The selected professor is Ronald D. Cohn, who is the president and CEO of The Hospital for Sick Children or SickKids. He contributed to the overall development of clinical approaches for children with degenerative neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). The techniques include whole-genome sequencing for enhancing clinical management and diagnostics in the field of pediatric medicine, targeted genome sequencing as a genetic test tool, and use of clinical genome and exome sequencing (CGES). In addition, he sees great prospects in applying Crispr/Cas9 for treatment developments for genetic disorders.
Localization and cloning of the DNA sequence of the dystrophin gene open up fundamentally new diagnostic possibilities for Duchenne muscular dystrophy (DMD), based on the study of mutant alleles in patients, members of their families, or in putative heterozygous carriers of pathological mutations. This applies equally to perinatal diagnosis, which can be carried out using molecular methods of analysis at the earliest stages of fetal development.
The same approaches are quite acceptable for preclinical diagnosis of the disease, which allows researchers and clinicians to develop and start a rational treatment tactic, as well as to effectively identify heterozygous carriers in high-risk families for the prevention of Duchenne myodystrophy. The decisive advantage of molecular diagnostics is its versatility, the ability to use any DNA-containing cells or tissues for analysis, and in principle, the analysis can be performed at any stage of ontogenesis, starting with the zygote stage.
A serious problem for the diagnosis of heterozygous carriage is the case of gonadal mosaicism – the presence in a somatic cell of the gonad of a woman who carries mutations of the dystrophin gene, which, in turn, leads to the appearance of several generations of germ cells or oocytes with mutant and normal dystrophin genes. Such mutations can occur even at the level of primary germ cells, that is, in the early stages of the future development of the mother.
It is practically impossible to assess the size of the aberrant clone of oocytes. If the mother of a patient with DMD cannot determine heterozygous carriage, then it becomes difficult to make a prediction regarding the health of the next child in the family. Modern molecular genetic methods can significantly improve diagnostics for determining the status of carriage in high-risk DMD families, making it more accurate and expanding the scope of its application.
In order to determine the presence of Duchenne dystrophy, molecular biological methods are used to determine the exact location and type of mutation. Integrating a patient’s phenotype as a part of clinical data plays a major role in identifying the correct variant of CGES analysis (Bowdin et al. 1079). A biopsy of the patient’s tissue, used in complex cases, allows determining the presence and localization of dystrophin and the state of muscle tissue. However, monitoring the patient’s condition is also necessary during the treatment process. Repeated sampling of a biopsy is not practiced, as it can be difficult for a patient to tolerate, moreover, a biopsy can assess the condition of only a small area of ​​one muscle.
At the same time, the functional test of walking allows medical specialists to assess the condition of only outpatients, and the result of the test depends on the patient’s care, his desire and ability to follow the technique. Therefore, the task of developing a minimally invasive and reliable method for assessing the dynamics of the condition of a patient with myodystrophy during and after therapy seems to be very urgent. The solution may be an approach similar to a liquid biopsy, that is, an analysis of exosome miRNAs obtained from the patient’s blood.
Attempts to use stem cells to treat various pathologies have been undertaken for a long time. In DMD models, stem cells derived from cardiac stroma are used. The main mechanism of stem cell treatment is believed to be due to their ability to stimulate differentiation and regeneration. It should be noted that in all these and discussed topics, little attention was paid to the possible side effects of exosome administration. Most often, the authors evaluate the change in the weight of experimental animals, the level of liver enzymes, blood urea nitrogen, but do not consider the reaction of the immune system.
Treatment of DMD with native exosomes seems promising, however, this approach is somewhat similar to the existing ones, since it is not aimed at solving a key problem – the absence of dystrophin. The use of exosomes as carriers of active substances would help to solve this problem. The idea of ​​delivering a full-sized protein or its gene, or at least a reduced form, looks very attractive. However, the length of the full-sized dystrophin exceeds the upper estimate of the exosome diameter. Therefore, in all likelihood, loading a protein or coding construct into exosomes will be ineffective. However, exosomes can be used to deliver low molecular weight compounds.
Whole-genome sequencing can also be a critical technique in improving the overall understanding of genetic disorders. It can also lead to complete identification of every type of variation based on one’s genes (Stavropoulos et al. 2). Based on methodological principles and approaches, DNA markers are defined as a combined group of methods based on a qualitative or quantitative assessment of genetic material in the analyzed biological samples.
Moreover, each type of marker can be characterized by basic genetic characteristics, such as the nature of inheritance, the mechanism of transmission, localization in the genome, and the level of variability. In addition, additional laboratory diagnostic criteria are distinguished, such as the resolution of the analysis. This also includes reproducibility of results – the degree of complexity of their interpretation, the dependence of the analysis on the type of tissue being analyzed, its age, degree of damage, the probability of obtaining erroneous results, and artifacts.
