Why Extracellular Vesicles Are Attractive Vehicles for RNA-Based Therapies?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Aqel F et al reviewed the origin and classification, composition, isolation, characterization, engineering and application in RNA delivery of extracellular vesicles (EVs). I think this manuscript needs major revision and is not suitable for publishing in SynBio in its current form. Here are some comments of this manuscript for authors.

 

1. I think the abstract should be rewritten and should mainly focus on extracellular vesicles (EVs).

 

2. The title of the manuscript is “Engineered Extracellular vesicles: A futuristic approach for targeted delivery of RNA therapeutics”, the origin and classification, composition, isolation and characterization occupy half of the manuscript. The authors should pay more attention to their application in RNA delivery and treatment of various diseases.

 

3. The authors should give the application field of RNA-loaded extracellular vesicles (EVs), for example what diseases can they be used to treat.

 

4. The section 3 of the manuscript should be re-organized and give a clear description about the application of extracellular vesicles (EVs).

 

5. The authors should carefully improve the English language throughout the manuscript.

Comments on the Quality of English Language

The authors should carefully improve the English language throughout the manuscript.

Author Response

Comment 1: I think the abstract should be rewritten and should mainly focus on extracellular vesicles (EVs). 

Response 1: We would like to thank the Reviewer for this point. We agree with the Reviewer that abstract should be rewritten. In the revised version of the manuscript updated version of the abstract with the focus on EVs is included:

Extracellular Vesicles (EVs) are a focus of intense research worldwide, with many groups exploring their potential for both diagnostic and therapeutic applications. Researchers have characterized EVs into various subtypes, modified common surface markers, and developed diverse isolation and purification techniques.

Beyond their diagnostic potential, EVs are being engineered as delivery vehicles for various molecules and therapeutics. RNA therapeutics have the potential to be a transformative solution for patients suffering from chronic and genetic disorders; and generally targeting undruggable targets. Despite the success of many RNA therapeutics in both in vivo studies and clinical trials, a significant challenge remains in effectively delivering these therapies to target cells. Many research groups have adopted the use of lipid nanoparticles (LNPs) and other nanocarriers to encapsulate RNA therapeutics, aiming to deliver them as stably as possible to ensure optimal bioavailability and efficacy. While LNPs have proven successful as delivery vehicles, their use is not without drawbacks, such as accumulation within the body. EVs could be a potential solution to many of the problems around LNPs and other nanocarriers.

 

 

 

Comment 2: The title of the manuscript is “Engineered Extracellular vesicles: A futuristic approach for targeted delivery of RNA therapeutics”, the origin and classification, composition, isolation and characterization occupy half of the manuscript. The authors should pay more attention to their application in RNA delivery and treatment of various diseases. 

Response 2: We would like to thank the Reviewer for this comment. The paper’s aim to first review what has been published on EVs, to describe them, and the way they are isolated and how could they be characterized and modified, because these potential carriers are very heterogenous, and there are a lot of hurdles when it comes to using them in therapeutics (e.g. purification might change functionality; quality control is important part for therapeutic product development and is discussed during the dedicated ISEV meetings) . We believe it is important to give a comprehensive overview of the EVs before we focus on RNA therapeutics. Then as we finish describing the EVs, we move on to other nanocarrier options such as LNPs, and then we describe the RNA therapeutics, their importance, and how they can be integrated into EVs. We have adjusted the title and text of the manuscript with the focus on application of RNA loaded EVs.

Comment 3: The authors should give the application field of RNA-loaded extracellular vesicles (EVs), for example what diseases can they be used to treat. 

Response 3: We thank the Reviewer for raising such an important question. The section 4.3 describing application of  RNA-loaded EV is added: Applications of RNA-loaded EVs   

Comment 4: The section 3 of the manuscript should be re-organized and give a clear description about the application of extracellular vesicles (EVs). 

Response 4:  Thank you very much for the constructive comment.  Sections 3 and 4 were reorganized. Titles were adjusted and we believe it is more coherent now.

Reviewer 2 Report

Comments and Suggestions for Authors

1.    Often the reference authors in the text and their number in the references are separated by several lines as only for example:

·      Radha Munagala (80)

·      Lin et al (81)

·      Coelho et al (93,94)

·      Sato et al (99)

2.    Often the extracellular vesicles, already has a cargo of small RNAs molecules, making it a usable target for molecular diagnosis. The authors did not address this fact or of any attempt to remove the preexisting cargo before loading it with any therapeutic molecules.

