Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy
Authors: Emily H Canessa
Peter P Nghiem
Joe N Kornegay
Stanislav Vitha
Cynthia Balog-Alvarez
Amanda K Bettis
Sara Mata López
Journal: PLoS ONE (2020)
Discipline: Multidisciplinary
Keywords: Duchenne muscular dystrophy; DMD gene; dystrophin protein; golden retriever muscular dystrophy dog; GRMD
Affiliations: Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States of America., Microscopy and Imaging Center, Texas A&M University, College Station, TX, United States of America., Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Johnson City, NY, United States of America.

Abstract:  Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression. 

Retracted: No

PubPeer comments:
 Dear Authors, We have discussed your paper u201cWidespread seasonal gene expression reveals annual differences in human immunity and physiologyu201d with great interest and excitement during our weekly literature meeting. However, as we have noticed a few things that we would have liked to see explained (or corrected) in future publications, we permit ourselves to present some suggestions for improvements: General comments: 1.tAuthors interpret their data as if seasonality of gene expression caused by the environment (changing seasons) is the reason for seasonality of human disease. However, correlation is not causation. Although the paperu2019s take-home message is a very tempting interpretation of the presented data, it is only a correlation, which does not take into account myriads of other factors. Humans have learnt how to overcome environmental constraints thousands of years ago (and probably a few human species ago), so they are no longer dependent on the environment to survive. However, their culture and behavior is dependent on seasonality. Additionally, as it was shown in many publications, it is usually the predators and or (in human case) pathogens that shape the specie evolution. Moreover, the pathogenic infections are also seasonal (as also mentioned in this paper). Therefore, the more convincing interpretation of the presented data would be that PATHOGENS cause the variation of gene expression in humans across the seasons. However, that is only the tip of the iceberg. What is causing the pathogens seasonality in the modern human environment? It is most likely strongly influenced by human customs and behavior. During the summer, cities are empty and people keep social distancing while taking holidays. The recent pandemic has clearly shown that social distancing significantly reduces pathogen seasonality, as there was no seasonal flu in Europe during lockdown. It is worth remembering that Australian summer holiday season is in December and January, which still fits the paperu2019s data. Iceland and Gambia are harder to interpret as both have critically different customs from Europe or Australia. Iceland is a relatively small community without big cities. Social distancing is not only a part of (Nordic) cultural habits but it is also unavoidable in the majority of the country. On the other hand, Gambia has a relatively dense population and highly occupied public transportation, which may not facilitate social distancing. Nevertheless, social distancing is only one of the factors of human behavior that could affect pathogen (and gene expression) seasonality. Others could be: schooling system, public transportation system, housing, social gatherings, waste management, food culture, poverty, healthcare system, public holidays, dominant religion, political system etc. 2.tAge and gender were used as confounding factors in the statistical analysis. Why did authors not use ethnicity? It would be especially interesting when comparing results between countries of mixed ethnicity (UK, Australia) with countries of homogenous ethnicity (Gambia, Iceland). 3.tThe major dataset (BABYDIET) used in the paper are from people with specific genetic background. T1D PBMC dataset came from people with type-1 diabetes. If the subjects of the study had disease predispositions, it could have enhanced the inflammatory phenotypes. Were there any healthy control datasets used in the study? 4.tStarting the paper from the detail (mRNA expression of circadian genes) and then moving to general description of the data feels a bit confusing. Wrapping the story up with figure 1 could have created a better narrative. 5.tAlthough the authors have analyzed the data patterns thoroughly, there is little analysis of the seasonal genes itself. The analysis presented in Supplementary Fig. 8 should have been summarized and included in Fig. 5. It would also be interesting to do and present a similar analysis only for the genes that were consistently seasonal in both children and adults. 6.tWere datasets searched for seasonal (summer winter) patterns only? Were there any other yearly patterns besides seasonal? 7.tDo the authors have any hypothesis for why some genes are not seasonal in adults? Did the authors look more closely at the genes that were seasonal only in children? 8.tIt would be interesting to analyze the current pandemic through the results presented in this paper. Has the pandemic and the measures taken against the pandemic changed the seasonal gene expression? Do seasons affect the pandemic development? Specific comments: 1.tBABYDIET samples were collected across many years. Were there any differences between babiesu2019 1st year and subsequent ages? Was it possible to follow specific patientsu2019 seasonal variation across the year? 2.tFig. 1b - Why is there no difference in the expression of ARNTL between morning and morning afternoon samples? Did any of the circadian clock genes present a difference in the expression between morning and morning afternoon samples? 3.tFig. 4b - Can the eosinophils seasonality be explained by the seasonality of the pollen? Was it reported previously? 4.tFig. 4c u2013 Standard units for hematological parameters are not known by everyone. It would be helpful for the reader if it was specified on each graph axis. 5.tFig. 5 title says that the data is representative for the whole Europe. However, for the sake of the paperu2019s major topic (seasonality) it would have been better to call it representative only for NORTHERN Europe. There were no datasets from the countries of southern Europe, which have very mild winters and possibly different seasonal pattern. 6.tFig. 5a u2013 The diagram is a bit confusing and out of the place as it is explaining data from Supplementary fig. 8. Using both colors and color names in one diagram should have been avoided. Associated pathways could have been named under the clusters to avoid using another color-coding. 7.tMost of the paragraph u201cSeasonal differences in human immunityu201d should have been placed in the discussion as it is not describing the results but interprets the data and presents the hypothesis 8.tFig. 6 u2013 genes presented here could also have been analyzed similarly to Supplementary Fig. 8. It is not only interesting which seasonal genes are shared between BABYDIET dataset and adipose tissue dataset, but it is also interesting which genes are specifically seasonal exclusively in adipose tissue. ,