Gene therapy is a fundamentally new approach, representing a combination of biomedical technologies for treating gene defects by introducing new genetic constructs into the body that can restore or replace a defective gene. Such therapy allows people to fix errors caused by mutations in the DNA structure or DNA damage by viruses. Treatment with this method is based on the principle of replacement or restoration of a damaged gene. Having received a copy of the gene, the cell is able to use it to synthesize the necessary proteins. Various mechanisms can be used in gene therapy, such as replacing a pathogenic gene with a healthy copy, turning off the gene that causes the disease and introducing a new or modified gene into the body to help treat the condition.
Viruses are used to introduce new genes, and they have the natural ability to deliver genetic material to cells. Before using the virus to transfer therapeutic genes into cells, it is modified to eliminate the ability to cause disease. After this, DNA is introduced into the non-pathogenic virus through a chemical reaction, and then human cells are infected with this virus, which leads to the movement of DNA into the nuclei of these cells. This method of correction began to be applied after approaches for obtaining isolated genes were developed. Another way to insert genes is to use liposomes, microscopic sacs containing DNA that are absorbed by human cells, thus delivering their DNA to the cell nucleus (Ramamoorth & Narvekar, 2015). Therefore, there are several available vectors for providing gene therapy.
The testing of the genetic correction of hereditary disease is carried out on the primary cultures of the patient’s cells, in which this gene is normally functionally active. On these cell models, the effectiveness of the selected exogenous DNA transfer system is evaluated, the expression of the introduced genetic construct is determined, its interaction with the cell genome is analyzed, and correction methods are developed at the biochemical level. Using cell cultures, it is possible to create a system for targeted delivery of recombinant DNA, but the reliability of this system can be checked only at the level of the whole organism.
The next step is to solve the problem of a vector that provides efficient, and if possible, even targeted gene delivery to target cells. Then, transfection is carried out, that is, transfer of the obtained construct to target cells, the transfection efficiency, the degree of correctability of the primary biochemical defect in cell cultures in vitro, and, most importantly, in vivo in animal biological models are evaluated. One of the main disadvantages of the method is that when the virus is introduced, a potential reaction to it similar to infection can occur (Dunbar et al., 2018). In addition, new normal DNA may be lost, or it may not be able to invade new cells after some time, which will lead to a relapse of a genetic disease.
Furthermore, gene therapy can target both germlines and somatic cells, but these approaches are different. Germline-focused therapies are primarily designed to prevent or treat genetic disorders and abnormalities in the next generation.
The process can be considered to be simpler than targeting somatic cells because there is a significant misbalance in the number of cells. By altering one’s germ cells, a person can ensure that his or her offspring will acquire the necessary genetic information, which will result in either a healthier child or one who is less prone to hereditary diseases. Although most gene therapy approaches are highly complicated and require a vast amount of knowledge of the overall genome, germline therapy is relatively simpler in comparison with somatic cell therapy. In order to produce healthy or genetically enhanced offspring, the necessary changes and modifications need to occur in one ovum and sperm. This makes gene therapy delivery systems a lot simpler due to the limited number of cells involved.
However, somatic gene therapy aims at treating an already developed individual. In general, in order to conduct effective gene therapy, one needs to acquire a patient’s genome and find genetic factors because preventative measures and treatments need to have a target. Somatic gene therapy is further complicated by the therapy delivery mechanisms because organisms consist of large quantities of somatic cells, which possess the same genetic code. Thus, it means that an issue in one gene will be present in all somatic cells. The most plausible approach is to use vectors of delivery, such as viruses. The latter process is highly challenging in itself, which makes somatic gene therapy more immediate but also more difficult.
One of the many applications of the Crispr/Cas9 system, which is characterized by significantly increased requirements for the specificity and accuracy of the modifications introduced, is the editing of somatic cell genomes in vivo for the treatment of human diseases with a genetic basis, or for studying the functions and interactions of genes in vivo.
In contrast to the work described above in this direction, it is not possible to analyze and select cells in which no non-targeted double-strand breaks were introduced, which necessitates the improvement of modification methods. These examples demonstrate that despite significant successes in the field of genome editing, one of the main needs of this direction is to improve methodological approaches and develop strategies for using these approaches to increase the accuracy and specificity of Crispr/Cas9.
However, there are new methodologies for modifying the approaches, such as Crispr/Cas9, through prime editing. Both of the mentioned methodological tools use Cas9 protein in order to make necessary cuts, but the latter can increase the specificity of the Cas9 enzymes by a significant margin (Ledford, 2019). Although it is not going to replace previous gene therapy methods, its high levels of specificity allow it to be safely used in humans.
The main reason is the fact that high specificity will reduce the overall risk of deleting or inserting necessary gene components in non-targeted genome locations, which results in lesser risks for complications. Each type of gene therapy methodological approach has its own purpose, mainly because not all genetic disorders are the consequence of small genetic changes or point mutations. Therefore, a certain number of genetic issues will inevitably require more multi-targeted and less specific gene therapy.
In conclusion, it is important to note that the overall development of a gene therapy program is preceded by a thorough analysis of tissue-specific expression of the corresponding gene, identification of the primary biochemical defect, the study of the structure, function, and intracellular distribution of its protein product, as well as biochemical analysis of the pathological process. All these data are taken into account when drawing up the appropriate medical protocol.
References
Dunbar, C. E., High, K. A., Joung, J. K., Kohn, D. B., Ozawa, K., & Sadelain, M. (2018). Gene therapy comes of age. Science, 359(6372), 1-10. Web.
Ledford, H. (2019). Super-precise new CRISPR tool could tackle a plethora of genetic diseases. Nature. Web.
Ramamoorth, M., & Narvekar, A. (2015). Non viral vectors in gene therapy – an overview. Journal of Clinical and Diagnostic Research: JCDR, 9(1), 1-6. Web.