Author Response

Comment 1: Often the reference authors in the text and their number in the references are separated by several lines as only for example:

• Radha Munagala (80)
• Lin et al (81)
• Coelho et al (93,94)
• Sato et al (99)

Response 1: We thank the Reviewer for the constructive comment. References were carefully edited and adjusted. We were also facing the problem after the formatting that some of the references were lost and now everything is corrected.

 

Comment 2: Often the extracellular vesicles, already has a cargo of small RNAs molecules, making it a usable target for molecular diagnosis. The authors did not address this fact or of any attempt to remove the preexisting cargo before loading it with any therapeutic molecules.

Response 2: We thank the Reviewer for raising such an important question. The dedicated paragraph "Native cargo of extracellular vesicles" was added to explain this topic as this is one of the most important questions related to the safety and efficiency of the EV as a therapeutic product. In general, the exosomes do not fully get emptied out of cargo before they are loaded, it is not feasible. However, EVs were tested for toxicity in general, therefore the initial cargo in EVs doesn't seem to be a huge problem. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Engineered Extracellular vesicles: A futuristic approach for targeted delivery of RNA therapeutics” tackles an interesting matter. However, as this topic is highly researched, several recent reviews have already exhaustively addressed exosomes and their characteristics, biogenesis, and isolation and purification methods. Furthermore, very recent reviews (2023/2024) focused on exosomes for RNA delivery: 10.3390/pharmaceutics15082042; https://doi.org/10.1016/j.ymthe.2024.02.025; https://doi.org/10.1038/s12276-024-01201-6; https://doi.org/10.3390/biologics4010007. Therefore, as it is, this manuscript lacks novelty and is a recollection of information already published in recent papers. Moreover, it is not well organized, lacks cohesion, and the written information is frequently misleading and incorrectly cited. Overall, the manuscript is not suitable for publication. Please refer to my specific comments below for more details on the reject decision.

> Two of the keywords are 'biomarker' and 'regenerative medicine'. Nevertheless, this is not presented/discussed in the manuscript.

> Line 28: The Introduction starts with references (3,4).

> Lines 36-37; 42-44: The authors mention that a major hurdle of delivery vehicles/nanoparticles is the high immunogenecity/low biocompatibility. However, in lines 30-31, it is stated that these drug delivery vehicles are biocompatible (do not elicit an immune response). 

> The authors focus on the methods of isolation and purification of exosomes; however, this is a subject that has been exhaustively addressed in the literature. The same applies to the characterization methodologies. 

> Lines 35-40: The cited references (7-11) do not contain the written information. The cited papers only refer to exosomes. Also, information in lines 42-44 is lacking reference(s).

> Line 46: the authors consider EVs the same as exosomes. However, exosomes are only one type of EVs. Throughout the manuscript, the authors often use EVs and exosomes interchangeably, which may be confusing to the reader.

> Lines 70-77: It is important to note that the clinical trial was terminated due to company bankruptcy, with no disclosure of results.

> Line 92: the authors should specify "protection against ischemia/reperfusion injury" and not simply "protection against injury". Also, reference (22) does not refer to immune modulation.

> Abbreviations in the text should be revised.

> Lines 96-97 are lacking reference(s).

> In line 52, the authors considered exosomes as having a size of 40-100 nm, while in line 99 is 40-100 nm.

> Line 110: the cited reference mentions that vesicular apoptotic bodies range from 500 nm–2 μm, and not 50-5000 nm.

> Line 116: the author's name is incorrect and the researchers assessed protein-to-lipid ratios, not lipid-to-protein.

> Lines 118-120: reference (29) does not have the written information. 

> Figure 2: EGFR, TNFR, actin, tubulin and MHC II are not described in the text that precedes the figure. Also, TNFR is not represented in the Figure. Lastly, 'ICAM' is not defined.

> Tables 1 and 2 have two titles each.

> Section 2.5. EVs engineering: It would be advantageous to specifically include studies for RNA loading/delivery strategies.

> Lines 355-358: the written information is not in cited reference.

> Line 359: '...hydrophobic outer membrane' is incorrect. Since it is a bilayer, the hydrophilic heads are arranged out and in, and the hydrophobic tails are arranged inside the bilayer.

