I thank the reviewer for having reviewed the article and for giving comments.

The reviewer’s main concern is that there is no direct evidence that VSMC dystrophin loss is the root cause of DMD. The reviewer suggests performing an experiment involving restoration of dystrophin expression in VSMC in mdx mice or specifically ablating ERBB2/4 in VSMC in WT mice. As I suggested in my response to reviewer 2’s comments, it may be better to knock-out dystrophin in skeletal muscle only using the HSA promoter (whose tissue specific expression is rather well characterized) thereby preserving dystrophin expression in all other tissues including VSM and thus preserving oxygenated blood and lymph flow to and from skeletal muscles. In the experiment suggested for specifically ablating ERBB2/4 in VSMC, one would not necessarily see dystrophic symptoms since the results from the current article suggest that lack of dystrophin in VSM is the root cause of DMD, i.e. the onset of dystrophic symptoms when there is systemic dystrophin deficiency (including lack of functional dystrophin in both skeletal muscle and VSM).

I did not fully understand the point regarding utrophin or other structural proteins compensating, structurally, for lack of dystrophin. As reported in the article and consistent with a published report ¹, utrophin expression is reduced to some extent when ERBB2/4 receptors are ablated. Furthermore, whatever compensation may be provided by other structural proteins does not prevent AChR cluster fragmentation neither in vitro nor in vivo ² due to a lack of dystrophin.

The scope of the current article is to provide evidence of a new concept/discovery, that dystrophin expression in muscles cells is dependent on NRG/ERBB signaling, and based on these experiments as well as previous published observation is the resulting conclusion that absence of dystrophin in VSM has a greater impact i.e. causes the onset of DMD than its absence in skeletal muscles only. Publication of this hypothesis is important as it will allow research groups who already have animal models that can be used to rapidly generate skeletal muscle specific dystrophin knock-out mice as described above, to substantiate (or refute) the results in the current article.

In the current article I demonstrated that NRG/ERBB signaling is essential for dystrophin expression in myotubes. We previously demonstrated acetylcholine receptor (AChR) cluster fragmentation ² in ERBB2/4 dKO myotubes and also (in vivo) in muscle fibers isolated from the (same) HSA-CRE/ERBB2/4 dKO mouse. Similarly AChR cluster fragmentation has been reported for the dystrophin deficient mdx mouse ³. We had reported that ACHR cluster fragmentation was due to a lack of phosphorylated alpha dystrobrevin 1 (α-DB1). However, we had also reported that in the absence of NRG/ERBB signaling there were reduced levels of the 75 kDa α-DB1 protein (that gets phosphorylated) in the dystrophin associated protein complex (DAPC) ². Similarly, in dystrophin deficient mdx mice there is a reduced amount of dystrobrevin at the sarcolemma ⁴. I have demonstrated in the current article that the reduced amount of the 75 kDa α-DB1 is due to a reduced association of it with the DAPC when dystrophin is lacking and not due to downregulation of gene expression. Hence our previous observation on AChR cluster fragmentation ² is most likely due to a lack of dystrophin in skeletal muscles (in ERBB2/4 dKO mice) and not due to a lack of phosphorylation of the 75 kDa α-DB1since the amount of α-DB1 available for phosphorylation, is reduced. This is similar to the situation in mdx mice.

It cannot be excluded that the AChR cluster fragmentation we observed ² in skeletal muscles (in ERBB2/4 dKO mice) is a combination of reduced α-DB1 levels and absence  of its phosphorylation. However, in view of the fact that there is no detectable dystrophin expression (protein nor messenger RNA, Figure 1 A–C) in the current article) in the absence of signaling through NRG/ERBB, the AChR cluster fragmentation we observed is most likely due to a lack of dystrophin rather than to a lack of phosphorylation of the reduced amount of the 75 kDa α-DB1. This is supported by published observations in mdx mice where the lack of dystrophin also results in a reduction in the amount of dystrobrevin associated with the sarcolemma and AChR cluster fragmentation, despite the presence of NRG/ERBB signaling that could phosphorylate (the reduced amount of) α-DB1. The observations in mdx mice substantiate the observations presented in the current manuscript, supporting the notion that the reduced amount of α-DB1, even if it can be phosphorylated as in the mdx mouse, is not sufficient to prevent AChR cluster fragmentation. Together the data strongly suggest that in ERBB2/4 dKO mice, the cause of the AChR cluster fragmentation we observed ² is due to a lack of dystrophin protein in skeletal muscle rather than due to a lack of phosphorylation of α-DB1.  The observed AChR cluster fragmentation in vitro in Erbb2/4 dKO myotubes, due to a lack of dystrophin, is also observed in vivo suggesting that the lack of dystrophin expression is not compensated for in vivo.

The main discovery in our previous paper ² is that signaling through NRG/ERBB results in the phosphorylation  of α-DB1 and as it had already been reported that phosphorylation of α-DB1 stabilizes AChR clusters at the NMJ ⁵, our discovery provided the missing link i.e. that ERBB2/4 receptors phosphorylated α-DB1. My contribution to that manuscript was not only to demonstrate NRG/ERBB signaling phosphorylated α-DB1 but also that α-DB1 in myotubes appears to be very rapidly phosphorylated and dephosphorylated in WT muscles which may be important for further downstream signaling mechanisms and/or in postsynaptic responses during neurotransmission in addition to, as already reported, stabilizing AChR clusters at the NMJ (this is my opinion and not necessarily that of the other co-authors on that paper).

In conclusion, even though dystrophin is lacking in skeletal muscles of ERBB2/4 dKO mice these mice do not show dystrophic symptoms suggesting that the VSM in these mice, which still  express dystrophin because the HSA promoter used to ablate dystrophin in these mice is not active in VSM, enable increased blood flow and lymph clearance, especially during exercise. The results presented in this article, together with published observations, support the notion that lack of dystrophin in skeletal muscle only does not result in DMD but rather that the lack of dystrophin in both, skeletal muscle and VSM results in DMD. Hence the root cause of DMD is the lack of dystrophin in VSM.

