Small Interfering RNA (siRNA) as a Targeted Therapy for Acute Respiratory Distress Syndrome: Evidence from Experimental Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review by Kiseleva on “Small Interfering RNA (siRNA) as a Targeted Therapy for 2 Acute Respiratory Distress Syndrome: Evidence from Experimental Models” provides background on the pathophysiology of direct and indirect ARDS, a number of different large animal models for the disease, and the mechanisms of siRNA action. It also provides discussion of several published papers using siRNAs to prevent or limit severity of ARDS in small animal models. While the discussions of animal models, ARDS pathophysiology, and siRNA activity are very well developed and informative, the discussion on siRNA use and results in ARDS settings lack the same depth and information. This is a letdown since the title of the review deals with the use of siRNA for ARDS.

 

The major limitation of the manuscript is the lack of detail and analysis of the papers presented that actually use siRNAs to knock down genes that may be relevant in ARDS. It would be highly informative to provide quantitative data and results from these papers showing exactly how effective the siRNAs are in these models and papers. How much knockdown is achieved and does this affect the “treatment”? what is the distribution of siRNAs/knock down genes in the lungs of the animals in the different papers? Further, discussions of controls (non-targeted siRNAs, etc) should also be included for each paper discussed and the main body of the review should be on the section dealing with the use of siRNAs for ARDS. There is a lack of discussion on how each siRNA is delivered (how much, delivery route, delivery agent, complexity of siRNA – single or pool of siRNAs, etc); this could be very important and having all the information in one review would be of great benefit for those thinking about embarking on RNA therapeutics for this disease.

 

Other points:

The section on animal models is very heavy in pig studies, which is great, but this is not comprehensive. Multiple pig models other than those presented have been developed and used for ventilatory and therapeutics studies that should be mentioned (e.g., Nieman and colleagues). Further, there is really no discussion of any mouse or rat model, despite all siRNA studies to date for ARDS have been carried out in these species. While a number of excellent reviews exist on the benefits and limitations to mouse models, at least a short description here would be beneficial.

 

There is no discussion of miRNAs or the relation of (similarities, differences, etc) siRNAs and miRNAs for knockdown of target genes in ARDS models, despite a number of published studies.

 

When discussing the various papers using siRNAs to prevent ARDS, quantitative results from the papers should be provided.

Author Response

Dear Reviewer 1,

Thank you for your careful reading of manuscript Small Interfering RNA (siRNA) as a Targeted Therapy for Acute Respiratory Distress Syndrome: Evidence from Experimental Models and for your valuable comments. Your suggestions have significantly helped us improve the quality of our work. We hope that all the changes made are sufficient and will fully address the shortcomings.

 

The review by Kiseleva on “Small Interfering RNA (siRNA) as a Targeted Therapy for 2 Acute Respiratory Distress Syndrome: Evidence from Experimental Models” provides background on the pathophysiology of direct and indirect ARDS, a number of different large animal models for the disease, and the mechanisms of siRNA action. It also provides discussion of several published papers using siRNAs to prevent or limit severity of ARDS in small animal models. While the discussions of animal models, ARDS pathophysiology, and siRNA activity are very well developed and informative, the discussion on siRNA use and results in ARDS settings lack the same depth and information. This is a letdown since the title of the review deals with the use of siRNA for ARDS.

Thank you for your feedback. A more detailed analysis of the studies has been added. And section 4 was reorganized.

 The major limitation of the manuscript is the lack of detail and analysis of the papers presented that actually use siRNAs to knock down genes that may be relevant in ARDS. It would be highly informative to provide quantitative data and results from these papers showing exactly how effective the siRNAs are in these models and papers. How much knockdown is achieved and does this affect the “treatment”? what is the distribution of siRNAs/knock down genes in the lungs of the animals in the different papers?

Thank you for the recommendation.The knockdown efficacy data are shown in Table 3. A discussion of these results has been added in Section 4.   Unfortunately, the authors of the studies described do not provide information on knockdown in organs other than the lungs.

