Proteins Substituting Missing or Mutated Cellular Proteins
Gene therapy is the introduction of new genetic material (e.g. nucleic acids) into the cells of an individual, which produces a therapeutic benefit for human patients. Therapeutic DNA and RNA are popularly used in this therapy. As time goes by, gene therapy has already become a powerful weapon in modern medicine's realm to help fight serious diseases such as cancer, diabetes, hemophilia, cystic fibrosis, acquired immunodeficiency syndrome, high blood pressure, peripheral vascular disease, coronary heart disease, neurodegenerative diseases, and other genetic disorders.
The fundamental basis of using nucleic acids in therapeutics is inhibition of DNA or RNA expression, thus halting the production of abnormal proteins related to disease while leaving other normal proteins unaffected. As reported, gene therapy was conceived with an intention to express proteins that are defective or missing, curing autosomal recessive and chromosome X-linked illnesses. The replaced proteins can exert their functions inside the cells (for instance, in the case of the treatment for muscular dystrophies) or on the cell membrane (for instance, the CFTR gene in cystic fibrosis), or be secreted into the bloodstream or extracellular environment (for instance, in the case of coagulation factors in hemophilia).
Mutated Cellular Proteins
Through genome-wide studies, it has been known that solid tumors usually contain 20-100 protein-encoding genes that are mutated. A small portion of these changes refers to drivers who are responsible for the development or progression of tumors. The remainders are passengers, offering no selective growth advantage. Theoretically, these proteins give rise to unparalleled opportunities for biomarker research. Unlike other protein markers such as prostate-specific antigen (PSA) or carcinoembryonic antigen (CEA), the mutant proteins are only derived from tumor cells. In addition, they're not simply correlated with tumors, as conventional markers, but in the context of driver gene mutations directly result in tumor generation.
Challenging to Identify Mutant Proteins
The detection of proteins encoded by mutated genes (i.e. mutant proteins) is straightforward when proteins are truncated by a nonsense mutation. This detection typically can be performed easily by western blotting (WB) of cellular extracts. Nevertheless, a large number of disease-caused mutations are missense mutations that alter encoded proteins just subtly. For example, in recent sequencing analysis of all protein-encoding genes in human cancers, more than 80% of somatic mutations are demonstrated to be missense. Although it's notionally possible to identify these abnormal proteins straightly with antibodies directing mutant epitopes, doing so might be difficult to accomplish in practice.
Because of many different mutations occurred in a single cancer-associated gene, it's extra necessary to develop a specialized antibody for each possible mutant epitope, enhancing the difficulty of the strategy. Another method is the measurement of the activity of mutant proteins. Maybe this approach is useful in several situations, but it's not often applicable due to no activity-based assays available for most proteins. And the proteins produced from mutated genes generally have activities that can only be quantitative, rather than qualitative. Consequently, there is an urgent need for assays that would generically permit quantification of mutant proteins.
Improved Approaches for Examination
Figure 1. Schematic of the approach. (Wang, 2019)
Cancer biomarkers are a current subject of intense research considering their potential utility for diagnosis, targeted therapy, and prognosis. In principle, the gene products expressed from somatic mutations are the final protein markers, being not simply related to tumors but actually responsible for tumorigenesis. One report has shown here that these changed protein products can be directly identified and quantified by mass spectrometry (MS). The peptides resulting from normal and mutant alleles are detected by selected reaction monitoring (SRM) of their productions through a triple-quadrupole MS. In addition to addressing basic questions about the relative level of genetically abnormal proteins in cancer, this approach proves valuable for clinically diagnostic applications.
Recent advances in MS allow sampling of a large fraction of normal and abnormal cellular proteomes in a special and an unbiased fashion. MS method has already become the main choice for quantifying protein expressions and several quantitative proteomics strategies for this purpose have been introduced. Intriguingly, MS has been used to sensitively detect and accurately quantify somatic mutations at DNA levels but not at protein levels. Indeed, one of the most commonly applied methods for quantifying these mutations in DNA depends on the measurement of the mass of oligonucleotides differing at a single base. Prior studies have revealed that it is possible to identify post-translationally altered proteins using MS, and to identify highly abundant abnormal proteins, for example, those for amyloidosis.
In Conclusion
Human gene therapy can produce effective therapeutic influences by bringing therapeutic nucleic acids into the body. Most human diseases are associated with heredity in origin, and virtually all diseases have a genetic component (except for trauma). Hence, the chance to treat such disorders by replacing the defective gene with a normal healthy gene offers a promising therapeutic approach for patients who suffer from these diseases. In gene therapy, DNA or RNA-based therapeutics have one of the significant advantages over currently available pharmaceuticals is their selective recognition of molecular targets and signal pathways, showing very specificity of action.
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Reference
- Wang, Q.; et al. (2019). Mutant proteins as cancer-specific biomarkers. PNAS. 108(6): 2444-2449.