It is noteworthy that there is no direct evidence to support the current “general consensus” that lack of dystrophin in skeletal muscle is the cause of DMD. DMD is a pathological state resulting from a systemic dystrophin deficiency. Thus in D/BMD, both skeletal muscle and VSM lack fully functional dystrophin. As far as can be determined from the current literature, there has been no study published that supports the notion that lack of dystrophin in skeletal muscle only causes DMD. In contrast, the findings in the current article argue against the current “general consensus” and instead suggest that although lack of dystrophin in skeletal muscle does not cause DMD, it is a prerequisite for the onset of DMD when dystrophin is also lacking in VSM. This does not mean that restoring dystrophin expression in VSM will rescue the dystrophic state in skeletal muscles in mdx mice that are already dystrophic (it may, but this will need to be determined experimentally). Our working hypothesis does predict that when dystrophin would be expressed early in VSM, hence when it would be expressed during normal developmental stages such as in WT mice, it would prevent or delay the onset of the dystrophic state (in mice where dystrophin is lacking in skeletal muscle only).

Therapeutic attempts have been reported to ameliorate the dystrophic phenotype in dystrophin deficient muscles but only when strategies were used that result in the expression of dystrophin or variants of it in both skeletal muscle and VSM (discussed in detail in the current article), which is in line with the observations in the current study. In view of the current article and lack of direct experimental evidence demonstrating DMD when dystrophin is lacking in skeletal muscles only, the general consensus may need to be revised i.e. that DMD is caused by a lack of dystrophin in both skeletal muscle and VSM (at least) and that lack of dystrophin in skeletal muscles only may not be sufficient to result in DMD. This is an important consideration for long lasting therapy as mentioned in the discussion of the current article.

Not only structural components (such as dystrophin) within the muscle fibers are essential to keep skeletal muscles healthy and prevent dystrophy during electrical stimuli/exercise, but also increased blood flow (to provide sufficient nutrients and oxygen to the muscle tissue) as well as a properly functioning lymphatic system (to remove resulting waste products). It is thus not surprising that even though dystrophin is lacking in skeletal muscles, such as in the Erbb2/4 dKO mice, the onset of dystrophic symptoms is only observed during exercise when blood flow (and subsequent lymph drainage) is affected as a consequence of the systemic absence of dystrophin in both skeletal muscle and VSM (compounded by inflammation/immune system involvement) such as in the case of the mdx mice or DMD patients.

Bibliography

1.  Khurana, T. S. et al. Activation of utrophin promoter by heregulin via the ets-related transcription factor complex GA-binding protein alpha/beta. Mol. Biol. Cell 10, 2075–2086 (1999).

2.  Schmidt, N. et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J. Cell Biol. 195, 1171–1184 (2011).

3.  Kong, J. & Anderson, J. E. Dystrophin is required for organizing large acetylcholine receptor aggregates. Brain Res. 839, 298–304 (1999).

4.  Blake, D. J. Dystrobrevin dynamics in muscle-cell signalling: a possible target for therapeutic intervention in Duchenne muscular dystrophy? Neuromuscul. Disord. 12 Suppl 1, S110–7 (2002).

5.  Grady, R. M. et al. Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions. J. Cell Biol. 160, 741–752 (2003).

I thank the reviewer for reviewing the revised version of the article.

The current article ¹ supports the notion that the root cause of Duchenne muscular dystrophy (DMD) is a lack of dystrophin in vascular smooth muscle (VSM). Thus it addresses what causes the onset of dystrophic symptoms in patients suffering from DMD where there is systemic dystrophin deficiency including in skeletal muscle. This is of importance since it suggests that therapeutic attempts should (also) target VSM, which is not always the focus of current therapies.

Loss of dystrophin expression as a consequence of abolishing neuregulin (NRG) signalling is directly demonstrated in Erbb2/4 dKO myotubes. Our results, together with previous published observations ² suggest that the absence of dystrophin in vivo in skeletal muscle in HSA-CRE/Erbb2/4 dKO mice is responsible for the observed fragmentation of acetyl choline receptor (AChR) clusters in these mice, similar to that observed in muscles of dystrophin deficient mdx mice. As in mdx mice ^3,4 (in reference 4, figure 4B, less dystrobrevin is detected in microsomal preparations from mdx), less α-dystrobrevin1 (α-DB1) is associated with the DAPC in Erbb2/4 dKO myotubes strongly suggesting that this is a direct consequence of the absence of dystrophin. Additional results in the current manuscript also show that the AChR cluster fragmentation observed in vitro in Erbb2/4 dKO myotubes and in vivo in both HSA-CRE/Erbb2/4 dKO and mdx mice is likely caused by a loss of dystrophin which results in the reduced association of α-DB1 with the DAPC ² and this reduced association is not due to a lack of phosphorylation of α-DB1 ¹.

Thus we observed that loss of NRG/ERBB signalling results in a loss of dystrophin expression, affecting normal DAPC assembly and AChR cluster formation similarly to that observed in dystrophin deficient mdx mice.  However, in contrast to mdx mice, impaired muscle generation is only seen in MCK-CRE/Erbb2 KO mice, where NRG/ERBB signalling and hence dystrophin expression is abolished in both skeletal muscle and VSM, but not in HSA-CRE/Erbb2 KO or HSA-CRE/Erbb2/4 dKO mice ⁵ where NRG/ERBB signalling is only abolished in skeletal muscles since the HSA promoter is not active in VSM ⁶.

In contrast to the comments of the reviewer, the current article does not claim that the lack of dystrophin in VSM only would cause muscular dystrophy. Instead, it argues that functional vascular smooth muscle cells with normal dystrophin expression are essential in preventing or delaying the onset of muscular dystrophy when dystrophin is absent in skeletal muscles. This is supported by the observation that dystrophic symptoms are not observed in skeletal muscle of HSA-CRE/Erbb2/4 dKO mice ¹ that otherwise display AChR cluster fragmentation and less dystrobrevin association with the DAPC as in dystrophin deficient mdx myotubes, or when dystrophin expression is prevented in skeletal muscles only ⁷. It is also in line with observations of improved muscle performance and reduced muscle degradation in mdx mice ⁸ or DMD patients ⁹ treated with PDE-5 inhibitors that restored normal blood vessel function and blood flow during exercise, in spite of the total absence of dystrophin.