> Lines 366-367: it is stated that the drug diffuses into the exosomes. However, the cited reference states that it is 'self-assembled into the lipid bilayer of exosomes'. There is no mention of concentration gradient, only the interaction between curcumin and the bilayer.

> Line 385: reference (73) refers to gene transfer into mouse lyoma cells by electroporation, not EVs.

> Lines 386-389 lack reference(s).

> Lines 417-419: the sentence is difficult to understand. The cited reference explores the biogenesis of exosomes and microvesicles, namely how cytoplasmic proteins are 'recruited' into these vesicles. It does not address EVs surface modification.

> Lines 419-421: cited reference does not mention EVs and endosomal escape. 

> Figure 5: in the text, figure 5A and B are not mentioned. Also, Figure 5C appears first than Figure 5B. The extrusion process also physically disrupts the bilayer. It should be included together with electroporation, sonication and freeze/thaw. 

> Section 2.6. Exosomes as delivery vehicles: this section should either be discarded or focus on RNA delivery. 

> Lines 518-524: this is repeated information form previous sections.

> Lines 539-542: Sato et al. does not exist in the reference list.

> In Figure 6, the authors list 'low immunogenecity' and 'low toxicity' as Cons for hybridosomes.

> Section 3.5 should be one with Scetion 3.3.

> It would be advantageous to display a Table with clinical trials of RNA-loaded exosomes.

> Line 586: Tien Vu is not the first author of the cited paper.

> Section 3.6. 'Limitations of the field' should be 'Section 4'. Also, the cons listed in Figure 6 should be further expanded in this subsection. Information in sections 'Endosomal escape' and 'Targeted delivery to specific cell type' relates to surface or other modifications. This should be placed in subsection 2.5.2. 'Surface modifications'.

> All references should be revised. E.g. (27) and (29) are the same, 30 and 38 are missing information...

 

Comments on the Quality of English Language

English language requires moderate revision.

Author Response

We thank the Reviewer for this critical and detailed revision of our work. 

Comment 1: The manuscript entitled “Engineered Extracellular vesicles: A futuristic approach for targeted delivery of RNA therapeutics” tackles an interesting matter. However, as this topic is highly researched, several recent reviews have already exhaustively addressed exosomes and their characteristics, biogenesis, and isolation and purification methods. Furthermore, very recent reviews (2023/2024) focused on exosomes for RNA delivery: 10.3390/pharmaceutics15082042; https://doi.org/10.1016/j.ymthe.2024.02.025; https://doi.org/10.1038/s12276-024-01201-6; https://doi.org/10.3390/biologics4010007. Therefore, as it is, this manuscript lacks novelty and is a recollection of information already published in recent papers. Moreover, it is not well organized, lacks cohesion, and the written information is frequently misleading and incorrectly cited. Overall, the manuscript is not suitable for publication. Please refer to my specific comments below for more details on the reject decision.

Response 1: This review focuses on the use of extracellular vesicles (EVs) as delivery vehicles for RNA therapeutics. EV-based therapeutics, particularly as carriers for RNA, represent an emerging and rapidly evolving field, with the seminal work on RNA transfer between cells reported by Valadi et al. in 2007 (DOI: 10.1038/ncb1596). Currently, no EV-based RNA therapeutic agents have been approved, largely due to the complexity of these systems and the lack of standardized criteria for their evaluation. Despite significant ongoing research, debates persist regarding the impact of EV purification methods on their functionality and safety, as well as the best approaches for EV characterization. The EV characterization field is advancing, with updated guidelines provided by the International Society for Extracellular Vesicles (in the form of MISEV and special working group publications) offering best practices for researchers. 

 

We believe that we have successfully addressed the main challenges and advantages in the field, rather than merely reiterating existing published information. Additionally, we have enhanced the quality of the manuscript by thoroughly addressing the reviewers' comments.

 

Comment 2:  Two of the keywords are 'biomarker' and 'regenerative medicine'. Nevertheless, this is not presented/discussed in the manuscript.

Response 2: Keywords were adjusted by removal of "biomarker" and "regenerative medicine"

Comment 3:> Line 28: The Introduction starts with references (3,4).

Response 3: We thank the Reviewer for highlighting this important point. We believe this happened during the formatting of the references. Now it is adjusted.