In the presented studies, due to the lack of standardized presentation of data on knockdown efficiency (relative protein amount according to Western blot analysis or relative gene expression level according to PCR), the analysis of the correlation between knockdown efficiency and therapeutic outcome is complicated, however, based on the available data, a rough estimate of dose-dependent efficacy was made. In studies where the knockdown level was >50%, a pronounced clinical effect was observed, namely, in the work of Krupa et al., a knockdown efficiency of 92% correlated with a strong decrease in lung injury indices (alveolar hemorrhage, edema, infiltration); 50% knockdown of infection-induced CCL2 mRNA led to a significant decrease in cell infiltration, viral load and improved histology; a decrease in the relative amount of HDAC7 protein >40% led to a significant decrease in the number of neutrophils, TNF-α in BAL and a 3-fold increase in survival; 50% knockdown efficiency of LCN2 resulted in comprehensive improvements including the control of ferroptosis. Thus, the knockdown level is a critical parameter for the effectiveness of siRNA therapy. Most targets require at least 40-50% suppression of expression to achieve a significant therapeutic effect. Analysis of the presented studies shows that the higher the knockdown level, the more pronounced the therapeutic effect. This seems logical, as interrupting key pathogenic cascades requires a significant reduction in the activity of the target protein.

Further, discussions of controls (non-targeted siRNAs, etc) should also be included for each paper discussed and the main body of the review should be on the section dealing with the use of siRNAs for ARDS.

Thank you for your comment. Discussions of controls were added.

Similar to the lack of standardization in reporting knockdown efficiency, the studies reviewed used different types of controls:

1. Scrambled/non-targeted siRNA (sh-NC): This is the "gold standard" control. These oligonucleotides have a random sequence, not complementary to any known genome, but are similar in length, charge, and chemical structure to the active siRNA. They allow us to separate sequence-specific effects (gene targeting) from non-specific effects of the RNA molecule itself, activation of immune receptors (e.g., TLRs), or components of the delivery system. In the tables, this control was used for TIMP1, HDAC7, NOX4, LCN2, and CCL2 (mock). This significantly increases the reliability of the data. The use of non-targeted siRNA against the background of ARDS induction in studies of BTK (Krupa et al.), NOX4, and CCL2 demonstrates that the therapeutic effect is not a consequence of non-specific interference of the siRNA itself or the carrier in the pathological process.
2. Control with only the delivery system (liposomes, nanoparticles) or solvent (PBS, saline): This control (mock, PBS control) allows us to assess the impact of the administration procedure and the carriers themselves on disease development. For example, in the TIMP1 study, there was a separate "2X3-DOPE only" group, which is appropriate, as liposomes themselves can exert an immunomodulatory effect. Similarly, in the TNF-α and TLR4 studies, PBS control was necessary to assess the underlying inflammation caused by LPS.
3. Sham group: Used in surgically induced models (CLP for BTK and NOX4). The Sham group undergoes all manipulations except the key damaging effect (the puncture itself and ligature), allowing for an assessment of the impact of surgical stress.

However, the study by Zhou et al. lacked a group with off-target siRNA, and comparisons were made only with the Mock (saline) and Sham groups [100]. This weakens the evidence base, as it does not completely exclude non-specific effects of naked siRNA administration. Also, many studies lack data on systemic toxicity or an assessment of off-target effects (with the exception of CCL2 and TLR4, where this was done). Although scrambled siRNAs partially control for off-target effects, the use of transcriptome analysis methods to identify them would be ideal. Nevertheless, in most of the presented studies, the design of control groups is adequate and includes key elements (scrambled siRNAs, vehicle control), which increases the reliability of conclusions about the specificity of the studied siRNAs.

 

There is a lack of discussion on how each siRNA is delivered (how much, delivery route, delivery agent, complexity of siRNA – single or pool of siRNAs, etc); this could be very important and having all the information in one review would be of great benefit for those thinking about embarking on RNA therapeutics for this disease.

Thank you for your comment. We have added a discussion on how and when each siRNA is delivered to Section 4.

 The presented studies demonstrate a wide range of approaches to siRNA delivery. Intranasal administration of siRNA offers the advantages of noninvasiveness, local delivery to the lungs, minimized systemic exposure, and the ability to target specific cells (neutrophils). However, it depends on the condition of the mucosa and mucociliary clearance and tends to be unevenly distributed. Intranasal administration in animals, analogous to nebulization in intubated patients, appears to be the most realistic route for clinical use of siRNA. Intratracheal administration, a relatively simple procedure in animals that is difficult to translate to humans, offers the significant advantage of direct delivery of siRNA to the lower respiratory tract. Use of the intravenous route is associated with a high probability of systemic exposure, the need for careful fine-tuning of the administration technique, and, most importantly, requires highly specific lung targeting systems (e.g., peptides, pulmonary endothelial antibodies). Without these, the risk of side effects is high.

