Standardized Methodologies to Utilize Exosome Treatment as Potential Nano Substances in Hearing Loss

Round 1

Reviewer 1 Report

Overall the author presents an interesting discussion of the application of exosomes for the treatment of hearing loss.

Major comments:

While the article is comprehensive in some aspects, I think there some areas that need to be expanded.

• Firstly, a good description of what exosomes are, how they can be used to deliver payloads, and what their potential payloads are should be included early in the discussion.
• Secondly, as the article concentrates mainly on the methodologies surrounding the isolation and delivery of exosomes, I would be tempted to change the title to include 'standardized methodologies to isolate and test exosome treatment applicability in hearing loss', or something similar.
• Finally, I would try to outline a few different biological mechanisms of hearing loss (chemical induced apoptosis, age related hair cell loss, inflammation) and speculate, with published evidence, how exosomes have been used to disrupt these processes. This does not need to be in depth, but it does need to be presented to try to convince the reader that exsosome treatment might be a viable option.

Minor comments:

• Line 16 - change 'tiny organ in humans' as it is too informal
• Line 18 - change 'mechanism of exsosomes'. Is this mechanism of exosome production or payload delivery?
• Line 31 - change 'increase in aging population' to 'aging population'.
• Line 75 - change 'repair damaged tissues by exosomes', to 'repair damaged tissues by administering exsosomes' or something similar.
• In the figure - change 'directly treatment' and 'indirectly treatment' to 'direct treatment' and 'indirect treatment'.
• Lines 105-107 should be part of an introduction about exosome properties earlier on in the manuscript.

Author Response

Reviewer #1.

Overall the author presents an interesting discussion of the application of exosomes for the treatment of hearing loss.

Major comments:

While the article is comprehensive in some aspects, I think there some areas that need to be expanded.

Point #1: Firstly, a good description of what exosomes are, how they can be used to deliver payloads, and what their potential payloads are should be included early in the discussion.

Response #1: Thank you for your comment. The payload of stem cell derived exosome means that specific molecules used for communication between cells such as miRNA or proteins. I have added specific sentence to explain payload in exosome derived from stem cells in early in the “Future prospects”, line 233-238, page 7.

“Stem cell derived exosomes are used for communication between cell to cell, and it is known that the payload changed depending on the origin of the stem cells. It has been reported that miRNAs and proteins generated in communication between donor cells and target cells are different. In other words, the author believe that it may be helpful to selectively deliver interest genes to cure tissue or cells in destination using exosome with different payload.”

Point #2: Secondly, as the article concentrates mainly on the methodologies surrounding the isolation and delivery of exosomes, I would be tempted to change the title to include 'standardized methodologies to isolate and test exosome treatment applicability in hearing loss', or something similar.

Response #2: Thank you for your advice. As you suggested, the title has been revised to include methodological approach.

“Standardized methodologies to utilize exosome treatment as potential nano substances in hearing loss”

Point #3: Finally, I would try to outline a few different biological mechanisms of hearing loss (chemical induced apoptosis, age related hair cell loss, inflammation) and speculate, with published evidence, how exosomes have been used to disrupt these processes. This does not need to be in depth, but it does need to be presented to try to convince the reader that exosome treatment might be a viable option.

Response #3: Thank you for your comment. The manuscript was corrected and Figure 2 was added. I have described the expected data from the analysis and observation of the auditory organ in vivo, line 175-181, page 5.

“To observe the anatomical phenotype of hearing loss, the morphology can be observed either through immune fluorescence staining (figure 2(a)) or electron micros-copy (figure 2(b)). The observation of changes in hair cells in the organ of Cotri is evidence of direct damage to the auditory organ, and the Myo7a staining, a representative hair cell marker, may prove a physical phenomenon of hearing abnormalities. In addition, in chemical hearing, it can be confirmed that there is an abnormality in electro-chemical transmission by comparing the dB reduction trend through ABR measurement (figure 2(c)).”

In addition, the figure 2 have added to explain from previously study.

Figure 2. Representative anatomical analysis strategy for in vivo experiments. (a) Analysis of whole auditory organ by immune fluorescence staining using Myosin 7a (Myo7a). (Myo7a: green, DAPI: blue), (b) Observation of organ of corti (OC) by trans-mission electron microscopy (8), (c) Comparison of hearing kinetics depending on 80 db to 20dB on 8000kHz.