Comment 4:  Lines 36-37; 42-44: The authors mention that a major hurdle of delivery vehicles/nanoparticles is the high immunogenecity/low biocompatibility. However, in lines 30-31, it is stated that these drug delivery vehicles are biocompatible (do not elicit an immune response). 

Response 4: Thank you for your comment. We recognize this information was not clear in our manuscript. The statement in lines 36–37 is intended as a general observation: "A major hurdle in drug delivery is the lack of efficient and non-immunogenic delivery vehicles". We have revised the paragraph to emphasize the advantages and challenges of targeted delivery

Comment 5: The authors focus on the methods of isolation and purification of exosomes; however, this is a subject that has been exhaustively addressed in the literature. The same applies to the characterization methodologies. 

Response 5:  We would like to thank the Reviewer for this comment. Despite the ongoing intensive efforts in the field, there are currently no established guidelines for the GMP manufacturing of EV-based therapeutics. One of the aims of this review is to provide a comparative summary of the advantages and limitations of purification and characterization techniques that are relevant for therapeutic development.

Comment 6: Lines 35-40: The cited references (7-11) do not contain the written information. The cited papers only refer to exosomes. Also, information in lines 42-44 is lacking reference(s).

Response 6: We thank the Reviewer for raising this point. We have revised reference list and corrected all related issues

Comment 7: > Line 46: the authors consider EVs the same as exosomes. However, exosomes are only one type of EVs. Throughout the manuscript, the authors often use EVs and exosomes interchangeably, which may be confusing to the reader.

Response 7: We thank the Reviewer for highlighting this important point. The manuscript has been carefully revised, and the authors have replaced potentially confusing terminology with "EVs" to ensure clarity and avoid any misunderstandings. As well as additional explanation in the paragraph was  added to section 2.1: "In theory, one can differentiate between EVs, umbrella-term, and exosomes, a subtype of EVs. However, in practice it is not easy to isolate a particular subtype by having it completely pure of the other subtypes".

Comment 8: Lines 70-77: It is important to note that the clinical trial was terminated due to company bankruptcy, with no disclosure of results.

Response 8:  We thank the Reviewer for pointing out this relevant aspect of our manuscript. The information has been added to the table. In the text we focused on preclinical data.

Comment 9:  Line 92: the authors should specify "protection against ischemia/reperfusion injury" and not simply "protection against injury". Also, reference (22) does not refer to immune modulation.

Response 9: Thank you for the suggestion the text is adjusted. 

Comment 10: Abbreviations in the text should be revised.

Response 10:  Thank you for your suggestion. Abbreviations were revised.

Comment 11: > Lines 96-97 are lacking reference(s).

Response 11: Reference is added.

Comment 12: > In line 52, the authors considered exosomes as having a size of 40-100 nm, while in line 99 is 40-100 nm.

Response 12:  Thank you for the critical comment. The text is revised and size of exosomes is adjusted to "30-100 nm". And comment on literature heterogeneity was added. There is a variability in the literature when it comes to size range of the subtypes of extracellular vesicles. Generally, exosomes are within the smallest size range, microvesicles are bigger than exosomes, and apoptotic bodies are the largest. Overlapping sizes among these 3 categories could be also found in the literature.

Comment 13: Line 110: the cited reference mentions that vesicular apoptotic bodies range from 500 nm–2 μm, and not 50-5000 nm.

Response 13: Thank you for this comment. The size of AB is adjusted. And another reference showing size hdiversity of AB is added  (Reference29)

Comment 14: > Line 116: the author's name is incorrect and the researchers assessed protein-to-lipid ratios, not lipid-to-protein.

Response 14: We apologize for the incorrect wording in the manuscript and have revised the text accordingly.

Comment 15: > Lines 118-120: reference (29) does not have the written information. 

Response 15: The reference was adjusted accordingly.

Comment 16:> Figure 2: EGFR, TNFR, actin, tubulin and MHC II are not described in the text that precedes the figure. Also, TNFR is not represented in the Figure. Lastly, 'ICAM' is not defined.

Response 16: Figure 2 was revised accordingly and information was added to the text.

Comment 17:> Tables 1 and 2 have two titles each.

Response 17: Thank you for pointing this formatting issue out. Tables were adjusted accordingly.

Comment 18:> Section 2.5. EVs engineering: It would be advantageous to specifically include studies for RNA loading/delivery strategies.

Response 18: Thank you for you comment. This information is included in the RNA packaging related section later in the manuscript.