For obvious reasons previous research has focused on the role of skeletal muscle in dystrophin deficient mdx mice or DMD patients since dystrophic symptoms are observed in skeletal muscle and not in VSM where absence of dystrophin results in aberrant vasoregulation. This aberrant vasoregulation is independent of whether skeletal muscles express dystrophin or not. The observation of the current study, supported by published data, suggests that muscular dystrophy in mice is only observed when dystrophin is lacking in both skeletal muscles and VSM and not in skeletal muscle only, suggesting that it is the lack of dystrophin in VSM, resulting in aberrant vasoregulation that causes the onset of DMD when skeletal muscles lack dystrophin.

Hence, absence of dystrophin in VSM only, as is the case for example in the study by Wasala N. et al. that the reviewer refers to ¹⁰, by artificially expressing dystrophin in only skeletal muscle of mdx mice (by using the HSA promoter), will not reveal this role of VSM in the onset of dystrophy observed in situations where dystrophin is absent in both skeletal muscle and VSM such as in mdx mice and DMD patients or MCK-CRE/Erbb2 KO mice that lack NRG/ERBB signalling in skeletal muscle and VSM.

Unfortunately, the paper by Wasala N. et al. ¹⁰ does not report how well the expression of a mini-dystrophin gene in only skeletal muscle ameliorates muscular dystrophy in mdx mice. Skeletal muscle pathology following expression of mini dystrophin driven by the HSA promoter (in skeletal muscle) was not compared to samples from wild type (wt) or mdx control mice. Instead, the authors refer to three previously published papers ^4,11,12 that report the rescue of the dystrophic phenotype when the same mini-dystrophin is expressed using a CMV promoter, which is different from the skeletal muscle specific HSA promoter they used. Thus mini-dystrophin would be expressed in skeletal muscle and VSM in all three previous studies and obviously results in the restoration of muscle function, but it cannot be assumed that the same restoration will be obtained when dystrophin is only expressed in skeletal muscle (by using the HSA promoter). Instead, Wasala N. et al. ¹⁰ only compare the skeletal muscle pathology between transgenic mice expressing the HSA-mini-dystrophin and transgenic mice where this mini-dystrophin is again removed after recombination with CRE recombinase.

Hence, when more severe dystrophic symptoms are observed by Wasala N. et al. ¹⁰ when mini-dystrophin expression is abolished through CRE mediated recombination, it can not only be interpreted, as the authors have done, that the loss of mini-dystrophin in skeletal muscle causes a dystrophic state, but also that the lack of dystrophin in VSM causing aberrant vasoregulation in combination with a lack of mini-dystrophin in skeletal muscle causes a dystrophic state which is actually similar to the situation in mdx mice.

An experiment to be performed, that is beyond the scope of the current study, would be to generate a conditional dystrophin KO mouse and cross it with the HSA-CRE transgenic mouse to genetically delete dystrophin in skeletal muscles only in order to confirm our results obtained with the HSA-CRE/Erbb2/4 dKO mice. A dystrophic pathology as observed when dystrophin is absent in both skeletal muscle and VSM, as in mdx mice, would not be expected to be seen in this mouse.

The lack of an obvious dystrophic pathology of dystrophin deficient skeletal muscles in mice with functional VSM, and consequently sufficient blood and lymph flow, will allow satellite cells and circulating myeloid cells ¹³ to support muscle regeneration since it has been demonstrated that satellite cells from mdx mice can regenerate muscle fibers when the host environment is not dystrophic ¹⁴

Given that, unlike in humans, satellite cells are not depleted in the DMD mouse model mdx, muscle regeneration can continue during the relatively short life span of the mouse, explaining the milder dystrophic symptoms in mdx mice as compared to DMD patients. In that regard it might even be more important for DMD patients to prevent/delay muscle degeneration by restoring dystrophin expression in VSM as well when therapeutic efforts to restore dystrophin in skeletal muscle are made.

The reviewer states that presence/absence of dystrophin should be analysed by for example immunostaining in muscle sections in the HSA-CRE/Erbb2/4 dKO mice.  For the reasons mentioned in the current article (juxtaposition of satellite cells expressing a high level of dystrophin and dystrophin expression in muscle fibers ¹⁵, and contaminating cells or tissues), it is practically not possible to resolve dystrophin expression in muscle sections or myotubes formed from primary myoblasts, although it was possible to observe AChR cluster fragmentation in vitro and in vivo as a consequence of the loss of dystrophin expression (demonstrated in myotubes), an observation that was also made in mdx mice. It would also not add much to the arguments presented in the current article to demonstrate dystrophin expression in VSM in these mice, since the HSA promoter is not active in VSM of HSA-CRE/Erbb2/4 dKO mice.

It is by now well accepted, perhaps considered a general consensus, that DMD is not just the consequence of the loss of structural support due to the loss of dystrophin in skeletal muscles, but rather also due to a combination of disruption of signalling events involving components of the DAPC and inflammation involving immune cells. Thus the concept that aberrant vasoregulation due to a loss of dystrophin expression in VSM, with the lymphatic system also being affected due to systemic dystrophin deficiency, is the root cause of DMD fits rather well with what is known so far. Thus a holistic approach is needed when determining the root cause of DMD and, as a consequence, treatment strategies that would be more complete and long lasting.

Bibliography

1.      Gajendran, N. The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se [version 1; referees: 2 not approved]. F1000Res. 7, 1321 (2018).

2.      Schmidt, N. et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J. Cell Biol. 195, 1171–1184 (2011).

3.      Blake, D. J. Dystrobrevin dynamics in muscle-cell signalling: a possible target for therapeutic intervention in Duchenne muscular dystrophy? Neuromuscul. Disord. 12 Suppl 1, S110–7 (2002).

4.      Lai, Y. et al. Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy. J. Clin. Invest. 119, 624–635 (2009).

5.      Escher, P. et al. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308, 1920–1923 (2005).

6.      McCarthy, J. J., Srikuea, R., Kirby, T. J., Peterson, C. A. & Esser, K. A. Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting. Skelet Muscle 2, 8 (2012).

7.      Ghahramani Seno, M. M. et al. RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology. Hum. Mol. Genet. 17, 2622–2632 (2008).