Minor comments:

Point #1: Line 16 - change 'tiny organ in humans' as it is too informal

Response #1: Thank you for your comment. I modified the words 'tiny organ in humans' to ‘an independent organ in humans’ in manuscript.

Point #2: Line 18 - change 'mechanism of exosomes'. Is this mechanism of exosome production or payload delivery?

Response #2: Thank you for your question. I modified the sentence in manuscript to explain detailly, line 20-22, page 1.

“It is necessary to select a research direction to assess direct or indirect effects on recipients based on the physiological mechanisms of exosomes that deliver useful molecules (called payloads) to recipient cells or tissues.”

Point #3: Line 31 - change 'increase in aging population' to 'aging population'.

Response #3: Thank you for your comment. I changed the words 'increase in aging population' to 'aging population' in manuscript.

Point #4: Line 75 - change 'repair damaged tissues by exosomes', to 'repair damaged tissues by administering exsosomes' or something similar.

Response #4: Thank you for your comment. I changed the words 'repair damaged tissues by exosomes' to 'repair damaged tissues by exosomes treatment' in manuscript.

Point #5: In the figure - change 'directly treatment' and 'indirectly treatment' to 'direct treatment' and 'indirect treatment'.

Response #5: Thank you for your comment. I changed the words in figure 1.    

Point #6: Lines 105-107 should be part of an introduction about exosome properties earlier on in the manuscript.

Response #6: Thank you for your comment. I agree with your opinion that this sentence is related with explanation of exosome biogenesis. However, to highlight the importance of cell-to-cell communication, I have have written this sentence describing the size and lipid membrane composition in the indirect exosome treatment section. This avoids overlapping the description of exosome biogenesis in the introduction of the existing literature, and supports the explanation in the paragraph, so I think it is correct to put it at the beginning of this section.

Author Response File: Author Response.pdf

Reviewer 2 Report

The review by Park describes a roadmap for researchers interested in using exosomes derived from stem cells to treat hearing loss. The author considers a strategy to create, evaluate and use an exosome platform for preclinical studies. Factors analyzed include: selecting strategy (direct or indirect), choosing an appropriate model (in vitro, ex vivo or in vivo), testing for toxicity, etc.

MAJOR CONCERNS

(1) In the Introduction, the author provides a very shallow description of the causes of hearing impairment that reveals a profound misunderstanding of his topic. The causes underlying age-related hearing loss (presbycusis), noise-induced hearing loss or hereditary hearing loss (which accounts for about 60% of cases of HL at birth) are very different. and so exosome treatment strategies should have to be also different. Moreover, some of this classes of HL may be more amenable to exosome therapy than others. However, the author does not make any distinctions and hence the reader is unable to understand which kind of hearing loss (HL) may be treated by exosomes. This should be clarified because some classes of HL will be more amenable to treatment than others.

(2) The author also does not seem to understand that the pathologic mechanisms underlying the different types of HL are very diverse. Nor he seems to be aware that HL in many instances is caused by death and subsequent disappearance of auditory cells. The windows of opportunity for interventions to treat HL are thus very short in some cases, and inexistent in others (e.g. noise-induced HL due to acoustic trauma, such as described in lines 47-48). The author should consider which class of HL is more amenable to treatment with exosomes. Certainly it should be a form in which target cells for therapy remain alive for a certain period after the onset of hearing loss.

(3) Another issue is the contrast between the title ("for preventing hearing loss") and the body of the review (most of the time, "treating hearing loss"). Interventions to prevent HL in noisy environments have so far focused in orally administered drugs. Are exosomes, with their complex delivery usually involving direct injection inside the cochlea and hence requiring surgery, as proposed by the author, really good candidates for such preventive therapies? Indeed, in the midterm, it is more likely that exosome strategies will be applied for repair rather than prevention.

(4) The author does not consider that, in many cases of HL, hair cells are the ones injured by the pathologic process. Unfortunately, hair cells are difficult to access because they are located inside the scala media in the cochlea. Exosomes would have to traffic through the epithelial cells of Reissner's  membrane or through the supporting cells on top of the basilar membrane to reach their target (hair cells). Is this feasible? Could we consider injection directly inside the scala media (with the concomitant injury to the endolymph compartment of the cochlea)?

MINOR CONCERNS.

(5) Lines 109-110 and 111-112. The same sentence appears twice.

(6) Lines 226-228. I do not understand the meaning of this sentence.

(7) Figure 1. There are several spelling errors ("ultrafilteration" should read "ultrafiltration", "directly treatment" should be "direct treatment", etc). Please revise.