Comment 19:> Lines 355-358: the written information is not in cited reference.

Response 19: The reference is adjusted accordingly.

Comment 20:> Line 359: '...hydrophobic outer membrane' is incorrect. Since it is a bilayer, the hydrophilic heads are arranged out and in, and the hydrophobic tails are arranged inside the bilayer.

Response 20: Thank you for this comment. The text is adjusted.

Comment 21:> Lines 366-367: it is stated that the drug diffuses into the exosomes. However, the cited reference states that it is 'self-assembled into the lipid bilayer of exosomes'. There is no mention of concentration gradient, only the interaction between curcumin and the bilayer.

Response 21: Thank you for this comment. The text is adjusted.

Comment 22: > Line 385: reference (73) refers to gene transfer into mouse lyoma cells by electroporation, not EVs.

Response 22: Thank you for this comment. The reference was adjusted accordingly. 

Comment 23:> Lines 386-389 lack reference(s).

Response 23: Thank you for the comment the reference list is extended.

Comment 24:> Lines 417-419: the sentence is difficult to understand. The cited reference explores the biogenesis of exosomes and microvesicles, namely how cytoplasmic proteins are 'recruited' into these vesicles. It does not address EVs surface modification.

Response 24: We thank the Reviewer for this relevant comment. EV surface engineering strategies also include fusion to plasma membrane anchors (strategy described in the cited reference). In the cited paper GFP fusion was used for visualization of the TyA protein targeting into secreted vesicles. Similar strategies are used for the EV surface engineering: selection of the membrane anchors, design of orientation of selected targeting moiety for the appropriate surface display.

Comment 25> Lines 419-421: cited reference does not mention EVs and endosomal escape. 

Response 25: Thank you for the comment. Corrected reference from Nakase group was added

 

Comment 26:> Figure 5: in the text, figure 5A and B are not mentioned. Also, Figure 5C appears first than Figure 5B. The extrusion process also physically disrupts the bilayer. It should be included together with electroporation, sonication and freeze/thaw. 

Response 26: Thank you for pointing this. The order and the text is adjusted accordingly.

Comment 27:> Section 2.6. Exosomes as delivery vehicles: this section should either be discarded or focus on RNA delivery. 

Response 27: Thank you for the comment. We intend to maintain the sequence in the text and have revised the section discussing endogenous cargo and its potential impact on the loaded cargo.

Comment 28:> Lines 518-524: this is repeated information form previous sections.

Response 28: Thank you for the comment. This section was reorganized.

Comment 29:> Lines 539-542: Sato et al. does not exist in the reference list.

Response 29: Thank you for pointing this issue. This is a result of  refences formatting. Everything is added correctly to the list.

Comment 30:> In Figure 6, the authors list 'low immunogenecity' and 'low toxicity' as Cons for hybridosomes.

Response 30: Thank you for your comment. The field is currently debating the benefits of hybrid systems. While they can significantly improve the immune response to delivery vehicles, considerable effort is still required to develop non-toxic and non-immunogenic alternatives. Consequently, hybrid systems still present certain disadvantages compared to extracellular vesicles.

Comment 30:> Section 3.5 should be one with Scetion3.3

Response 30: Thank you for the important comment. We have rearrange the structure of the manuscript

Comment 31:> It would be advantageous to display a Table with clinical trials of RNA-loaded exosomes.

Resposne 31: We thank the Reviewer for this relevant suggestion. Due to the limited number of RNA-loaded extracellular vesicles in clinical trials, we have included them in Table 2.

Comment 32> Line 586: Tien Vu is not the first author of the cited paper.

Response 32:  Thank you for the comment. The authors adjusted the text based on the first author.

Comment 33:> Section 3.6. 'Limitations of the field' should be 'Section 4'. Also, the cons listed in Figure 6 should be further expanded in this subsection. Information in sections 'Endosomal escape' and 'Targeted delivery to specific cell type' relates to surface or other modifications. This should be placed in subsection 2.5.2. 'Surface modifications'.

Response 33: Thank you very much for your suggestion. The authors have reorganized the manuscript, and the limitations of the field have been made an independent Section 5. However, 'endosomal escape' and 'efficient targeted delivery' remain key challenges in RNA therapeutics, and we believe they should continue to be included within the limitations section.

Comment 34> All references should be revised. E.g. (27) and (29) are the same, 30 and 38 are missing information...

