common | 329; 329 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT05103332] |
| Koren et al. (2022) | Olpasiran | Elevated LP(a) | Common | 48; 16 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT03626662] |
| Nissen et al. (2022) | Zerlasiran | Elevated LP(a) | Common | 24; 8 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT04606602] |
| Wiegman et al. (2025) | Inclisiran | HoFH | Rare | 9; 4 | 12 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT04659863] |
| Gottlieb et al. (2016) | ALN-RSV01 | BOS by RSV infection in LTx recipients | Common | 45; 42 | 1 | Unmodified | Unmodified | 2 | Discontinued [NCT01065935] |
| Gane E. J. et al. (2023) | VIR-2218 | Chronic HBV infection | Common | 24; 8 | 12 | GalNAc | 2′-O-MOE, 2′-F, S-GNA, PS | 2 | Ongoing [NCT03672188] |
| Ray et al. (2025a) | Solbinsiran | Mixed hyperlipidemia | Common | 42; 14 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT04644809] |
| Ray et al. (2025a) | Solbinsiran | Mixed hyperlipidemia | Common | 147; 57 | 9 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Ongoing [NCT05256654] |
| Fabbrini et al., 2024‡ | JNJ-75220795 | MAFLD | Common | 6; 3 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT05039710] |
| Fabbrini et al., 2024‡ | JNJ-75220795 | MAFLD | Common | 39; 16 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 1 | Ongoing [NCT04844450] |
| Zamora et al. (2011) | ALN-RSV01 | BOS by RSV infection | Common | 16; 8 | 1 | Unmodified | Unmodified | 2 | Discontinued [NCT00658086] |
| Suzuki et al. (2017) | STNM01 | Chron’s disease | Common | 9; 9 | 1 | Undisclosed | Undisclosed | 1 | Ongoing |
| Yuen et al., 2020§ | ARC-520 | Chronic HBV infection | Common | 38; 20 | 6 | ARC-EX1 | 2′-OMe, 2′-F, PS | 2 | Terminated [NCT02604199] |
| Yuen et al., 2020§ | ARC-520 | Chronic HBV infection | Common | 21; 11 | 6 | ARC-EX1 | 2′-OMe, 2′-F, PS | 2 | Terminated [NCT02604212] |
| Srivast et al. (2023) | Fitusiran | Severe haemophilia a or B | Rare | 79; 40 | 9 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (Qfitlia) [NCT03417245] |
| Young et al. (2023) | Fitusiran | Severe haemophilia a or B | Rare | 41; 19 | 9 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (Qfitlia) [NCT03417102] |
| Hou et al. (2024) | Xalnesiran (RG6346) | Chronic HBV infection | Common | 60; 35 | 24 | GalNAc | 2′-OMe, 2′-F, PS | 2 | Discontinued [NCT04225715] |
| Doi et al. (2023) | TRK-250 | Idiopathic pulmonary fibrosis | Rare | 24; 10 | 1 | Unspecified | Undisclosed | 1 | Ongoing [NCT03727802] |
| Koren et al. (2024) | Inclisiran | ASCVD | Common | 234; 216 | 11 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT04929249] |
| Taub et al. (2025) | Inclisiran | Elevated LDL-C | Common | 174; 87 | 6 | GalNAc | 2′-OMe, 2′-F, PS | 3 | Approved (LEQVIO) [NCT05763875] |
Baseline characteristics of the 57 included trials.
*, †, ‡, § = Studies that include 2 trials in one article.
Tx (Treatment group), Co (Comparison group), mo (Month), HoFH (Homozygous Familial Hypercholesterolemia), HeFH (Heterozygous Familial Hypercholesterolemia), ASCVD (Atherosclerotic Cardiovascular Disease), AKI (Acute Kidney Injury), IgA nephropathy (Immunoglobulin A nephropathy), PH1 (Primary Hyperoxaluria Type 1), PH2 (Primary Hyperoxaluria Type 2), PH3 (Primary Hyperoxaluria Type 3), Lp(a) (Lipoprotein(a)), LDL-C (Low-Density Lipoprotein Cholesterol), FCS (Familial Chylomicronemia Syndrome), hATTR amyloidosis (hereditary transthyretin amyloidosis), ATTR-CM (Transthyretin Amyloidosis with cardiomyopathy), hATTR-CM (hereditary transthyretin amyloidosis with cardiomyopathy), HBV (Hepatitis B Virus), BOS (Bronchiolitis Obliterans Syndrome), RSV (Respiratory Syncytial Virus), LTx (Lung Transplantation), MAFLD (Metabolic dysfunction-associated steatotic liver disease), 2′-OMe (2′-O-methyl), 2′-F (2′-deoxy-2′-fluoro), PS (Phosphorothioate), 2′-O-MOE (2′-O-methoxyethyl), S-GNA ((S)-glycol nucleic acid), GalNAc (N-acetylgalactosamine).
