Gene therapy

Limitations of traditional therapy

In modern medical practice, there are more than four thousand known human hereditary diseases. Out of this vast number, only a few are amenable to ongoing therapy. The traditional approach to managing such patients is replacement therapy. Its principle lies in the regular use of an exogenous (i.e., externally introduced) product that, in a healthy body, is coded by the very gene that is defective in the patient. A classic and most well-known example is the administration of insulin for the treatment of diabetes mellitus.

However, replacement therapy has strict limitations. Treatment via the simple administration of the missing metabolite proves effective exclusively when this metabolite is a small molecule. Only small molecules are capable of unimpeded transport through the circulatory system and penetration into target tissues.

When it comes to enzyme replacement therapy, a number of critical problems arise:

• Instability: most enzymes are extremely rapidly degraded in the biological fluids of the body.
• Delivery difficulties: it is practically impossible to deliver an enzyme precisely to its site of action while preserving its active form.
• Permeability: cell membranes are completely impermeable to large macromolecules, which include all proteins and enzymes.

Due to the macromolecule delivery problem, it becomes obvious: proteins must be synthesized directly within the cells where they are necessary for normal vital activity.

Basic concepts of gene therapy

To overcome the barriers of traditional medicine, gene therapy was developed. The essence of this advanced method consists of introducing a normal copy of a gene into the genome of a patient carrying defective alleles.

Within the framework of this field, specific terms are used:

• Transgene — a normal gene that is artificially introduced into the patient's cell and organism.
• Transgenic organism — an organism that carries the introduced gene in its genome.

The main goal of the procedure is to ensure that, following successful integration, the transgene begins to independently synthesize the missing product. This allows for the complete restoration of the patient's normal phenotype.

Clinical example: severe immune deficiency

One of the striking examples of the successful application of gene technologies is the treatment of severe combined immunodeficiency (SCID). The etiology (cause) of this disease lies in the congenital absence of a crucial enzyme — adenosine deaminase (ADA). A direct consequence of this deficiency is a profound impairment of the function of key immune cells — T lymphocytes and B lymphocytes.

The gene therapy technique in this case is performed outside the patient's body (the ex vivo approach) and includes several successive stages:

1. Material collection: bone marrow cells are extracted from the patient, from which leukocytes (white blood cells) are then carefully isolated.
2. Vector preparation: a special retrovirus is created in the laboratory. It contains vector DNA into which a normal allele of the required gene (ADA+ gene) has already been inserted.
3. Transfection (infection): the previously isolated leukocytes are infected with the engineered retrovirus.
4. Integration: the viral DNA fragment, along with the normal gene, is physically incorporated into the chromosomes of the patient's leukocytes.
5. Cultivation and control: the modified cells are grown in a special culture. At this stage, the function of the inserted gene is mandatory checked — the expression of the ADA+ transgene is evaluated.
6. Reinfusion: cells with recombinant DNA that have proven their functionality are injected back into the patient's circulatory system.

Treatment outcome

Upon entering the bloodstream, the renewed leukocytes begin to actively synthesize the ADA enzyme, leading to the restoration of their full immune function. However, it is important to understand that the duration of the therapeutic effect is strictly limited to the lifespan of these modified leukocytes. Over time, they die off naturally, so to maintain immunity, the treatment may require periodic repetition.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Mechanisms of retroviral DNA integration into host cell chromosomes
• Differences between ex vivo and in vivo gene therapy strategies
• Methods for laboratory control of transgene expression in cell culture
• Pathogenesis of T and B lymphocyte dysfunction in adenosine deaminase deficiency

Other topics in the "Medical Genetics" section

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.