The strategy of targeting neutrophils with siRNA (via anti-Gr-1) in ARDS is promising and effective in clinical trials [99]. However, clinical translation requires addressing the immunogenicity of antibody conjugates, cost, and the search for even more specific markers of activated neutrophils in ARDS. Analysis of the presented studies also demonstrates the need for carriers: experience with naked siRNA demonstrated that even direct intratracheal administration is effective, but this is insufficient for clinical use [100]. The development of safe, effective, and stable nanocarriers such as lipid nanoparticles and polymer complexes is a prerequisite for moving to clinical trials. All studies used single siRNAs. Pooled siRNAs (a mixture targeting different regions of a single mRNA) could theoretically improve knockdown efficiency, but they complicate the regulatory process and increase the risk of off-target effects.

A serious problem with translating the results to the clinic, identified in the described papers, is the prophylactic or very early therapeutic administration of siRNAs in most experiments. For example, an anti-Timp1 siRNA was administered 8 days prior to LPS-induced injury, an anti-Btk siRNA (Zhou et al.) was administered 1 hour prior to CLP, and an anti-HDAC7 siRNA was delivered 72 hours prior to bacterial instillation [98,100,103]. This approach shows that suppressing these genes can prevent or markedly reduce the development of lung damage. However, it does not adequately model the clinical reality of ARDS, when treatment is initiated after the syndrome already has a vivid colic picture and the inflammatory cascade is fully active. This represents a major obstacle to clinical implementation, as the prophylactic efficacy of siRNA therapy cannot directly predict its efficacy in saving tissues from ongoing damage. The inflammatory environment of advanced ARDS, characterized by massive neutrophil infiltration, protease release, and vascular leakage, has the potential to disrupt exposed siRNA or interfere with the effectiveness of delivery systems, thereby reducing the therapeutic effect. From a methodological perspective, prophylactic administration of siRNA is rigorous and informative. It allows for clear proof of a causal relationship between the target gene and the pathogenesis of ARDS. If gene knockdown, performed before the onset of injury, prevents the development of the syndrome, this serves as direct evidence of the gene's key role in initiating the cascade. This approach allows for the assessment of the maximum therapeutic potential of siRNA under idealized, controlled conditions, where the delivery system and siRNA molecules do not encounter barriers of established inflammation, and for the optimization of delivery and dosing parameters in models with a more predictable background. However, the translational interpretation of such data is extremely limited. The clinical reality of ARDS is that the patient is admitted to the intensive care unit with an already manifested, often rapidly progressing, syndrome. The physician is dealing not with the risk of development, but with an active, advanced pathological process. Consequently, the model of prophylactic siRNA administration does not adequately simulate the clinical scenario. Success in preventing injury does not guarantee success in reversing or halting it. This creates a significant gap between preclinical efficacy and clinical applicability, representing one of the key factors contributing to the high failure rate when moving from animal models to clinical trials (the so-called "valley of death" of translational medicine).

Therapeutic (post-traumatic) designs, where intervention occurs after the initiation of the pathological process, have greater clinical relevance. For example, in the study by Krupa et al., siRNA was administered 8 hours after the first "hit" (LPS) but before the second (immune complexes) [99]. This model intervenes during the priming and progression phases of inflammation. In the study by Heloísa Athaydes Seabra Ferreira, siRNA to CCL2 was administered 1 and 3 days after SARS-CoV-2 infection, that is, during the period of active viral replication and developing inflammation [104]. In the study by Xiaodong Wang, a plasmid containing shRNA to LCN2 was administered 1 hour after LPS, that is, during the incipient systemic inflammatory response [101]. These studies have immeasurably greater translational value. They demonstrate that siRNA therapy can modulate an already active inflammatory cascade, which is critically important, as in ARDS, many mediators are already released, cells are activated, and barriers are damaged. They also demonstrate the effectiveness of siRNA in developing ARDS, characterized by an abundance of proteases, nucleases, oxidative stress, and cellular debris, which can degrade siRNA and nanocarriers. Furthermore, these studies demonstrate that siRNA influences survival and damage resolution, not just its prevention.