Quality assessment
The McHarm score ranged from 7 to 14, with a median score of 13 (Supplementary Material S3). Of the 53 reports, 51 (96%) reports specified the number of deaths in each study group, and 50 reports (94%) provided a precise definition of serious adverse events either in the main article or the protocol.
Safety outcomes
A meta-analysis of safety outcomes was performed on the 57 RCTs. Among the 57 RCTs, one study did not report data on mortality (Suzuki et al., 2017). Consequently, the analysis included data from 56 studies for death, 55 for SAEs, 32 for severe adverse events, and 48 for discontinuation because of adverse events. Compared to the control group, the siRNA treatment group did not have an increased risk of death or severe adverse events (RR 0.77, 95% CI 0.62–0.95, I2 = 0% and RR 0.85, 95% CI 0.76–0.95, I2 = 0%, respectively). The risk of SAEs and treatment discontinuation due to adverse events did not show a significant increase in the treatment group compared with the control group (RR 0.94, 95% CI 0.88–1.00, I2 = 0% and RR 0.86, 95% CI 0.66–1.13, I2 = 0%, respectively) (Figure 2).
FIGURE 2
Publication bias
Visual inspection of the funnel plot for the 56 studies reporting on death revealed no significant asymmetry, suggesting a low likelihood of publication bias (Supplementary Material S4).
Subgroup analysis of death outcome
Subgroup analysis based on disease type showed that siRNA treatment did not increase the risk of death compared with the control in either the rare disease subgroup (RR 0.76, 95% CI 0.58–1.00) or the common disease subgroup (RR 0.78, 95% CI 0.54–1.12). Consistent results were observed in subgroup analyses by drug delivery technology (GalNAc vs. non-GalNAc) and clinical trial phase (Phase 1, 2, or 3), with siRNA treatment not increasing the risk of death in any subgroup. When analyzed by development status (marketed, ongoing, or discontinued/terminated), the risk of death was not increased in the marketed and ongoing development subgroups (RR 0.76, 95% CI 0.59–0.96 and RR 0.47, 95% CI 0.22–0.98, respectively). The discontinued/terminated subgroup showed a risk of death similar to that of the control group (RR 1.01, 95% CI 0.60–1.71) (Table 2).
TABLE 2
| Subgroups | No. Of trials (Tn/Cn) | RR (95% CI) |
|---|---|---|
| Disease category | ||
| Rare disease | 20 (1,265/888) | 0.76 (0.58–1.00) |
| Common disease | 36 (5,795/3,785) | 0.78 (0.54–1.12) |
| Delivery mechanism | ||
| GalNAc | 44 (6,307/4,094) | 0.79 (0.62–1.01) |
| Others | 12 (753/579) | 0.66 (0.40–1.11) |
| Study stage | ||
| Phase 1 | 15 (426/163) | 0.40 (0.15–1.06) |
| Phase 2 | 24 (3,138/1,395) | 0.61 (0.35–1.06) |
| Phase 3 | 17 (3,496/3,115) | 0.84 (0.66–1.06) |
| Product development status | ||
| Marketed | 22 (4,038/3,245) | 0.76 (0.59–0.96) |
| Ongoing development | 24 (2,488/1,049) | 0.47 (0.22–0.98) |
| Discontinued or terminated | 10 (534/379) | 1.01 (0.60–1.71) |
Subgroup analysis of death according to disease category, delivery mechanism, study stage, and development status.
No (number), Tn (Treatment group), Cn (Comparison group), OR (Odds ratio), CI (Confidence interval), GalNAc (N-acetylgalactosamine).