8.      Kobayashi, Y. M. et al. Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature 456, 511–515 (2008).

9.      Nelson, M. D. et al. PDE5 inhibition alleviates functional muscle ischemia in boys with Duchenne muscular dystrophy. Neurology 82, 2085–2091 (2014).

10.    Wasala, N. B. et al. Genomic removal of a therapeutic mini-dystrophin gene from adult mice elicits a Duchenne muscular dystrophy-like phenotype. Hum. Mol. Genet. 25, 2633–2644 (2016).

11.    Zhang, Y. et al. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy. Hum. Mol. Genet. 22, 3720–3729 (2013).

12.    Zhang, Y. & Duan, D. Novel mini-dystrophin gene dual adeno-associated virus vectors restore neuronal nitric oxide synthase expression at the sarcolemma. Hum. Gene Ther. 23, 98–103 (2012).

13.    Camargo, F. D., Green, R., Capetanaki, Y., Jackson, K. A. & Goodell, M. A. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat. Med. 9, 1520–1527 (2003).

14.    Boldrin, L., Zammit, P. S. & Morgan, J. E. Satellite cells from dystrophic muscle retain regenerative capacity. Stem Cell Res. 14, 20–29 (2015).

15.    Dumont, N. A. et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat. Med. 21, 1455–1463 (2015).

αI thank the reviewer for reading the article and for giving comments/suggestions.

The reviewer refers to previous studies that are challenged by this article. This comment was also made by reviewer 1 to which I have responded in detail below referring to published studies. Briefly, although several of the previous studies to restore dystrophin expression targeted skeletal muscle, dystrophin or mini dystrophin would also be expressed in vascular smooth muscle (VSM) due to the delivery route, promoter used for expression of dystrophin or gene editing components, or the vehicle used for delivery and hence it is neither possible to conclude that restoring dystrophin expression in skeletal muscle is sufficient to rescue the dystrophic pathology nor to exclude the role of VSM in the onset of dystrophic symptoms. Furthermore, studies that reported restoring dystrophin expression in skeletal muscle will result in the dystrophin associated protein complex (DAPC) to be restored and enable signalling from this complex to be re-established, but this does not mean that on the long term the aberrant vasoregulation due to a lack of dystrophin in VSM will not cause again the onset of dystrophy in skeletal muscle.

As the reviewer is aware, the current article concludes that the root cause of DMD is the lack of dystrophin in VSM but it does not claim that restoring dystrophin expression in VSM will restore the dystrophic pathology in skeletal muscle to the normal state although it may ameliorate it.  If lack of dystrophin in VSM is responsible for the onset of DMD as the current article concludes, then from what is known so far, the lack of dystrophin in skeletal muscle may exacerbate the dystrophic pathology caused by aberrant vasoregulation, possibly due to structural defects as well as the interruption of signalling events from the dystrophin associated protein complex (DAPC). This hypothesis is supported by a published study where they demonstrated that satellite cells from mdx mice retained their regenerative capacity and that the host environment was critical for satellite cell function ¹. Hence if the host environment is in a dystrophic state (onset of which may have been due to aberrant vasoregulation), such as in mdx skeletal muscle, impaired muscle regeneration is observed. Thus the current article suggests that treatment strategies should (also) target VSM for any long term benefit for DMD patients.

The reviewer comments that the manuscript (article) fails to show if NRG/ERBB signaling had a functional role in muscular dystrophy by identifying downstream signalling targets as written at the end of the introduction. Clearly the results in Fig. 1 shows that dystrophin is a signalling target of NRG/ERBB and the fact that, based on HSA promoter activity, ERBB2/4 dKO mice do not show dystrophic symptoms (explained below) is due to ERBB2/4 and hence also dystrophin still being expressed in VSM (and possibly endothelial cells). Thus NRG/ErbB signalling is essential for dystrophin expression, and the systemic lack of dystrophin causes muscular dystrophy.

The conditional HSA-CRE/ErbB2 KO mouse ² (is the same ErbB2 lox P flanked mouse used in breeding to generate the HSA-CRE/ErbB2/4 dKO mouse) shows similar pathology to the HSA-CRE/ErbB2/4 dKO mouse. However when dystrophin is lacking in skeletal muscle and VSM as in the conditional MCK-CRE/ErbB2 KO mouse ³ where CRE expression is driven by the muscle creatine kinase promoter, it shows impaired muscle regeneration similar to the mdx mouse that lacks dystrophin and hence would be expected to show a more severe pathology compared to the MCK-CRE/ErbB2 KO mouse.

The reviewer commented that only a very short dataset is provided. Studies supporting the conclusion of the current article that the lack of dystrophin in VSM is the root cause of DMD are:

1) Ablation of ErbB2/4 receptors results in a lack of dystrophin in myotubes;

2) Skeletal muscle specific HSA-CRE/ErbB2/4 dKO mice that would lack dystrophin in skeletal muscle but express dystrophin in VSM do not show a dystrophic pathology ⁴;

3) HSA-CRE/ErbB2 KO ² mice used in breeding to generate HSA-CRE/ErbB2/4 dKO show a similar pathology to the latter;

4) MCK-CRE/ErbB2 mice ³ that would lack dystrophin in skeletal muscle and VSM (as MCK promoter is active in both), show impaired muscle regeneration;

5) Lack of dystrophin in skeletal muscle fibers through RNAi knock down did not show an overt dystrophic pathology ⁵;

6) expression of dystrophin in VSM, even at significantly lower levels compared to wild type controls, improved aberrant vasoregulation in mdx mice ⁶;

7) Taken together, the current article and several published studies involving rescue of dystrophic muscle would suggest, that aberrant vasoregulation is the cause of the onset of DMD (current article) and that lack of dystrophin in skeletal muscle exacerbates the dystrophic pathology.

Other data are available in the publication of the patent filing ⁷ (patent link: WO 2017/036852 A1, link also in legend to Fig. 2) reporting that signalling (stimulation/upregulation) from NRG/ERBB is most likely mediated by the intracellular domain of ERBB4 (4ICD) whereas signalling from α-dystrobrevin 1 (α-DB1) downregulates dystrophin expression since genetic deletion of dystrobrevin results in increased dystrophin and ERBB4 expression with most of the ERBB4 being cleaved to generate 4ICD. Data in the published patent filing also show that restoring α-DB1 in DB KO myotubes downregulates dystrophin expression to levels found in myotubes formed from C2C12 cells. These data, although clearly demonstrate signalling events occur from the DAPC, are not necessary to support the conclusion in the current article and hence were not included.