The distinction between preventive and therapeutic designs for siRNA studies in ARDS is not simply a methodological nuance, but a fundamental question of translational relevance. While preventive studies prove the concept and identify key targets, they say little about actual clinical utility. The future of preclinical development of siRNA therapeutics for ARDS must be inextricably linked to strict adherence to the principle of clinical realism in experimental design. Only studies in which the intervention simulates a real-world clinical situation (treatment of an already established syndrome) will be able to generate reliable data justifying the transition to expensive and risky human clinical trials. Shifting the paradigm from "can this prevent disease?" to "can this cure active disease?" is essential for bridging the gap between promising laboratory results and the development of effective treatments for critically ill patients.

Other points:

The section on animal models is very heavy in pig studies, which is great, but this is not comprehensive. Multiple pig models other than those presented have been developed and used for ventilatory and therapeutics studies that should be mentioned (e.g., Nieman and colleagues). Further, there is really no discussion of any mouse or rat model, despite all siRNA studies to date for ARDS have been carried out in these species. While a number of excellent reviews exist on the benefits and limitations to mouse models, at least a short description here would be beneficial.

Thank you for this note. Section 3 has been reorganized. Additionally, Figure 1 ("Clinically Relevant Models of ARDS in Large Animals") has been added.

There is no discussion of miRNAs or the relation of (similarities, differences, etc) siRNAs and miRNAs for knockdown of target genes in ARDS models, despite a number of published studies.

Thanks for the suggestion. References to seminal works on this topic have been added, and Table 1 ("Main Differences Between siRNA and microRNA") has been included.

 A detailed comparison of microRNA and siRNA as potential therapeutic agents is described in the papers by Lam JK et all and Wang P et all therefore below is a table 2 reflecting the main differences between these molecules [31–33].

Table 2. Main differences between siRNA and microRNA.

siRNA	miRNA
Origin [34,35]
Generally exogenous (from outside the cell, e.g., introduced experimentally or viral RNA)	Endogenous (naturally encoded by the genome as non-coding RNA)
Structure [36,37]
Double-stranded RNA, ~21-23 nucleotides with 2-nucleotide 3' overhangs	Single-stranded from a hairpin precursor; mature miRNA ~19-25 nucleotides, forms imperfect duplex
Biogenesis [36,37]
Derived from long double-stranded RNA processed by Dicer	Transcribed as primary miRNA, processed to pre-miRNA hairpin, then cleaved by Dicer
Target binding [33,38]
Perfect or near-perfect complementarity to a single mRNA target	Partial complementarity, mainly binding 3' UTR of multiple mRNAs
Number of targets [33]
One specific mRNA target per siRNA	Multiple mRNA targets per miRNA, can regulate hundreds of genes
Mechanism of gene silencing [33]
Cleaves target mRNA, leading to its degradation	Represses translation or destabilizes mRNA without direct cleavage
Function in the cell [33]
Defense against viruses, transposons, or experimental gene silencing	Endogenous regulation of gene expression and fine-tuning of biological pathways

When discussing the various papers using siRNAs to prevent ARDS, quantitative results from the papers should be provided.

Thank you for your comment. Key information on the numerical parameters has been added to Table 4 (“Control groups and Main effect on the lung of siRNA used in vivo to treat ALI/ARDS”).