Discussion
This systematic review and meta-analysis assessed the safety of siRNA therapeutics by analyzing 57 randomized clinical trials covering 28 different agents and a total of 11,757 participants. We evaluated the safety outcomes and further investigated the safety profile through subgroup analyses based on disease type, clinical trial phase, drug development status, and the use of GalNAc conjugation. The overall quality of safety data reporting was high, with a median McHarm score of 13, and 38 reports (71.7%) scored 12 or higher. This indicates that the quality of adverse event reporting was sufficient to justify an evaluation of the safety of siRNA agents using the available data.
Our analysis revealed that the siRNA therapeutic group did not show a significant increase in the incidence of key safety outcomes (death, serious adverse events, severe adverse events, and treatment discontinuation due to adverse events) compared with the control group. In fact, the incidence of death and severe adverse events was significantly lower in the treatment group. Nevertheless, this finding from the analysis of the overall group does not preclude the possibility of potential toxicity in individual patients. Some clinical trials have reported serious hepatic or electrocardiogram abnormalities that led to participant withdrawal (Balwani et al., 2020; Baum et al., 2023). Although rare, these cases underscore the necessity of rigorous monitoring for adverse events where a causal link to the drug cannot be excluded.
The risk-benefit assessment for siRNA therapeutics in common diseases may differ from that for rare, often genetic, diseases (Goyenvalle et al., 2023; Andersson, 2022). This difference is partly because multiple therapeutic alternatives often exist for common conditions (Goyenvalle et al., 2023). Therefore, the safety threshold is particularly high. Even with superior efficacy, siRNA therapeutics may not achieve widespread clinical adoption without a well-established safety profile. Our subgroup analysis for common diseases showed that siRNA treatment did not increase mortality risk (RR = 0.78), suggesting that its use in this population is unlikely to be associated with high risk. However, this finding has limitations, as most of the included studies enrolled patients who had failed standard therapies and thus had higher disease severity. Therefore, an extrapolation to patient populations with lower disease severity should be done with caution. To address this, trials like the VICTORION-MONO study are evaluating the strategy of administering inclisiran earlier before other lipid-lowering therapies are used (Taub et al., 2025). Such study designs will enable a more accurate risk-benefit assessment of siRNA therapeutics in a broader population with common diseases.
From the perspective of delivery mechanisms, GalNAc conjugation, which is widely applied to both marketed and investigational drugs, demonstrated an acceptable safety profile in terms of mortality. Notably, therapeutics using non-GalNAc delivery technologies also did not show an increase in mortality (RR: 0.66, 95% CI: 0.40–1.11). However, this latter finding must be interpreted with caution. It is possible that non-GalNAc drugs with unfavorable safety profiles were discontinued in early clinical or preclinical phases and the data were never published. This potential for publication bias limits the ability to conclude that non-GalNAc delivery methods have a similarly acceptable safety profile as GalNAc delivery methods. When stratified by development status, marketed siRNA drugs demonstrated no increased mortality compared with placebo, reaffirming their clinical utility. Agents currently in development also showed a favorable mortality profile without increased risk (RR 0.47), indicating a promising safety profile. Given that most of the data for these investigational agents comes from early-phase trials (9 Phase 1 and 14 Phase 2 studies), with only one Phase 3 study (Watts et al., 2025), a more precise safety profile will be established as data from large-scale Phase 3 trials accumulate.