The reviewer comments that the ref provided (ref. number 20) in the article does not include any information about a lack of dystrophin in muscles. As I wrote in my comment to the report of reviewer 1, the supplemental data for that paper includes information that reports the absence of centralized nuclei and lack of atrophic fibers thus there was no indication to look for dystrophin expression. The paper ⁴ also reports that sustained muscle strength was not affected nor was a myasthenic condition observed. These mice did show a hind limb extension reflex which was not reported in the paper. As I also wrote to in my comment to the report of reviewer 1, it was not possible to resolve dystrophin expression (nor phosphorylation of α-DB1 ⁸) in vivo or in muscle lysates of ErbB2/4 dKO mice for the reasons explained in my comment to the report of reviewer 1 below.

The reviewer also comments that “…data demonstrating that the dystrophin expressed in smooth muscle cells (or other blood vessel cells) only (i.e. not in myofibers) is sufficient to rescue the pathology should be provided. This experiment has been published whereby dystrophin was expressed in VSM that improved aberrant vasoregulation in mdx mice even though the amount of dystrophin was significantly lower ⁶ than that in wild type control mice. Although the current article concludes that lack of dystrophin in VSM is the root cause (onset) of DMD, this does not necessarily mean that expressing dystrophin in VSM when the skeletal muscle is already is in a dystrophic state (due to systemic dystrophin deficiency) would rescue the dystrophic phenotype. Thus experiments designed to express full length dystrophin in VSM only at embryonic day 9.5 (when dystrophin is normally expressed) is not feasible given the size of the dystrophin cDNA and complicated by the fact that expression levels, even of a mini dystrophin, may not be sufficient. Hence an alternative experiment to obtain a situation in vivo where dystrophin is expressed in VSM as well as in other cells and tissues but not in skeletal muscle is to prevent NRG signalling by ErbB2/4 dKO and hence dystrophin expression in skeletal muscle i.e. the HSA-CRE/ErbB2/4 dKO mouse mentioned in the current study.

The loss of dystrophin when ErbB2/4 receptors are ablated by CRE expression driven by the HSA promoter in skeletal muscle does not result in dystrophic symptoms (when dystrophin is expressed in VSM and other cells and tissues) is consistent with published observations whereby RNAi knock-down of dystrophin in skeletal muscles did not show an overt dystrophic pathology ⁵.

I would like to invite the reviewer to read my comments below to reviewer 1 as several of the concerns raised were similar. As I will submit a revised version that would address the concerns raised by reviewer 1 (report and comment) and reviewer 2, if the reviewer has any additional concerns that is communicated as a comment to this response, I would try and address those as well.

Bibliography

1.  Boldrin, L., Zammit, P. S. & Morgan, J. E. Satellite cells from dystrophic muscle retain regenerative capacity. Stem Cell Res. 14, 20–29 (2015).

2.  Leu, M. et al. Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development. Development 130, 2291–2301 (2003).

3.  Andrechek, E. R. et al. ErbB2 is required for muscle spindle and myoblast cell survival. Mol. Cell. Biol. 22, 4714–4722 (2002).

4.  Escher, P. et al. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308, 1920–1923 (2005).

5.  Ghahramani Seno, M. M. et al. RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology. Hum. Mol. Genet. 17, 2622–2632 (2008).

6.  Ito, K. et al. Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice. Hum. Mol. Genet. 15, 2266–2275 (2006).

7.  Gajendran, N. ERBB4 RECEPTOR MODULATORS FOR USE IN THE TREATMENT OF DYSTROPHIN-ASSOCIATED DISEASES. (2017). WO 2017/036852 A1

8.  Schmidt, N. et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J. Cell Biol. 195, 1171–1184 (2011).

I thank the author for replying to my concerns. As I mentioned when revising the manuscript, I do consider interesting the concept that non-skeletal muscle cells have a role in DMD. This is plausible and, as the author states, there are several published studies showing that cells other than myofibres have a role in DMD but the general consensus is that the main issue is a lack of dystrophin in myofibres and that the lack of dystrophin in other cell types exacerbate the disease (which is pretty much the opposite of the author’s claim). For example the paper Ito et al 2006 concludes that “these data suggest that dystrophin in VSMCs may play an important role in the local autocrine regulation of α-adrenergic constriction, and that the loss of this regulatory mechanism may exacerbate muscle fiber necrosis”.

However, I am not criticizing the claim of the current manuscript per se, my only concern is that this claim is not supported by experimental evidences (as also observed by the other reviewer). The author mentioned several publications supporting the conclusions of this manuscript but, clearly, the manuscript must sustain the claim with solid original data. Showing that in vitro “Ablation of ErbB2/4 receptors results in a lack of dystrophin in myotubes” is simply not enough to persuade the readers that the lack of dystrophin in smooth muscle cells is “The root cause of Duchenne muscular dystrophy”. The manuscript should stand on its own and observations from other published studies supporting the conclusion are valuable but should not replace original data.

I would be pleased to see a revised version that addresses these concerns, but I advise the author to include on it solid in vivo data to support its claims.

I thank the reviewer for taking the time to review the article and for the comments.

I realise that the role of neuregulin in muscle, especially in transcription of synapse specific genes and NMJ formation has been controversial. The reviewer suggests that in vivo NRG/ErbB signalling could be compensated by another signalling pathway (when ERBB2/4 receptors are ablated) to stimulate dystrophin expression ¹ since, the reviewer comments, agrin compensates for this (NRG signalling through ERBB2/4) at the NMJ ². However, in the article that is referred to by the reviewer and that describes the ErbB2/4 dKO mice ², the aim was to determine if neuregulin (NRG) signaling through ERBB2/4 receptors in muscle was required for synapse specific transcription and NMJ formation. That Agrin plays an important role in acetylcholine receptor clustering and NMJ formation was demonstrated by the fact that mice carrying a mutation in the agrin gene lacked NMJs ³. Thus the original article on the ErbB2/4 dKO mice concluded that NRG was not required for synapse-specific gene transcription at the NMJ and that development and maintenance of neuromuscular synapses were only marginally affected. Hence, the article does not conclude that NRG signalling is compensated for by Agrin, but rather that NRG is not required for synapse specific gene expression and NMJ formation. In addition, unlike NRG/ERBB signalling stimulating dystrophin expression in muscle ¹, transcription of synapse specific genes and the formation of the NMJ involves reciprocal interactions between muscle fibers and motor neurons.