 

Author Response

The review by Park describes a roadmap for researchers interested in using exosomes derived from stem cells to treat hearing loss. The author considers a strategy to create, evaluate and use an exosome platform for preclinical studies. Factors analyzed include: selecting strategy (direct or indirect), choosing an appropriate model (in vitro, ex vivo or in vivo), testing for toxicity, etc.

MAJOR CONCERNS

(1) In the Introduction, the author provides a very shallow description of the causes of hearing impairment that reveals a profound misunderstanding of his topic. The causes underlying age-related hearing loss (presbycusis), noise-induced hearing loss or hereditary hearing loss (which accounts for about 60% of cases of HL at birth) are very different. and so exosome treatment strategies should have to be also different. Moreover, some of this classes of HL may be more amenable to exosome therapy than others. However, the author does not make any distinctions and hence the reader is unable to understand which kind of hearing loss (HL) may be treated by exosomes. This should be clarified because some classes of HL will be more amenable to treatment than others.

Response #1: Thank you for your opinion. I agree with your insight. In this review, I tried to establish a research strategy for therapeutic application studies on auditory organ using exosomes. Currently, there are few reports of clinical studies using exosomes in the field of hearing loss. It means that close research between physiologists and engineers is required for the treatment of the auditory organ as an independent organ. Our institution is analyzing the mechanisms of the auditory organ through exosome processing, with added references for strategies (42, 54, 55). The advantages and strategies of exosome treatment are being studied using the standard follow progressing proposed in "4. Strategies for exosomes application." The biggest advantage of exosome therapy is that it is nano-sized particles made of lipid membranes, and interesting payload expression and knockout technologies that we want are being developed. Therefore, according to the strategy of this paper, we plan to conduct research for the treatment of hearing loss based on the applied technology of exosomes.

(2) The author also does not seem to understand that the pathologic mechanisms underlying the different types of HL are very diverse. Nor he seems to be aware that HL in many instances is caused by death and subsequent disappearance of auditory cells. The windows of opportunity for interventions to treat HL are thus very short in some cases, and inexistent in others (e.g. noise-induced HL due to acoustic trauma, such as described in lines 47-48). The author should consider which class of HL is more amenable to treatment with exosomes. Certainly it should be a form in which target cells for therapy remain alive for a certain period after the onset of hearing loss.

Response #2: Thank you for your comment. Your opinion is correct. As with your insights, studies using exosomes should be applicable to a variety of hearing loss models, including models of noise-induced hearing loss. We are currently conducting research using animal models owned developed by our institution. The Acute drug toxicity animal model by aminoglycoside and the age-related hearing loss animal model are covered (6, 8, 37). Also, it was confirmed that exosomes have various payloads recently (42,55), we confirmed that the internal substances of exosomes are changed through co-culture of stem cells and auditory cells of different origins in our laboratory (55). This is evidence that it can be applied to a variety of hearing loss animal models. Therefore, we plan to develop new hearing loss animal models and conduct preclinical testing.

(3) Another issue is the contrast between the title ("for preventing hearing loss") and the body of the review (most of the time, "treating hearing loss"). Interventions to prevent HL in noisy environments have so far focused in orally administered drugs. Are exosomes, with their complex delivery usually involving direct injection inside the cochlea and hence requiring surgery, as proposed by the author, really good candidates for such preventive therapies? Indeed, in the midterm, it is more likely that exosome strategies will be applied for repair rather than prevention.

Response #3: Thank you for your comment. According to references, there are many reports that the death of auditory hair cells is prevented by exosomes' Heat shock protein 70 (HSP70). Our laboratory has also confirmed that the death of auditory hair cells is protected through in vitro experiments (54). Factors such as HSP 70 have been observed to delivery from stem cells to auditory hair cells in ex vivo test. On the other hands, interestingly, when analyzing proteins and miRNAs in exosomes, many proliferative factors are expressed from the stem cells. In other words, I think the research direction will be determined by the development of treatment technology after the development of prevention technology using exosomes therapy.

(4) The author does not consider that, in many cases of HL, hair cells are the ones injured by the pathologic process. Unfortunately, hair cells are difficult to access because they are located inside the scala media in the cochlea. Exosomes would have to traffic through the epithelial cells of Reissner’s membrane or through the supporting cells on top of the basilar membrane to reach their target (hair cells). Is this feasible? Could we consider injection directly inside the scala media (with the concomitant injury to the endolymph compartment of the cochlea)?

