Small Extracellular Vesicle Enrichment of a Retrotransposon-Derived Double-Stranded RNA: A Means to Avoid Autoinflammation?
and Ken C. Pang 5,6,7,8,*
Round 1
Reviewer 1 Report
This is a hight interesting manuscript in which the authors show that dendritic cells (DC)-derived small extracellular vesicles (SEVs) are enriched in a long noncoding RNA (lncRNA), VL30, compared to their parental cells. SEV-enriched isoforms of VL30 RNA display a tandemly repeated motif that is preferentially loaded into SEVs. However, the overexpression of this motif alone in cells results toxic. In conclusion, authors identify an “exo-motif” by which cells remove the potentially toxic retroviral RNA while avoiding the damage. The article by Marilou H. Barrios and colleagues deals with a high interesting aspect, providing further information on how therapeutic RNAs can be more efficiently packaged into SEVs by the addition of an exo-motif in the RNA sequence of interest.
The paper is well organised and written, although some minor changes must be corrected.
- Abbreviations should be explained what it refers to from the first time it is mentioned.
- Line 1: SEVs is defined in the abstract but not in the introduction.
- lncRNA is not clarified in the text and suddenly appears in line 206. Introduce the abbreviation in line 59.
- Line 208: BMDCs is not previously defined. Introduce the abbreviation in line 81.
- Cell/gene abbreviations are differently written in the text and in the figures:
- Line 240: WEHI-231 and NIH-3T3 while in Figure 2: W231 and NIH/3T3.
- Line 309 and figure legend 5 (IFIT1, IRF7 and MDA5) while in Figure 5 (IFIT-1, IRF-7 and MDA-5).
- The “material and method” section is adequately described although some minor details should be clarified:
- Line 79: Introduce the ethical code number approved by the Animal Ethical Committee.
- Line 101: Specify the commercial company of the RNase employed in the experiment.
- Section 2.11.
- Line 183: eliminate the “(“located before "with".
- Unify the writing of the spaces to refer to the units (nM, <, >), separately or together with the numbers, throughout the paragraph (i.e. lines 183 and 185), and the results (i.e. lines 224, 230, 231…)
- Line 246: Correct the reference.
- Line 307: eliminate the “9” introduced before induces.
- Line 252: remove the “s” between SEVs and contain.
Questions:
- Is five minutes enough time for RNase treatment? Please check this point as the most protocols are 15 to 30 minutes.
- Figure 2: what does mean mB-actin (Y-axis)? What is the different with B-actin studied in figure 4B?
Suggestions:
I find the experiments well designed. The figures are concise, comprehensive with figure legends self-explanatory. Authors identify one lncRNA-motif sequence that promotes RNA-SEV loading. To further confirm this hypothesis through different approach and/or point of view, I suggest make a VL30-construct without the “motif sequence” and study its incorporation under the same conditions performed with the full length VL30 lncRNA and the motif alone.
Line 50 “…certain motifs govern the sorting of miRNAs into exosomes…” can also be supported by a citation of a recent manuscript (doi: 10.1111/bph.15421. PMID: 33644849) in which the authors mention certain proteins and specific sequence motifs that seem to facilitate the selective miRNA sorting into extracellular vesicles.
I consider that the conclusion reflected in point 5 is supported by the results.
“Personal suggestion”: To explain the cell toxicity induced in cells by the incorporation of huge copies of motif-only VL30, it would be interesting discuss about disorders (some neuropathologies, including Huntington´s and/or Kennedy´s diseases) occasioned by tandem repeated sequences which, in some way, results harmful to the cells and therefore to the organism. Therefore, the "exo-motif" studied in this manuscript may not be toxic by itself but the immunostimulatory response may be produced by the tandem repeat originating a secondary structure that may also be produced by other tandem repeat sequences not being exclusively of this motif. Moreover, although the overexpression of this tandemly-repeated motif promotes auto-inflammatory response in-vitro, could not reflect the response in an in-vivo model. I understand that this point is outside of this manuscript scope, but I find a good point to keep in mind.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In the present work, the authors identified a 26-nucloetide RNA motif that is efficiently incorporated in SEVs. These results might be of great interest for researchers involved in the study of SEVs as RNA delivery vehicles.
The manuscript is very intersting and very well presented.
Some minor typo errors only (please chack also lines 246-247).
I would also suggest to remove borders from panels in Figure 1.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript by Barrios et al. identified a repetitive 26-nucleotide motif that directs RNA loading into small extracellular vesicles (SEVs). This motif is present in a long noncoding RNA of retroviral origin (VL30). The loading mechanism by which specific RNAs are significantly enriched in the exosome fraction is poorly understood and subject to intense investigation. In this regard, the results obtained might provide a way to load therapeutic RNAs into SEVs during their production by cells. Thus, the manuscript is interesting and has important implications for the field of therapeutics.
The manuscript is very well written, a pleasure to read. The results are clearly presented, the methodology is robust, and the experiments were carefully done. The SEVs were thoroughly characterized according to the ISEV guidelines. The discussion is adequate and clearly states the implications and limitations of the study.
I only have a few comments to help improve the readability of the manuscript:
Methods section (line 85). What concentration of GM-CSF was used in the culture to obtain dendritic cells?
Figure 2. In my opinion, it would help to cut the y-axis in order to see the value for VL30 expression in cells. In this way, it will be possible to appreciate approximately the fold increase. For example, the text says that “abundance in SEVs range from 500- to 3000-fold higher than in parental cells”, but in Figure 2c seems much higher than that as cells value cannot be read. I have a similar comment for Figure 4, b and c.
Line 246. It says “(Error! Reference source not found.)” ???
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