We subsequently demonstrated that neuromuscular synapses being marginally affected in the ErbB2/4 dKO mice was due to a lack of phosphorylation of alpha-dystrobrevin 1 (α-DB1) ⁴. To demonstrate phosphorylation of α-DB1 by signalling through ERBB2/4 receptors, I used ErbB2/4 dKO myotubes  (as I did in this article) as well as myotubes formed from C2C12 and primary myotubes formed from myoblasts from wild type (wt) mice (Schmidt etal. Fig. 7 a & b) ⁴. We observed phosphorylation of α-DB1 in samples prepared from myotubes formed from freshly purified primary myoblasts from ErbB 2/4 dKO mice. This phosphorylation of α-DB1 could be blocked by ERBB inhibitors (Schmidt et al. Fig. 7a) ⁴ demonstrating  that: (i) signalling through ERBB2/4 receptors was responsible for phosphorylation of α-DB1, (ii) the observed phosphorylation of α-DB1 in myotubes formed from ErbB2/4 dKO primary myoblasts was likely caused by the presence of contaminating cells in the myoblast preparation. Contaminating cells in the myoblast preparation is obviously lost after extended culture periods as confirmed by the lack of phosphorylation of α-DB1 in ErbB2/4 dKO myotubes in the absence of any ERBB inhibitor (Schmidt et al. Fig. 7c) ⁴ For the same reason I used the ErbB2/4 dKO myotubes instead of muscle tissue, to demonstrate a lack of dystrophin expression ¹.

Once we discovered that signalling through ERBB2/4 receptors phosphorylated α-DB1 (Schmidt et al. Fig. 7 a-c) ⁴ we realised that this explained the fragmentation of acetylcholine receptor (AChR) clusters at the neuromuscular synapse in ErbB2/4 dKO mice. This was  subsequently confirmed by experiments in cultured myotubes (wild type and α-DB1 KO) as well as with and without α-DB1 expression constructs (Schmidt et al. Fig. 6, results were not presented in order of discovery) ⁴. We could not show absence of α-DB1 phosphorylation in vivo in the muscles of ErbB2/4 dKO mice for the same reasons mentioned above.

Due to the presence of tissues other than skeletal muscle in muscle lysates and cells other than myoblasts in primary myoblast purifications from ErbB2/4 dKO mice, it was not possible to resolve either the lack of phosphorylation of α-DB1 or the lack of dystrophin in the absence of ERBB2/4 signaling in the dKOs. In particular satellite cells present in muscle lysates (and in primary myoblast purifications) were shown to express a high level of dystrophin ⁵ and will still express dystrophin in ErbB2/4 dKO mice since the HSA promoter driving CRE expression is not active ⁶,and hence ERBB2/4 receptors would not be ablated in these cells. The juxtaposition of dystrophin expression in satellite cells and in myofibers makes it very difficult to distinguish dystrophin expression in satellite cells from that in muscle fibers ⁵. It may well be possible that researchers who may have looked for the dystrophin expression in muscle specific ErbB2 and/or ErbB4 KO may have run into this problem and not observed a lack of dystrophin in muscle or muscle sections. Satellite cells, however, appear to have a limited potential to regenerate skeletal muscle ⁶ and hence would not fully explain the lack of dystrophic symptoms in ErbB2/4 dKO mice.

Dystrophin and utrophin were detected in loxP-flanked ErbB2/4 myotubes (Gajendran et al., Fig 1.) ¹ formed from myoblasts, even after the extended culture period to generate them, and it was only after transient CRE transfection and ablation of ErbB2/4 receptors (confirmation of recombination checked by PCR) dystrophin expression was not observed but utrophin was, in different clones. Understandably, it was not possible to either demonstrate a lack of phosphorylation of α-DB1 or the lack of dystrophin expression when using lysates of total muscle tissues due to the presence of other tissues and cells, including the smooth muscle of blood vessels and satellite cells where ErbB2/4 receptors were not ablated (the HSA promoter driving CRE expression is not active there). In addition to dystrophin detection, any other component of the DAPC present in vivo in the muscle that we wanted to detect in myotubes such as utrophin, a-dystrobrevin, and a-syntrophin were also detected on western blots following immunoprecipitation of the dystrophin associated protein complex (DAPC) .

If the absence of NRG/ErbB2/4 signaling, and as a consequence absence of dystrophin in skeletal muscle, could be compensated in vivo by another mechanism, even possibly through another ligand other than NRG1, then this compensation mechanism would be independent of ERBB2/4 receptors as they are ablated in the skeletal muscles in these mice.

From experiments using α-DB1 KO mytobes in combination with α-DB1 expression constructs, it also became clear that signaling from α-DB1 downregulates dystrophin expression but not utrophin expression (results available at WO 2017/036852 A1 under documents “09.03.2017 Initial Publication with ISR (A1 10/2017)”) ⁷. Thus opposing signals from α-DB1 (downregulation) and NRG/ERBB2/4 (upregulation) regulates dystrophin expression. This regulation appears to be mediated by the cleaved intracellular domain of ERBB4 (4ICD) since increased dystrophin levels in dystrobrevin KO myotubes are accompanied by a strong upregulation of ERBB4 expression most of which is cleaved to generate 4ICD (see figures at WO 2017/036852 A1) ⁷.

Together with a wealth of published information, the data presented in this article, in my opinion, is essential and sufficient to conclude that expression of dystrophin is stimulated by NRG/ERBB signalling. It follows that in ErbB2/4 dKO mice that did not show dystrophic symptoms (see below), the absence of dystrophin in skeletal muscle but not in smooth muscle of blood vessels, clearly argues that the root cause of DMD is due to the lack of dystrophin in the smooth muscle of blood vessels. Naturally it is possible that dystrophin expression in the endothelial cells, between the smooth muscle and the lumen of blood vessels, in ErbB2/4 dKO mice also plays a role in vasodilation during exercise, assuming that the HSA promoter used to ablate ERBB2/4 receptors is not active in these cells.