It should be noted that among the whole variety of technologies and methods of DNA analysis, there is still no single universal marker that would allow solving problems related to the analyzed object. Therefore, in each case, it is necessary to choose the method that is most suitable for solving current problems. There are a large number of direct and indirect approaches to the analysis of information presented in DNA that differ according to the above criteria and the list of problems.
Among them, there are markers based on the use of PCR amplification technology, such as intensive cloning of genetic material in vitro using a set of reagents that support the process of replication of DNA fragments, have gained the most popularity. To determine the boundaries of the cloned fragment in a long single DNA chain, artificially synthesized specific oligonucleotide molecules are used that attach to specific places in the long DNA chain, thereby indicating the replication region for the DNA polymerase enzyme.
This allows a researcher to clone fragments located in any part of a long single strand of DNA. In general, based on the methodological features of PCR amplification, information on the structure of primers, the size and location of cloned fragments are the basis for most indirect methods for analyzing the nucleotide structure of DNA. The limitations of all indirect approaches are associated with the ability to work only with objects for which the studied genome sections have been previously deciphered.
In addition, the analysis of mouse models for metabolites can be useful in increasing the overall understanding of muscular dystrophies. For example, improper polyamine metabolism can be a critical factor in muscular disorder in mice, which can be partially improved by the expression of Smox and Amd (Kemaladewi et al. 1908). In addition, Crispr/Cas9 might prove to be useful as a therapeutic approach. For example, the tool can be used to gene expression modulation by upregulating utrophin in skeletal-muscle cells for patients with DMD (Wojtal et al. 92). Therefore, these instruments are essential for the overall improvement of clinical diagnostics and treatments.
Whole-genome sequencing is based on the evaluation of all coding sequences of the genome. The latter approach has the greatest prospects in the given field. Despite the fact that the total length of exons, that is, sections of DNA encoding proteins, makes up a small part of the genome, the vast majority of mutations of medical significance are localized precisely in exons. Proceeding from this, cheaper exome sequencing has advantages over genomic for solving diagnostic problems. For instance, whole-genome sequencing can identify a mutation causing the disorder in 2-3 years without additional testing procedures (Lionel et al. 439).
However, this approach implies that all genes whose mutations are associated with a risk of developing a disease of interest are already known. Whole-genome sequencing is devoid of this restriction since it not only allows the detection of defects in known genes but also provides the chances of discovering new genetic elements that cause disease. Combining diagnostic and research capabilities, this technology becomes the most important tool for studying hereditary pathology.
It should be noted that the high-performance sequencing procedure was simultaneously developed by various companies, each of which offers its own technological solutions. In any case, the technique involves the simultaneous sequencing of millions of short DNA fragments, followed by the assembly of individual readings into the genome or exome.
In conclusion, despite great efforts, effective treatment for Duchenne dystrophy has not yet been developed. Existing methods are limited, ineffective, and mostly symptomatic. Exosomes, due to their low immunogenicity and potential for surface modification, can become a means of delivering active substances. At the same time, analysis of the patient’s exosomes can provide diagnosis and monitoring of the course of treatment.
It should be noted that methods for working with exosomes require further optimization. Attention should be paid to the immunogenicity of exosomes, which has not yet been sufficiently studied. A combination of approaches, such as the use of exosomes with regenerative function, loading, and modification of surface proteins with muscle-specific peptides, looks interesting and promising. In addition, more detailed attention needs to be given to Crispr/Cas9 as a therapeutic instrument, which can target key genes and alter the expression rate.
References
Bowdin, Sarah, et al. “Recommendations for the Integration of Genomics into Clinical Practice.” Genetics in Medicine, vol. 18, no. 11, 2016, pp. 1075-1084.
Lionel, Anath C., et al. “Improved Diagnostic Yield Compared with Targeted Gene Sequencing Panels Suggests a Role for Whole-Genome Sequencing as a First-Tier Genetic Test.” Genetics in Medicine, vol. 20, no. 4, 2017, pp. 435-443.
Kemaladewi, Dwi, et al. “Increased Polyamines as Protective Disease Modifiers in Congenital Muscular Dystrophy.” Human Molecular Genetics, vol. 27, no. 11, 2018, pp. 1905-1912.
Stavropoulos, Dimitri J., et al. “Whole-Genome Sequencing Expands Diagnostic Utility and Improves Clinical Management in Paediatric Medicine.” Nature Partner Journals: Genomic Medicine, vol. 1, no. 15012, 2016, pp. 1-9.
Wojtal, Daria, et al. “Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders.” AJHG, vol. 98, no. 1, 2016, pp. 90-101.