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors This review provides a comprehensive and well-organized overview of siRNA-based therapeutic strategies for ARDS, with particular strength in its detailed discussion of animal models and target selection. However, a major conceptual limitation is that most of the cited siRNA interventions are administered prophylactically or very early in disease induction, which only partially reflects the clinical reality of ARDS, where treatment is initiated after disease onset. This issue is particularly relevant for interpreting the translational value of targets such as TIMP1, BTK, and HDAC7, as preventive knockdown may overestimate therapeutic efficacy. The authors should more explicitly acknowledge this limitation and, where possible, distinguish between studies that model true therapeutic intervention versus those that demonstrate mechanistic proof-of-concept. Addressing this point would strengthen the clinical relevance of the review and provide clearer guidance for future preclinical study design.   1. Major Comments: Translational relevance of prophylactic siRNA administration A key limitation of the current literature summarized in this review is that many siRNA interventions are administered prophylactically or very early relative to ARDS induction, rather than after disease establishment. While these studies are valuable for mechanistic validation, they may overestimate therapeutic efficacy compared with real-world clinical scenarios, where ARDS treatment typically begins after diagnosis. This issue is evident in several highlighted targets (e.g., TIMP1, BTK, HDAC7), where siRNA delivery precedes or coincides closely with injury induction. The authors are encouraged to explicitly distinguish between preventive and therapeutic study designs and to discuss how this distinction affects translational interpretation and future preclinical trial design. Strengthening this discussion would substantially enhance the clinical relevance of the review.   2. Minor Comments Clarification of dosing and timing inconsistencies In Table 2, the timing and dosing of siRNA administration are not consistently reported across studies. Where original publications lack precise quantitative information, this limitation should be explicitly stated to avoid the impression of missing data. Standardization of terminology The manuscript alternates between “ALI” and “ARDS” in some sections describing animal models. Although related, these terms are not interchangeable, and clearer differentiation would improve conceptual precision. Delivery-related immune activation While innate immune activation by siRNA and lipid nanoparticles is discussed, a brief summary comparing pulmonary versus systemic delivery routes in terms of immunogenicity and off-target inflammation would improve coherence. Figure/Table readability Table 2 is informative but dense. Consider simplifying column headings or splitting it into two tables (e.g., gene targets vs. delivery strategies) to improve readability.  

Author Response

Dear Reviewer 2,

Thank you for your careful reading of manuscript Small Interfering RNA (siRNA) as a Targeted Therapy for Acute Respiratory Distress Syndrome: Evidence from Experimental Models and for your valuable comments. Your suggestions have significantly helped us improve the quality of our work. We hope that all the changes made are sufficient and will fully address the shortcomings.

1. Major Comments: Translational relevance of prophylactic siRNA administration A key limitation of the current literature summarized in this review is that many siRNA interventions are administered prophylactically or very early relative to ARDS induction, rather than after disease establishment. While these studies are valuable for mechanistic validation, they may overestimate therapeutic efficacy compared with real-world clinical scenarios, where ARDS treatment typically begins after diagnosis. This issue is evident in several highlighted targets (e.g., TIMP1, BTK, HDAC7), where siRNA delivery precedes or coincides closely with injury induction. The authors are encouraged to explicitly distinguish between preventive and therapeutic study designs and to discuss how this distinction affects translational interpretation and future preclinical trial design. Strengthening this discussion would substantially enhance the clinical relevance of the review.

Thank you for this suggestion. Discussion of timing siRNA administration was added at section 4.

The presented studies demonstrate a wide range of approaches to siRNA delivery. Intranasal administration of siRNA offers the advantages of noninvasiveness, local delivery to the lungs, minimized systemic exposure, and the ability to target specific cells (neutrophils). However, it depends on the condition of the mucosa and mucociliary clearance and tends to be unevenly distributed. Intranasal administration in animals, analogous to nebulization in intubated patients, appears to be the most realistic route for clinical use of siRNA. Intratracheal administration, a relatively simple procedure in animals that is difficult to translate to humans, offers the significant advantage of direct delivery of siRNA to the lower respiratory tract. Use of the intravenous route is associated with a high probability of systemic exposure, the need for careful fine-tuning of the administration technique, and, most importantly, requires highly specific lung targeting systems (e.g., peptides, pulmonary endothelial antibodies). Without these, the risk of side effects is high.

The strategy of targeting neutrophils with siRNA (via anti-Gr-1) in ARDS is promising and effective in clinical trials [99]. However, clinical translation requires addressing the immunogenicity of antibody conjugates, cost, and the search for even more specific markers of activated neutrophils in ARDS. Analysis of the presented studies also demonstrates the need for carriers: experience with naked siRNA demonstrated that even direct intratracheal administration is effective, but this is insufficient for clinical use [100]. The development of safe, effective, and stable nanocarriers such as lipid nanoparticles and polymer complexes is a prerequisite for moving to clinical trials. All studies used single siRNAs. Pooled siRNAs (a mixture targeting different regions of a single mRNA) could theoretically improve knockdown efficiency, but they complicate the regulatory process and increase the risk of off-target effects.

