Broad-spectrum immune suppression encoded in self-amplifying RNA enables non-cytotoxic, non-immunostimulatory, externally controllable transgene expression

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

Weaknesses:

(1) Impact on Cellular Translation:

The authors demonstrate that modified saRNAs with additional components enhance transgene expression by inhibiting dsRNA-sensing pathways. However, it is unclear whether these modifications influence global cellular translation beyond the expression of GFP and mScarlet-3 (which are encoded by the saRNA itself). Conducting a polysome profiling analysis or a puromycin labeling assay would clarify whether the modified saRNAs alter overall translation efficiency. This additional data would strengthen the conclusions regarding the specificity of dsRNA-sensing inhibition.

(2) Stability and Replication Efficiency of Long saRNA Constructs:

The saRNA constructs used in this study exceed 16 kb, making them more fragile and challenging to handle. Assessing their mRNA integrity and quality would be crucial to ensure their robustness.
Furthermore, the replicative capacity of the designed saRNAs should be confirmed. Since Figure 4 shows lower inflammatory cytokine production when encoding srIkBα and srIkBα-Smad7-SOCS1, it is important to determine whether this effect is due to reduced immune activation or impaired replication. Providing data on replication efficiency and expression levels of the encoded anti-inflammatory proteins would help rule out the possibility that reduced cytokine production is a consequence of lower replication.

(3) Comparative Data with Native saRNA:

Including native saRNA controls in Figures 5-7 would allow for a clearer assessment of the impact of additional genes on cytokine production. This comparison would help distinguish the effect of the encoded suppressor proteins from other potential factors.

(4) In vivo Validation and Safety Considerations:

Have the authors considered evaluating the in vivo potential of these saRNA constructs? Conducting animal studies would provide stronger evidence for their therapeutic applicability. If in vivo experiments have not been performed, discussing potential challenges - such as saRNA persistence, biodistribution, and possible secondary effects-would be valuable.

(5) Immune Response to Viral Proteins:

Since the inhibitors of dsRNA-sensing proteins (E3, NSs, and L*) are viral proteins, they would be expected to induce an immune response. Analyzing these effects in vivo would add insight into the applicability of this approach.

(6) Streamlining the Discussion Section:

The discussion is quite lengthy. To improve readability, some content - such as the rationale for gene selection-could be moved to the Results section. Additionally, the descriptions of Figure 3 should be consolidated into a single section under a broader heading for improved coherence.

Reviewer #2 (Public review):

Summary:

Lim et al. have developed a self-amplifying RNA (saRNA) design that incorporates immunomodulatory viral proteins, and show that the novel design results in enhanced protein expression in vitro in mouse primary fibroblast-like synoviocytes. They test constructs including saRNA with the vaccinia virus E3 protein and another with E3, Toscana virus NS protein and Theiler's virus L protein (E3 + NS + L), and another with srIκBα-Smad7-SOCS1. They have also tested whether ML336, an antiviral, enables control of transgene expression.

Strengths:

The experiments are generally well-designed and offer mechanistic insight into the RNA-sensing pathways that confer enhanced saRNA expression. The experiments are carried out over a long timescale, which shows the enhance effect of the saRNA E3 design compared to the control. Furthermore, the inhibitors are shown to maintain the cell number, and reduce basal activation factor-⍺ levels.

Weaknesses:

One limitation of this manuscript is that the RNA is not well characterized; some of the constructs are quite long and the RNA integrity has not been analyzed. Furthermore, for constructs with multiple proteins, it's imperative to confirm the expression of each protein to confirm that any therapeutic effect is from the effector protein (e.g. E3, NS, L). The ML336 was only tested at one concentration; it is standard in the field to do a dose-response curve. These experiments were all done in vitro in mouse cells, thus limiting the conclusion we can make about mechanisms in a human system.

Author response:

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

We thank Reviewer #1 for their thorough review and for recognizing both the significance of our work and the potential of our strategy to expand saRNA applications beyond vaccines.

Weaknesses:

(1) Impact on Cellular Translation:

The authors demonstrate that modified saRNAs with additional components enhance transgene expression by inhibiting dsRNA-sensing pathways. However, it is unclear whether these modifications influence global cellular translation beyond the expression of GFP and mScarlet-3 (which are encoded by the saRNA itself). Conducting a polysome profiling analysis or a puromycin labeling assay would clarify whether the modified saRNAs alter overall translation efficiency. This additional data would strengthen the conclusions regarding the specificity of dsRNA-sensing inhibition.

