Gene therapy

Recombinant DNA technology made it possible so that now we can think about the therapy of those diseases which could be treated only by directly modifying the genetic material.

Although it was proposed in the 70s that exogenous "good" DNA could be used to replace the defective DNA in those who suffer from genetic defects, significant advance in gene therapy has been achieved only in the 90s, and approved drugs made by gene therapy is still not available.

The aim of gene therapy is to insert, alter, or remove genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutated gene; other forms involve directly correcting the mutation or modifying normal gene that enables for example a viral infection. Theoretically, it is suitable to correct genetic disorders, metabolic syndromes or neurological defects, kill cancer cells, modify the immune response or for immunization against pathogens. Target for gene therapy could be any of the body‘s cells (somatic gene therapy) or germ line cells. In case of germ line gene therapy, germ cells are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. With germ line gene therapy, not only genetic diseases could be treated but also several serious ethical problems arise, thus at least for now somatic gene therapy remains as an option. Most of somatic gene therapy procedures manipulate cells ex vivo: they take out cells from the patient‘s body, treat them in sterile environment, than put them back. Another possibility is to introduce the gene-containing material directly in vivo into the tissues to be treated, for example heart or brain.

Mutant genes can be corrected in different ways:

a) functional gene is inserted into a non-specific place of the genome;

b) change the mutant gene with homologous recombination;

c) selective reverse mutation;

d) modification of the regulation of defective gene (enhancing or silencing).

The latest method includes antisense RNA and RNA interference techniques and will be discussed in a following chapter.

Today we know nearly 3000 disorders that are caused by a mutation in a single gene (for example hemophilia and muscular dystrophy). Gene therapy studies are in an advanced stage in the therapy of cardiovascular diseases, inherited blindness, diabetic retinopathy, macular degeneration; most recently successful gene therapy of HIV-infection has been published [Lalezari 2011].

3.1. Technical problems related to gene therapy

Short “half life” - DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with the insertion of therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits and patients will have to undergo multiple rounds of gene therapy.

Immune response - Anytime a foreign object is introduced into human body, the immune system attacks the invader. The risk of stimulating the immune system in a way that reduces the effectiveness of gene therapy is always a potential risk. Furthermore, the immune system's enhanced response to invaders, it has seen before, makes it difficult for gene therapy to be repeated in the same patient.

Viral vectors – Viruses are the carrier of choice in most gene therapy studies but present a variety of potential problems to the patient: toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Many of the most commonly occurring disorders, such as heart disease, high blood pressure, arthritis, Alzheimer's disease and diabetes, are caused by the combined effects of variations in many genes. Such multifactorial disorders would be especially difficult to treat effectively using gene therapy.

3.2. Human vaccines made by recombinant DNA technology

Using rDNA technologies, a disease causing agent can be isolated, reduced to its basic components, its genetic makeup can be studied, and the agent can be modified so that it no longer causes disease but still induces a strong immune response. Vaccine development using

antigens critical for inducing protection. In addition, it is important to understand the pathogenicity of the invader and the immune response of the host, to ensure that the vaccine induces the appropriate immunological reaction. Recombinant vaccines are designed to be safer, more efficient and effective and/or less expensive than traditional ones.

Recombinant vaccines fall into three basic categories: live genetically modified organisms, recombinant inactivated vaccines, and genetic vaccines [Ellis 1999].

The first recombinant vaccine was developed against Hepatitis B virus (HVB). The vaccine contains one of the viral envelope proteins, hepatitis B surface antigen (HBsAg).

Recombinant HBV vaccines are safer than the attenuated type which through mutations can cause hepatitis or hepatic cancer. HBV vaccine is available in combination with Haemophilus influenzae vaccine (Comvax).

The first vaccine against cancer was approved in 2006 under the name Gardasil and it is used in the prevention of Human papillomavirus (HPV) types 6, 11, 16 and 18 [Lowy 2006]. HPV types 16 and 18 cause an estimated 70% of cervical cancers, and are responsible for many cancer cases of the genital tract; HPV types 6 and 11 cause an estimated 90% of genital warts cases. It does not treat existing infection, therefore, to be effective it must be given before HPV infection occurs. Gardasil cannot be added to patients on immunosuppressive therapy, during treatment with alkylating, cytotoxic or corticosteroid agents, or in immune deficiency.

It is contraindicated in blood clotting disorders because it raises the risk of bleeding and hematoma formation.

Table 8. Recombinant vaccines on the market

Generic name Brand

name Immune response Indication Side effects

Haemophilus B/

Hepatitis B vaccine

Comvax Haemophilus influenzae type b capsular polysaccharide

Clinical trials are ongoing with recombinant influenza vaccine but it is not available on the market. Recombinant influenza vaccines consist solely of hemagglutinin proteins produced by cell culture.

In the future, the development of recombinant vaccines will focus on bioavailability, increased efficiency and safety, and delivery methods will be made easier (oral administration). Trials are ongoing to develop vaccines against non-infectious diseases, first of all in autoimmune disorders and transplantation.