The FDA’s draft guidance for the preclinical development of siRNA agents outlines the required in vitro and in vivo assessments, such as on/off-target toxicities, PK/PD, general toxicity, and carcinogenicity (USFaD, 2024). The toxicities considered during this phase can be broadly classified into three types: (Chaud et al., 2025): hybridization- and sequence-dependent, (Levin, 2017), hybridization-independent but sequence-dependent, and (Ranjbar et al., 2023) hybridization- and sequence-independent (Goyenvalle et al., 2023; Andersson, 2022). Hybridization- and sequence-dependent toxicities, such as off-target effects, are managed preclinically via in silico analysis (Lindow et al., 2012; Rider et al., 2022). Hybridization- and sequence-independent toxicities, which occur when plasma concentrations exceed a certain threshold, can be managed by adjusting dosage and frequency (Goyenvalle et al., 2023). The remaining category—hybridization-independent but sequence-dependent effects includes proinflammatory effects and toxicity to high-exposure organs like the liver and kidneys (Goyenvalle et al., 2023). Proinflammatory effects, such as injection site reactions (ISRs), can still occur despite chemical modifications (Chi et al., 2017; Mustonen et al., 2017). ISRs were the most frequently reported adverse event in the included RCTs. However, the vast majority were of mild-to-moderate severity and resolved without intervention. Similarly, toxicity to high-exposure organs, assessed via laboratory results (e.g., AST/ALT, creatinine), was largely characterized by changes that were not clinically relevant or were transient, mild elevations that resolved spontaneously. The acceptable safety profile observed in the RCT outcomes, combined with the finding that most ISRs and lab abnormalities were predictable and manageable, implies that the adverse events reported in clinical trials generally fell within the anticipated range of toxicities considered during preclinical safety assessments. It is important, though, to interpret this with caution. Because our meta-analysis relies exclusively on published clinical data, preclinical or early toxicity profiles remain unreported. Therefore, it should be conservatively interpreted as reflecting the improved clinical tolerability of newer siRNA platforms.
Despite preclinical frameworks to anticipate risks, incompletely understood toxicity mechanisms could lead to unforeseen safety issues in the clinical setting (Ranjbar et al., 2023). This is particularly challenging in intractable diseases, where determining the specific cause of death and its association with an intervention can be difficult. Among several potential causes, imperfections in the chemical composition of agents have been identified, and efforts to address this are ongoing (Ali Zaidi et al., 2023; Zhang et al., 2021). Of the four studies in our analysis that were discontinued because of safety concerns, three were halted because the delivery vehicle, rather than the siRNA molecule itself, was found to cause mortality in non-human primates (Turner et al., 2018; Yuen et al., 2020). The fourth study, involving revusiran, was terminated because of a mortality imbalance (23 deaths in the treatment group vs. 7 in the placebo group) by the end of the safety follow-up period (Judge et al., 2020). While a definitive cause for the imbalance could not be identified, the presence of imbalances in other adverse events suggested that drug-mediated toxicity was likely a contributing factor (Judge et al., 2020). Revusiran is a first-generation GalNAc-siRNA conjugate with “standard template chemistry,” where 2′-OMe and 2′-F modifications alternate on every ribose (Judge et al., 2020). Vutrisiran, which targets the same indication, is a second-generation compound with “enhanced stabilized chemistry” (ESC) that modulates the amount of ribose modification, leading to enhanced resistance to degradation (Foster et al., 2018). This difference in structural stability results in markedly different drug exposures: the administration of revusiran involves 500-mg subcutaneous injection weekly, whereas vutrisiran is administered as a 25-mg subcutaneous injection once every 12 weeks (Fontana et al., 2025; Judge et al., 2020). All other approved and most ongoing siRNA agents now use second-generation ESC modifications. The favorable safety results of these other ongoing therapies suggest that improvements in delivery systems and chemical modifications, such as GalNAc conjugation and ESC, have translated into enhanced clinical safety.
This study has several limitations. First, by including only RCTs, our analysis may lack studies with very large sample sizes or long follow-up periods. Among the included RCTs, only three had a follow-up duration of 2 years or more (Huo et al., 2024; Clark et al., 2024; Fontana et al., 2025). For rare diseases with small recruitment pools, the statistical power to properly evaluate uncommon outcomes like death may be limited. Phase 1 and 2 trials involve smaller cohorts and shorter follow-ups, which may dilute the overall pooled estimates or limit the detection of rare adverse events. Nevertheless, we exclusively included RCTs because they are the gold standard for statistically verifying differences in safety outcomes between intervention and control groups (Chou et al., 2010). Given the ethical challenges of maintaining a long-term placebo-controlled phase for patients with high unmet medical needs, tracking safety outcomes in open-label extension (OLE) phases is needed for assessing long-term safety. A 5-year OLE study of inclisiran found no new safety signals and reported that most instances of elevated liver enzymes, a potential issue with repeated dosing, were of mild or moderate severity (Ray et al., 2023). Second, as safety was often a secondary endpoint in many RCTs, safety may not have been reported with the same level of detail as the primary efficacy endpoint. In fact, severe adverse events were not explicitly reported in 24 of the 53 reports. However, several factors support the reliability of the safety data. Most RCTs specified safety assessment plans in their protocols and provided precise definitions for adverse events. For death and SAEs, protocols mandated immediate reporting (e.g., within 24 h), minimizing the chance of missed reports. Furthermore, the use of standardized dictionaries like Medical Dictionary for Regulatory Activities and Common Terminology Criteria for Adverse Events for collecting and classifying adverse events ensured data consistency. We therefore conclude that the reporting of safety outcomes was systematic and reliable. Lastly, while we attempted to address the heterogeneity of diverse trials through subgroup analyses based on disease type, drug development status, and the use of GalNAc conjugation, our study could not evaluate all pharmacological variables. Due to limited number of trials and low adverse event rates across highly specific subgroups, we were unable to conduct further stratified analyses by dosing schedules, or distinct non-GalNAc delivery systems such as lipid nanoparticles (LNPs). Future meta-analyses with larger pools of standardized data are needed to evaluate how these pharmacological factors influence the safety profile of siRNA agents.