Response #4: Thanks for your great point. The structural issues of the auditory organs are also considered. Currently, our institution is trying the classic method of injecting drugs. First, we are considering injecting the eardrum, and second, we are considering injecting a round window. Direct injection into Scalar media was not good method because media overflows from the auditory organ. We believe that the change in media pH depending on the pH of exosomes will not yield good results. It would be good for developing exosome treatment technique on next phase which is targeting peptides may be required for exosomes to be safely delivered to the organ of Corti. Therefore, in this review, we describe the broadly strategies to treat exosome to auditory organ as potential nano sized substance. We will report the details of the injection method after confirming the results of the study to treatment method to auditory organ

 

MINOR CONCERNS.

(5) Lines 109-110 and 111-112. The same sentence appears twice.

Response #5: Thank you. I have deleted duplicate sentences.

(6) Lines 226-228. I do not understand the meaning of this sentence.

Response #6: Thank you for your point. I tried to explain that there are ESCRT dependent and independent pathways. The text has been edited to make it easier to understand.

(7) Figure 1. There are several spelling errors ("ultrafilteration" should read "ultrafiltration", "directly treatment" should be "direct treatment", etc). Please revise.

Response #7: Thank you for your comment. I modified the figure 1.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The author has not provided any compelling answer to the four major concerns raised in the previous review. Specifically:

• No comments are made about the kinds of hearing loss that the author wishes to address.
• The author mentions that supplying exosomes with HSP70 avoids cell death in cell culture models. However, cell death in the inner ear may happen before birth in many hereditary types of hearing loss, and thus exosome treatment cannot be any help unless delivered intrauterinally. No explanation is provided for this preventive therapy for noise-induced hearing loss, either. It is possible to prevent this kind of hearing by intervention in subjects exposed to noisy working environments, but it is not possible to do this for traumatic noise-induced hearing loss.

 

Author Response

Response: Thank you for your comment. I agree with your opinion. I tried to explain the benefits of exosomes, but I didn’t describe in the manuscript how to respond to the types of hearing loss. There are reported types of hearing loss includes noise-induced, genetic, sudden, ototoxicity, and age-related hearing loss. The application of exosomes for prevention or treatment can be classified according to the type of hearing loss, suggesting that they may help restore abnormalities in chemical/biological mechanism. We believe that the fundamental recovery of inner hair cells by exosome is difficult, because both noise-induced and sudden hearing loss occur due to physical stimulation of hair cell death. In addition, the application of exosomes for prevention and treatment for hearing restoration is difficult because genetic hearing loss is caused by developmental disorders in the embryonic period. However, there is an applicability of exosomes to treat the involvement of exosomes in the imbalance of signaling systems due to aging and changes in electronic mechanisms caused by these toxic drugs. It can be substantiated based on previous studies and reports that exosome-specific proliferative and protective factors can recover damaged tissues or cells. Although this field of exosome application to the hearing loss is small, but we believe that the analysis through physiological mechanism of exosome will be possible if the study is going through to various field utilizing exosomes. I have inserted text that is easy for readers to understand, line 73-85, page 2.

“There are reported types of hearing loss includes noise-induced, genetic, sudden, ototoxicity, and age-related hearing loss. The application of exosomes for prevention or treatment can be classified according to the type of hearing loss, suggesting that they may help restore abnormalities in chemical/biological mechanism. The fundamental recovery of inner hair cells by exosome is difficult, because both noise-induced and sudden hearing loss occur due to physical stimulation of hair cell death. In addition, the application of exosomes for prevention and treatment for hearing restoration is difficult because genetic hearing loss is caused by developmental disorders in the embryonic period. However, there is an applicability of exosomes to treat the involvement of exosomes in the imbalance of signaling systems due to aging and changes in electronic mechanisms caused by these toxic drugs. It can be substantiated based on previous studies and reports that exosome-specific proliferative and protective factors can recover damaged tissues or cells.”

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

The author has finally answered some of the points that I raised in the first and second reviews, and therefore the manuscript presents a more balanced view of the problems that exosome therapies may encounter for treating hearing loss. This field may hold promise for future application, but careful selection of the kinds of hearing loss to be investigated and a deeper understanding of the associated pathological processes are required for designing an efficient startegy for treating heraing loss with stem-cell-derived exosomes.

Author Response

I appreciated with your comment. Our institute plans to conduct research using future applicable models based on pathological understanding based on your opinion.