Published data on DMD, together with the data presented in this article, would suggest that the lack of dystrophin in skeletal muscle exacerbates the onset of dystrophy caused by a lack of increased blood flow during exercise. This observation is also supported by a very early observation in mdx muscles that only some fibers, and not all, in skeletal muscle initially show dystrophic symptoms ⁸ and also by the fact that expressing the full length dystrophin in smooth muscles in mdx mice, even though it was expressed at significantly lower levels than the level of dystrophin found wild type mice, improved aberrant vasoregulation ⁹. The fact that skeletal muscle in those mice still showed some atrophy could be due to insufficient dystrophin expression in the smooth muscle causing dystrophic symptoms as a consequence of reduced blood flow during exercise and exacerbated due to the lack of dystrophin in skeletal muscle (Reviewed in Ennen JP et al.) ¹⁰. The exacerbation of dystrophic symptoms in skeletal muscle, apart from structural defects, may also be as a result of disruption of signalling events from the DAPC due to the absence of dystrophin and this is further supported by the observation that α-DB KO results in increased dystrophin and ERBB4 expression in myotubes ⁷.

As far as could be determined from the literature, there are no compensatory signals in vivo that can ameliorate the effects caused by deficient ERBB2 or ERBB4 signalling such as defective muscle spindle formation ¹¹, dilated cardiomyopathy (DCM) following Herceptin (antibody against HER2/ErbB2) treatment ¹², DCM in conditional ErbB2 ^13,14 or ErbB4 ¹⁵ KO mice, AChR cluster disintegration (also observed in mdx mice) in muscle specific ErbB2/4 dKO mice ⁴, and the embryonic lethality observed in ErbB2 or ErbB4 KO mice ^16,17. NRG and its ERBB receptors appear to exert their function in a spatio-temporal fashion in development and maintenance (in adults).

The reviewer is correct that no comments were made about dystrophin in the ErbB2/4 dKO mice ². However in ErbB2/4 dKO mice nuclei in muscle fibers were examined extensively (see figures in supplemental data of that publication) ² and centralized nuclei, a hallmark of DMD ^18,19 were not observed. It was also reported in the supporting online text (supplemental data) ² that atrophic fibers could not be observed. Hence, based on these observations, there was no indication to look for dystrophin expression. It was also mentioned in the article that sustained muscle strength was not affected and that the ErbB2/4 dKO mice did not have a myasthenic condition. However these mice showed a hind limb extension reflex which was not reported in the article.

I trust that I have addressed the reviewers concern sufficiently, and if the reviewer would be willing to review a revised version of this article, I would welcome the opportunity to submit one that would include some of the explanations/clarifications presented here.

Bibliography

1.      Gajendran, N. The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se [version 1; referees: 1 not approved]. F1000Res. 7, 1321 (2018).

2.      Escher, P. et al. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308, 1920–1923 (2005).

3.      Gautam, M. et al. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85, 525–535 (1996).

4.      Schmidt, N. et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J. Cell Biol. 195, 1171–1184 (2011).

5.      Dumont, N. A. et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat. Med. 21, 1455–1463 (2015).

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7.      Gajendran, N. ERBB4 RECEPTOR MODULATORS FOR USE IN THE TREATMENT OF DYSTROPHIN-ASSOCIATED DISEASES. (2017). WO 2017/036852 A1

8.      Engel, W. K. Muscle biopsies in neuromuscular diseases. Pediatr. Clin. North Am. 14, 963–995 (1967).

9.      Ito, K. et al. Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice. Hum. Mol. Genet. 15, 2266–2275 (2006).

10.    Ennen, J. P., Verma, M. & Asakura, A. Vascular-targeted therapies for Duchenne muscular dystrophy. Skelet Muscle 3, 9 (2013).

11.    Leu, M. et al. Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development. Development 130, 2291–2301 (2003).

12.    Sparano, J. A. Cardiac toxicity of trastuzumab (Herceptin): implications for the design of adjuvant trials. Semin. Oncol. 28, 20–27 (2001).

13.    Ozcelik, C. et al. Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc. Natl. Acad. Sci. USA 99, 8880–8885 (2002).

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The author has responded with some reasonable arguments but the issue with the paper is lack of evidence. No new evidence is provided.  In vivo rescue of the dystrophic phenotype in dystrophic deficient mice and dogs by delivery to skeletal muscle is a compelling argument for the role of dystrophin in skeletal muscle. If there is a role in smooth muscle, then this needs to be shown by in vivo experiments not a cell line.

I thank the reviewer for responding to my comments.

Although the reviewer does not question the discovery described in the manuscript, that NRG signalling is required for dystrophin expression as demonstrated in myotube cultures, concerns remain regarding in vivo evidence to confirm that the root cause of muscular dystrophy is due to the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle.

The reviewer’s concern is understandable given the vast number of publications that focused on the lack of dystrophin in skeletal muscle as the cause of muscular dystrophy. These reports are indeed in contrast to our observation that  ErbB2/4 dKO mice don’t show dystrophic symptoms ¹. In these mice, NRG signalling, and as a consequence also dystrophin expression (based on our in vitro results/ new findings), is abolished in skeletal muscles but not in vascular smooth muscle (VSM).

Ideally, to address the reviewers concern, a conditional transgenic mouse where either ERBB2 (or ERBB4) is knocked out in both skeletal and VSM should be examined to have a situation where dystrophin would be lacking in both.

On the other hand it would also be good to demonstrate that dystrophin absence in skeletal muscle does not cause dystrophic symptoms, confirming our observations on the ErbB2/4 dKO mouse.

I address below the reviewers comments.

