Biotechnology - Yu.O. Sazykin 2006

General Biotechnology
Genomics and Proteomics
The Human Genome - Gene Therapy

By comparing genes, scientists will be able to identify links between various genetic variations, Mutations, and a wide range of diseases.

Progress in Human Genetics has led to The Emergence of Gene Therapy, a critically important Structure/182.html">Practical Application of Genomics in medicine. It offers a way to treat numerous hereditary disorders, which are classified into monogenic and polygenic diseases.

At THE MOLECULAR LEVEL, Monogenic Diseases stem from a defect in a single cellular protein—whether an enzyme, a transport protein, or a structural protein. First, the protein may be deficient in quantity; second, its function may be impaired. Thus, a mutation altering The activity of a specific enzyme can result in either the accumulation of a toxic substrate or a shortage of a compound essential for normal cellular function. Similarly, a mutation in a gene encoding a structural protein can lead to severe damage in Cells, Tissues, or Organs.

Furthermore, a mutation in a gene expressed in one tissue can have severe repercussions in another, triggering a cascade of symptoms. For instance, a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase blocks The conversion of phenylalanine to Tyrosine, leading to elevated levels of endogenous phenylalanine in the Blood, defective myelination of axons in the Central Nervous system, and, consequently, severe intellectual disability.

The polygenic nature of a disease means that multiple cellular Proteins carry certain defects. Although each body tissue expresses a unique subset of the total gene pool, certain mutations cause diseases that literally affect all organs and tissues: Muscles, eyes, Liver, bones, The Heart, and others. Notably, conditions such as Cancer and Hypertension are considered polygenic. Certain non-hereditary and infectious diseases, particularly those of viral Etiology, are also classified as polygenic.

Naturally, gene therapy yields the best results when applied to Monogenic Disorders. In such cases, the target gene must be both mapped and identified (its function fully understood). To date, approximately one thousand genes involved in the onset and development of monogenic Hereditary diseases have been mapped, but only a few hundred have been identified.

Gene therapy requires the preliminary creation of a recombinant genetic construct containing a normal, "healthy" copy of the defective gene, along with a vector capable of transporting it into the Organism's cells. For the gene to function properly, specific cis- and trans-regulatory sequences are required. The former (cis-acting) are located on the same chromosome and may be directly adjacent to the gene or at some distance, acting as a promoter; the latter (trans-acting) are located on other Chromosomes.

While Methods for introducing genes into target cells in gene therapy are diverse, they are often insufficiently effective. This is largely because foreign DNA is integrated into The Genome of only a small percentage of tissue cells, and is frequently degraded by Nucleases. However, encouraging results have been achieved using genes "packaged" in Liposomes.

Currently, the most promising approach for gene transfer in gene therapy involves incorporating genes into vectors derived from Retroviruses or Adenoviruses. Naturally, this raises primary concerns regarding the safety of such vectors. The Viruses are genetically modified so that they retain their ability to enter cells while losing the capacity for autonomous Replication.

To ensure targeted delivery of the engineered sequence, researchers take advantage of the varying tropism of different viruses for specific tissue types. For example, adenoviruses exhibit high tropism for respiratory epithelial cells, whereas the herpes virus is highly tropic for central nervous system Neurons. Looking ahead, future gene therapy aims to utilize entire recombinant chromosomes, enabling the simultaneous manipulation of multiple genes and their regulatory sequences.

Modern gene therapy is strictly focused on somatic cells rather than germline (reproductive) cells.

Ex Vivo Gene therapy involves introducing a normal copy of a gene into somatic cells previously extracted from the patient's body. These corrected cells are cultured in vitro and then reintroduced to the patient via transfusion or transplantation. It is recommended to use the patient's own cells and return their "corrected" progeny back to them, which eliminates the risk of immune rejection mediated by innate Immunity. Nevertheless, relying exclusively on autologous cells narrows The Scope of gene therapy; consequently, various methods—such as The Use of immunosuppressants—have been developed to protect against the Immune Response triggered by non-autologous cells.

In In Vivo Gene therapy, delivery of the normal gene is performed directly into human tissues (specifically into the cells of targeted tissues). In this approach, the gene promoter must be trans-specific.

The roster of hereditary DISEASES ASSOCIATED WITH enzymatic deficiencies continues to grow as their biochemical mechanisms are unraveled. Accordingly, approaches to harnessing the theoretical potential of gene therapy are attracting increasing attention and becoming more concrete. A prime example is the application of gene therapy in the Treatment of cystic fibrosis. The cystic fibrosis gene—the cystic fibrosis transmembrane conductance regulator (CFTR)—encodes a membrane protein known as the CFTR protein. The primary function of CFTR is to form a chloride channel regulated by cyclic 3',5'-adenosine monophosphate (cAMP). Mutations in this gene alter the quantity or STRUCTURE OF THE protein, disrupting The transport of chloride ions and Water across the epithelial Cell membranes of several organs. As a result, the mucus secreted by exocrine glands becomes dehydrated and increasingly viscous. This triggers inflammation and the proliferation of infectious agents (secondary pathology). In cystic fibrosis, the Lungs (Bronchi) are the most severely affected.

The foundation for applying gene therapy to cystic fibrosis was laid by positive results obtained in cell cultures. Introducing just a single copy of the normal gene into a cell with a defective genome proved sufficient to restore ion transport. Even more encouraging was the finding that "correcting" as little as 10% of the total cells in a monolayer was enough to normalize chloride Transport Across the entire monolayer (likely due to Ion Exchange between neighboring cells). Furthermore, it turned out that strictly regulated expression of the normal foreign gene encoding the CFTR protein is not strictly necessary, as this protein is non-toxic even when overexpressed.

Following thorough preclinical studies, gene therapy for cystic fibrosis advanced to clinical trials. The normal gene, housed within modified adenoviruses, was delivered to lung epithelial cells using liposomes. However, the clinical outcomes were less stellar than anticipated: out of several hundred cases, only a handful of trials were successful. Nevertheless, the very transition from experimental gene therapy for cystic fibrosis to clinical application is a major milestone. In practice, future gene therapy for cystic fibrosis will likely be combined with antibiotic and enzyme replacement therapies, which currently extend patients' lives but do not achieve a complete cure.

Gene therapy is steadily capturing the attention of popular science publications and the mass media. Dozens of gene therapy technologies targeting various diseases have undergone clinical evaluation in thousands of patients and volunteers across the United States, the UK, France, and other nations. In several clinics, these trials have proceeded smoothly. As is well known, Phase I clinical trials are designed to test the safety of a new therapeutic agent or method. Notably, there have been reports of leukemia-like conditions emerging following the clinical testing of certain gene therapy technologies, all of which reportedly utilized retrovirus-based vectors. Additionally, isolated cases have been documented where the introduced gene was expressed for a shorter duration than planned.

However, even though early clinical trials have been less successful than preclinical data suggested, and the clinical use of certain gene therapy modalities has been temporarily halted, research and technological refinement continue overall. This is especially true given that in desperate medical scenarios, a physician—with the patient's consent or at their request—may proceed with experimental treatments even when certain doubts remain from preclinical testing.