Response 34: Thank you very much for your comments regarding the references. We encountered a reference formatting issue, but all references have been revised and corrected accordingly.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed most of my concerns.

Author Response

Thank you!

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed most of the reviewer's questions. However, the manuscript still lacks novelty.

> In my previous review, I underlined the extensive literature reports on extracellular vesicles/exosomes. The authors replied "This review focuses on the use of extracellular vesicles (EVs) as delivery vehicles for RNA therapeutics." However, the authors chose to maintain the extensively described EVs characterization, biogenesis, isolation and purification methods, not specifically focusing on the use of EVs as RNA delivery tools.

> The authors also focused on LNPs as delivery tools, despite the title refering to extracellular vesicles. Why not also describe other nanosystems for RNA delivery, including liposomes, viruses and polymeric nanoparticles? What are the specificities of EVs that make them "attractive" tools for RNA delivery? The authors state that EVs can be modified to enhance their targeting ability. With this, synthetic molecules would be introduced in this naturally-occuring delivery system, changing its inherent properties. In addition to the complex process of isolation and purification of exosomes, wouldn't this extra step add another bottleneck to clinical translation?

> One of my comments refered to the fact that, in Figure 6, the authors listed for hybridosomes "Low toxicity" and "Low immunogenicity" as disadvantages. Once again, how are low toxicity and low immunogenecity disadvantages? Should nanocarriers display high toxicity and elicite immune responses?

Comments on the Quality of English Language

Minor English language changes.

Author Response

We thank the Reviewer for this additional revision of our work. 

Comment 1:  In my previous review, I underlined the extensive literature reports on extracellular vesicles/exosomes. The authors replied "This review focuses on the use of extracellular vesicles (EVs) as delivery vehicles for RNA therapeutics." However, the authors chose to maintain the extensively described EVs characterization, biogenesis, isolation and purification methods, not specifically focusing on the use of EVs as RNA delivery tools.

Response 1: Thank you once again for this important comment. The authors are active members of the EV community and well-versed in the ongoing discussions in the field of EV-based therapeutics. This review goes beyond merely summarizing existing purification and characterization methods; it highlights key advancements and gaps within the field. A significant challenge in the development of EV-based therapeutics lies in the manufacturing process, particularly achieving GMP-compliant purification and characterization, which remains unstandardized and necessitates extensive dialogue with regulatory authorities. Our review examines these processes through the lenses of complexity, cost-efficiency, scalability, and other critical factors that arise during the manufacturing stage.

Comment 2: > The authors also focused on LNPs as delivery tools, despite the title refering to extracellular vesicles. Why not also describe other nanosystems for RNA delivery, including liposomes, viruses and polymeric nanoparticles? What are the specificities of EVs that make them "attractive" tools for RNA delivery? The authors state that EVs can be modified to enhance their targeting ability. With this, synthetic molecules would be introduced in this naturally-occuring delivery system, changing its inherent properties. In addition to the complex process of isolation and purification of exosomes, wouldn't this extra step add another bottleneck to clinical translation?

Response 2: Thank you for this valuable comment. Authors mentioned all viral and non-viral delivery vehicles in the introduction. LNPs were used in comparison to EVs as the most advanced non-viral vectors or the gold standard for delivering RNA therapeutics. We have now updated the subtitle to 'Packaging Systems – Lipid Nanoparticles as the Gold Standard for Non-Viral RNA Delivery.

Any form of EV modification would require additional effort in purification and characterization. However, the advantages gained through these modifications could potentially enhance current treatment strategies for a broad range of diseases and improve cost efficiency of the process.

Comment 3: > One of my comments refered to the fact that, in Figure 6, the authors listed for hybridosomes "Low toxicity" and "Low immunogenicity" as disadvantages. Once again, how are low toxicity and low immunogenecity disadvantages? Should nanocarriers display high toxicity and elicite immune responses?

Response 3: Thank you once again for highlighting such an important question. The authors share the same opinion as the reviewer. Low toxicity and low immunogenicity are advantageous, except when compared to 'no toxicity' and 'non-immunogenicity. In this context, we highlight the low toxicity and low immunogenicity of hybridosomes in comparison to exosomes, which have been proven to be non-toxic and non-immunogenic. Based on unpublished data presented during EV-dedicated conferences the therapeutic window for hybridosomes is improved compared to LNPs; however, further improvements are still needed to reduce toxicity.