Despite these limitations, this study holds significant value as the first systematic review and meta-analysis to quantitatively evaluate key safety outcomes of siRNA therapeutics across a diverse range of diseases. A further strength is the use of subgroup analyses based on specific criteria, such as disease rarity and development status, to compare safety under different conditions. Finally, this research offers a unique contribution by using clinical trial safety outcomes to reflect on the adequacy of the preclinical safety framework for siRNA therapeutics.
Conclusion
This systematic review and meta-analysis confirmed that siRNA therapeutics do not significantly increase the risk of safety outcomes compared with controls. This finding supports that the advancements of chemical modifications and drug delivery systems, introduced to reduce the toxicity of early-generation siRNAs, has successfully translated into enhanced clinical safety. Therefore, these results underscore the critical importance of maintaining and refining these toxicity mitigation strategies in the development of next-generation siRNA therapeutics. Nevertheless, as unforeseen safety issues can still manifest during clinical development, thorough safety validation throughout the early clinical trial phases remains essential. While short-term safety profile is reassuring, extended follow-up surveillance of delayed toxicities remain necessary to elucidate the long-term safety profile of these agents.
Statements
Data availability statement
The data analyzed in this study is subject to the following licenses/restrictions: If a researcher requests data, it can be obtained through the corresponding authors. Requests to access these datasets should be directed to Hyunsuk Jeong, suejeong@catholic.ac.kr.
Author contributions
CA: Data curation, Formal Analysis, Investigation, Methodology, Software, Writing – original draft. HJ: Conceptualization, Data curation, Formal Analysis, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – review and editing. HY: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – review and editing. NK: Data curation, Resources, Writing – review and editing. SH: Investigation, Methodology, Supervision, Validation, Writing – review and editing. IO: Investigation, Methodology, Resources, Supervision, Validation, Writing – review and editing.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Publisher’s note
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fdsfr.2026.1855572/full#supplementary-material
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Summary
Keywords
clinical trials, meta-analysis, safety, small interfering RNAs, systematic review
Citation
Ahn C, Jeong H, Yim HW, Kim NJ, Han S and Oh I (2026) Safety profile of small interfering RNA therapeutics: a systematic review and meta-analysis of randomized controlled trials. Front. Drug Saf. Regul. 6:1855572. doi: 10.3389/fdsfr.2026.1855572
Received
16 April 2026
Revised
28 May 2026
Accepted
09 June 2026
Published
10 July 2026
Volume
6 - 2026
Edited by
Juandy Jo, University of Pelita Harapan, Indonesia
Reviewed by
Nata Pratama Hardjo Lugito, University of Pelita Harapan, Indonesia
Jeremiah Hilkiah Wijaya, Monash University, Australia
Yen-Tzu Chang, University of Texas MD Anderson Cancer Center, United States
Chih-Wei Chu, University of Texas MD Anderson Cancer Center, United States
Updates
Copyright
© 2026 Ahn, Jeong, Yim, Kim, Han and Oh.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Hyunsuk Jeong, suejeong@catholic.ac.kr; Hyeon Woo Yim, y1693@catholic.ac.kr
† These authors have contributed equally to this work
Disclaimer
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