We thank the reviewer for this helpful insight and suggestion. We aim to conduct a puromycin labelling assay to clarify the effect of the various saRNA constructs on translation efficiency.

(2) Stability and Replication Efficiency of Long saRNA Constructs:

The saRNA constructs used in this study exceed 16 kb, making them more fragile and challenging to handle. Assessing their mRNA integrity and quality would be crucial to ensure their robustness.

Furthermore, the replicative capacity of the designed saRNAs should be confirmed. Since Figure 4 shows lower inflammatory cytokine production when encoding srIkBα and srIkBα-Smad7-SOCS1, it is important to determine whether this effect is due to reduced immune activation or impaired replication. Providing data on replication efficiency and expression levels of the encoded anti-inflammatory proteins would help rule out the possibility that reduced cytokine production is a consequence of lower replication.

This is another very helpful comment. We will conduct an analysis of saRNA integrity and quality by denaturing gel electrophoresis. To examine replicative capacity of the saRNA constructs, we aim to conduct RT-qPCR experiments.

(3) Comparative Data with Native saRNA:

Including native saRNA controls in Figures 5-7 would allow for a clearer assessment of the impact of additional genes on cytokine production. This comparison would help distinguish the effect of the encoded suppressor proteins from other potential factors.

Thank you for your suggestion. We will implement this change in the next version of the manuscript.

(4) In vivo Validation and Safety Considerations:

Have the authors considered evaluating the in vivo potential of these saRNA constructs? Conducting animal studies would provide stronger evidence for their therapeutic applicability. If in vivo experiments have not been performed, discussing potential challenges - such as saRNA persistence, biodistribution, and possible secondary effects-would be valuable.

(5) Immune Response to Viral Proteins:

Since the inhibitors of dsRNA-sensing proteins (E3, NSs, and L*) are viral proteins, they would be expected to induce an immune response. Analyzing these effects in vivo would add insight into the applicability of this approach.

We recognize the importance of in vivo studies and immune cell responses and plan to incorporate in vivo imaging in future studies to investigate these interactions, as well as examining delivery of various cargoes via saRNA to determine potential therapeutic benefits in different animal models of inflammatory pain, but such studies are beyond the scope of this current investigation. As suggested by the reviewer, we will incorporate a section on potential challenges of in vivo saRNA work in the revised manuscript.

(6) Streamlining the Discussion Section:

The discussion is quite lengthy. To improve readability, some content - such as the rationale for gene selection-could be moved to the Results section. Additionally, the descriptions of Figure 3 should be consolidated into a single section under a broader heading for improved coherence.

Thank you for your suggestions, we will make these changes in the next revision.

Reviewer #2 (Public review):

Summary:

Lim et al. have developed a self-amplifying RNA (saRNA) design that incorporates immunomodulatory viral proteins, and show that the novel design results in enhanced protein expression in vitro in mouse primary fibroblast-like synoviocytes. They test constructs including saRNA with the vaccinia virus E3 protein and another with E3, Toscana virus NS protein and Theiler's virus L protein (E3 + NS + L), and another with srIκBα-Smad7-SOCS1. They have also tested whether ML336, an antiviral, enables control of transgene expression.

Strengths:

The experiments are generally well-designed and offer mechanistic insight into the RNA-sensing pathways that confer enhanced saRNA expression. The experiments are carried out over a long timescale, which shows the enhance effect of the saRNA E3 design compared to the control. Furthermore, the inhibitors are shown to maintain the cell number, and reduce basal activation factor-⍺ levels.

We thank Reviewer #2 for their detailed assessment and recognition of the mechanistic insights provided by our study.

Weaknesses:

One limitation of this manuscript is that the RNA is not well characterized; some of the constructs are quite long and the RNA integrity has not been analyzed. Furthermore, for constructs with multiple proteins, it's imperative to confirm the expression of each protein to confirm that any therapeutic effect is from the effector protein (e.g. E3, NS, L). The ML336 was only tested at one concentration; it is standard in the field to do a dose-response curve. These experiments were all done in vitro in mouse cells, thus limiting the conclusion we can make about mechanisms in a human system.

We agree that these are weaknesses of our work. We plan to address some of these weaknesses by performing a dose response curve for ML336, examining saRNA integrity through denaturing gel electrophoresis, and will also aim to provide additional evidence for effects of effector proteins through RT-qPCR. We are also looking into testing these constructs in patient-derived FLS.