A serious problem with translating the results to the clinic, identified in the described papers, is the prophylactic or very early therapeutic administration of siRNAs in most experiments. For example, an anti-Timp1 siRNA was administered 8 days prior to LPS-induced injury, an anti-Btk siRNA (Zhou et al.) was administered 1 hour prior to CLP, and an anti-HDAC7 siRNA was delivered 72 hours prior to bacterial instillation [98,100,103]. This approach shows that suppressing these genes can prevent or markedly reduce the development of lung damage. However, it does not adequately model the clinical reality of ARDS, when treatment is initiated after the syndrome already has a vivid colic picture and the inflammatory cascade is fully active. This represents a major obstacle to clinical implementation, as the prophylactic efficacy of siRNA therapy cannot directly predict its efficacy in saving tissues from ongoing damage. The inflammatory environment of advanced ARDS, characterized by massive neutrophil infiltration, protease release, and vascular leakage, has the potential to disrupt exposed siRNA or interfere with the effectiveness of delivery systems, thereby reducing the therapeutic effect. From a methodological perspective, prophylactic administration of siRNA is rigorous and informative. It allows for clear proof of a causal relationship between the target gene and the pathogenesis of ARDS. If gene knockdown, performed before the onset of injury, prevents the development of the syndrome, this serves as direct evidence of the gene's key role in initiating the cascade. This approach allows for the assessment of the maximum therapeutic potential of siRNA under idealized, controlled conditions, where the delivery system and siRNA molecules do not encounter barriers of established inflammation, and for the optimization of delivery and dosing parameters in models with a more predictable background. However, the translational interpretation of such data is extremely limited. The clinical reality of ARDS is that the patient is admitted to the intensive care unit with an already manifested, often rapidly progressing, syndrome. The physician is dealing not with the risk of development, but with an active, advanced pathological process. Consequently, the model of prophylactic siRNA administration does not adequately simulate the clinical scenario. Success in preventing injury does not guarantee success in reversing or halting it. This creates a significant gap between preclinical efficacy and clinical applicability, representing one of the key factors contributing to the high failure rate when moving from animal models to clinical trials (the so-called "valley of death" of translational medicine).

Therapeutic (post-traumatic) designs, where intervention occurs after the initiation of the pathological process, have greater clinical relevance. For example, in the study by Krupa et al., siRNA was administered 8 hours after the first "hit" (LPS) but before the second (immune complexes) [99]. This model intervenes during the priming and progression phases of inflammation. In the study by Heloísa Athaydes Seabra Ferreira, siRNA to CCL2 was administered 1 and 3 days after SARS-CoV-2 infection, that is, during the period of active viral replication and developing inflammation [104]. In the study by Xiaodong Wang, a plasmid containing shRNA to LCN2 was administered 1 hour after LPS, that is, during the incipient systemic inflammatory response [101]. These studies have immeasurably greater translational value. They demonstrate that siRNA therapy can modulate an already active inflammatory cascade, which is critically important, as in ARDS, many mediators are already released, cells are activated, and barriers are damaged. They also demonstrate the effectiveness of siRNA in developing ARDS, characterized by an abundance of proteases, nucleases, oxidative stress, and cellular debris, which can degrade siRNA and nanocarriers. Furthermore, these studies demonstrate that siRNA influences survival and damage resolution, not just its prevention.

The distinction between preventive and therapeutic designs for siRNA studies in ARDS is not simply a methodological nuance, but a fundamental question of translational relevance. While preventive studies prove the concept and identify key targets, they say little about actual clinical utility. The future of preclinical development of siRNA therapeutics for ARDS must be inextricably linked to strict adherence to the principle of clinical realism in experimental design. Only studies in which the intervention simulates a real-world clinical situation (treatment of an already established syndrome) will be able to generate reliable data justifying the transition to expensive and risky human clinical trials. Shifting the paradigm from "can this prevent disease?" to "can this cure active disease?" is essential for bridging the gap between promising laboratory results and the development of effective treatments for critically ill patients.

 

2. Minor Comments Clarification of dosing and timing inconsistencies In Table 2, the timing and dosing of siRNA administration are not consistently reported across studies. Where original publications lack precise quantitative information, this limitation should be explicitly stated to avoid the impression of missing data.