In vivo evidence for a role for dystrophin in smooth muscle:

Mice where ErbB2 is conditionally knocked out in skeletal muscle, HSA-CRE/ErbB2, following CRE expression driven by the HSA promoter ² show a similar phenotype as ErbB2/4 dKO ¹ mice mentioned in this article (in both mouse strains the same HSA promoter is used to express CRE). As the HSA promoter is not active in smooth muscle, ERBB2 receptors would not be ablated and dystrophin will still be expressed in VSM, in contrast to skeletal muscles in these mice. The loxP flanked ErbB2 mouse is the same mouse that was used in breeding with an ERBB4 conditional  KO mouse and an HSA-CRE transgenic to finally generate the ErbB2/4 dKO mouse ¹. The abnormal muscle spindle formation reported for the HSA-CRE/ErbB2 mouse was not looked at in the ErbB2/4 dKO mouse probably because it had already been described in the HSA-CRE/ErbB2 KO mouse. In addition the study describing the ErbB2/4 dKO mouse was focused on the role of NRG/ERBB signaling in synapse specific transcription and NMJ formation.

Interestingly an ErbB2 conditional knock-out mouse with CRE expression driven by the muscle creatine kinase (MCK) promoter, MCK-CRE/ErbB2 was made that was different from the one mentioned above  as it showed impaired muscle regeneration and a requirement of ERBB2 for survival of muscle spindles and myoblasts ³. As VSM express both the brain and muscle isoforms of creatine kinase ⁴, the MCK promoter would be active in VSM and hence ERBB2 would be ablated resulting in a loss of dystrophin expression in VSM in these mice, explaining the different histopathology characters between the two ErbB2 KO mice that differ  in the promoter driving CRE expression. The muscle phenotype described for the MCK-CRE/ErbB2 KO mouse was not observed in the HSA-CRE/ERBB2 KO mouse where the HSA promoter is not active in VSM, and hence ErbB2 would not be ablated and therefore dystrophin would be expressed. MCK-CRE/ERBB2 KO mouse primary myoblasts lacking ErbB2, were reported to undergo extensive apoptosis when differentiating into myofibers ³, an abnormality that I did not observe that when using ErbB2/4 dKO myotubes ⁵.

In another study, siRNA mediated silencing of dystrophin expression in the muscles of adult mice was meticulously analysed ⁶. The authors concluded that in spite of the clear absence of dystrophin in the skeletal muscle, they did not observe any of the histopathology characters observed in mdx mice ⁶. This paper describes silencing of dystrophin expression in skeletal muscles of adult mice, which results in a delay before the existing dystrophin is depleted, and the authors suggest that the dystrophic pathology observed in dystrophin deficiency may be developmentally regulated. However in the ERBB2/4 dKO mice, dystrophin would be ablated early during development in skeletal muscles since the HSA promoter driving CRE expression is active on E9.5, when dystrophin would also start to be expressed ⁷, which would argue against the dystrophic pathology being developmentally regulated.

In vivo rescue of the dystrophic phenotype in mice and dogs may be due to delivery of the therapeutic (construct, anti-sense oligoribonucleotides, gene editing components) into skeletal and vascular smooth muscle

There have been numerous attempts to express dystrophin in skeletal muscle that ameliorated the dystrophic pathology to some extent, but none have led to applicable therapy. As the focus of most therapies are to restore  skeletal, and sometimes also cardiac muscle it was not of concern whether VSM would or would not be targeted during therapy. Gene therapy using Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoters ⁸ would also result in the  expression of dystrophin, or smaller versions of it, in surrounding VSM after intra-muscular (IM) injection or particle bombardment ⁹. In a Phase I study of gene therapy for Duchenne/Becker muscular dystrophy (D/BDM) the CMV promoter was also used ¹⁰ and hence also here dystrophin would be restored  in VSM. Systemic delivery of antisense oligoribonucleotides ¹¹, or restoration of dystrophin expression by (intravenous) stem cell transplantation ¹², would also result in dystrophin restoration in VSM.

Although in vivo rescue of the dystrophic phenotype in dystrophin deficient mice and dogs focused on delivery of the construct to skeletal muscle, it would also target VSM and hence it is neither possible to exclude the role of VSM nor to conclude that delivery to skeletal muscle is sufficient, to rescue the dystrophic phenotype. Furthermore, simply expressing dystrophin in skeletal muscle fibers lacking dystrophin would restore the dystrophin associated protein complex (DAPC) without saying much about whether muscle function is restored. If indeed muscle function is restored, then it should be looked at whether dystrophin may also have been expressed in VSM through leaking of the therapeutic into those tissues, sometimes through the circulation especially when using viral based therapies as described below.

Gene editing of mutated dystrophin using adeno-associated viruses (AAV) to deliver CRISPR components is a promising therapeutic strategy and has recently been demonstrated to function in a canine model of Duchenne muscular dystrophy ¹³. But it still needs to be determined if the gene editing will be sustainable over long term. In this study an AAV serotype 9 that shows tropism for heart and skeletal muscle was used to deliver the CRISPR components. To drive the expression of Cas9 (one of the gene editing components), a muscle specific creatine kinase regulatory cassette was used that should also result in gene editing of dystrophin in VSM especially when the viruses are injected intravenously for systemic delivery. In a second experiment in the same study, 6 weeks following delivery through direct injection into muscles, dystrophin expression and assembly of the DAPC in dystrophic muscles was observed. Dystrophin expression in contralateral muscles that were not injected was also observed and it was attributed to leakage of AAV9 into circulation.

In contrast to the siRNA silencing of dystrophin mentioned earlier, which did not affect muscle function, a very high amount of virus carrying the gene editing components was used in the case of CRISPR mediated gene editing in the canine, which will increase the likelihood for some of it leaking into the circulation and transducing other tissues, including VSM.

Given that therapy for Duchenne patients is largely focused on skeletal muscle, this article would suggest that development of therapeutics should (also) focus on VSM (and possibly also vascular endothelial cells) for any long term therapeutic benefit for patients with Duchenne and Becker muscular dystrophy. In addition the demonstration that NRG/ERBB signalling stimulates dystrophin expression would help to improve treatment regimens with Herceptin/Trastuzumab to ameliorate cardiomyopathy. Finally, this article suggests that investigation of changes in functional dystrophin levels as a possible cause of disease states associated with NRG/ERBB signalling is warranted.

I trust I have addressed the concerns the reviewer raised.

Bibliography

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13.    Amoasii, L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science (2018). doi:10.1126/science.aau1549