Thanks for this observation. Table 2 was reorganized and corrected.

Standardization of terminology The manuscript alternates between “ALI” and “ARDS” in some sections describing animal models. Although related, these terms are not interchangeable, and clearer differentiation would improve conceptual precision.

Thank you for your comment. When writing this article, the authors have maintained the terminology used in the original studies.

 

Delivery-related immune activation While innate immune activation by siRNA and lipid nanoparticles is discussed, a brief summary comparing pulmonary versus systemic delivery routes in terms of immunogenicity and off-target inflammation would improve coherence.

Thanks for comment. A discussion of direct (intranasal/intratracheal) and systemic routes of siRNA administration in terms of activation of the immune response has been added to the text.

It is also important to note that direct administration of siRNA into the respiratory tract (via instillation, inhalation, or nebulization) enables high local drug concentrations to be achieved directly in epithelial cells, alveolar macrophages, and other resident lung cells. In contrast, intravenous administration results in systemic siRNA distribution. Achieving therapeutic concentrations in the lungs then requires either very high doses or the use of complex targeting systems (e.g., those directed at the pulmonary endothelium)[52,53]. Thus, while local administration minimizes systemic exposure, it carries a significant risk of immunogenicity, as double-stranded RNA can be recognized by toll-like receptors (TLRs), particularly TLR3, TLR7, and TLR8, on the surface of pulmonary epithelium and alveolar macrophages. Unmodified or poorly modified siRNA, as well as drug contaminants, can activate these receptors. This triggers the local production of type I interferons (IFN-α/β) and proinflammatory cytokines (e.g., TNF-α, IL-6) directly at the site of inflammation [108]. In ARDS, this is tantamount to "adding fuel to the fire," potentially exacerbating an existing cytokine storm. Such localized inflammation can increase pulmonary edema and cellular infiltration, which may mask the therapeutic effect or even worsen the patient's condition. Notably, however, the risk of systemic off-target inflammation is low with this route, and severe manifestations like fever or systemic cytokine release are less likely than with intravenous administration. Proper siRNA design (using chemical modifications such as 2'-OMe or 2'-F) and biocompatible carriers can help mitigate these local side effects [46,98]. The key advantage remains the localization of the effect to the lungs, thereby avoiding systemic off-target effects in organs like the liver or kidneys. Conversely, systemic administration exposes siRNA and its carrier to a vast pool of immune cells throughout the body, including monocytes and macrophages in the liver and spleen, dendritic cells, B cells, and circulating neutrophils [109]. Activation via TLR7/8 in these cells can trigger a massive, systemic release of interferons and cytokines, leading to a condition resembling a "flu-like syndrome" (fever, myalgia, fatigue). For a critically ill ARDS patient on the verge of decompensation, this additional systemic inflammatory load can be particularly dangerous. Furthermore, intravenous administration is characterized by a pronounced "first-pass" effect through the liver, where most intravenously administered nanoparticles accumulate. This can lead to significant local immune activation in hepatic cells (hepatocytes, Kupffer cells), posing a potential risk of hepatotoxicity, as documented for some lipid nanoparticle (LNP) formulations[110]. A single, yet significant, advantage of systemic siRNA administration is its ability to target extrathoracic lesions in conditions like sepsis-induced ARDS. If the pathogenesis is triggered by a distant source (e.g., peritonitis), systemic administration could theoretically modulate inflammation at its origin. However, this approach demands absolute target specificity for the pathological process to avoid widespread adverse effects.

 

Figure/Table readability Table 2 is informative but dense. Consider simplifying column headings or splitting it into two tables (e.g., gene targets vs. delivery strategies) to improve readability.

Thank you for your comment. To improve readability, Table 2 has been split into two tables as per your suggestion.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Attached

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 3,

Thank you very much for your positive assessment  regarding our literature review, "Small Interfering RNA (siRNA) as a Targeted Therapy for Acute Respiratory Distress Syndrome: Evidence from Experimental Models."

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed previous comments raised by the reviewers and provide a more comprehensive and complete review of the literature. The resulting revised manuscript is much improved.

Reviewer 3 Report

Comments and Suggestions for Authors

Acceptable