## Ex vivo gene therapy

The first disease treated with gene therapy was so-called severe combined immunodeficiency (SCID). Patients with SCID have a severely weakened immune system. Several cases were described in which patients had to be isolated in ventilated rooms or personal bubbles because otherwise there was a great risk that even the mildest infection could be fatal. Bone marrow transplantation or gene therapy are the only chance for such patients to lead a normal life. So far, two genes are known whose mutations lead to the manifestation of the disease – a mutation in the IL2RG gene, which codes for the gamma interleukin receptor (located on the X chromosome, the disease is called X-linked SCID) and a mutation in the ADA gene, which codes for the enzyme adenosine deaminase (located on chromosome 20, ADA-SCID). In both cases, the mutations lead to the inactivation or complete loss of T and B lymphocytes. The first attempt at gene therapy for this disease was made in 1990. A four-year-old Ashanti DeSilva was diagnosed with the ADA-SCID variant. Blood samples were taken and white blood cells were isolated. These cells were transformed with a retrovirus carrying a healthy copy of the ADA gene. The transformed cells were returned to the patient's body, where they began to produce a functioning enzyme. Ashanti DeSilva thus became the first patient to successfully undergo gene therapy. Three years later, Andrew Gobea, who was only 5 days old, underwent a similar therapy. The difference was that he was administered modified germ cells from the mother's placental blood and umbilical cord. The administration was successful, but in the first years the therapy had to be combined with additional administration of the ADA enzyme. As mentioned earlier, there is a major risk associated with the use of retroviruses in the form of integration of DNA into important parts of the genome. This was also observed with this therapy – nine patients developed leukemia within six years of administration of the drug. Fortunately, a much safer therapy is now available for ADA-SCID, using a modified HIV virus.

An intuitive target for ex vivo gene therapy is blood diseases, such as beta-thalassemia and sickle cell anaemia. In both cases, the disease is caused by mutations in the gene encoding the β-chain of haemoglobin (HBB). Haemoglobin is responsible for binding oxygen and carbon dioxide in red blood cells. There are several mutations that can lead to a disruption of this function, which in the case of sickle cell anaemia is also reflected in an altered structure of the blood cells. Patients suffering from beta-thalassemia or sickle cell anaemia are dependent on frequent blood transfusions or a bone marrow transplant. The disadvantage of a bone marrow transplant is that it may not be accepted by the patient's body and is therefore attacked by the immune system. For that reason, a suitable alternative is therapy with the patient's own hematopoietic germ cells modified with a lentiviral vector carrying the human HBB gene. This is a one-time therapy for beta-thalassemia patients older than 12 years who are dependent on blood transfusions. Another promising example of ex vivo gene therapy is the so-called CAR-T therapy in the treatment of cancer patients (Figure 10.5). In this therapy, T lymphocytes are taken from the patient and then genetically engineered to have a chimeric antigen receptor (CAR) on their surface. CAR enables T lymphocytes to bind to specific sites – epitopes on the surface of target cells. Binding occurs only when the structures of the epitope and the CAR receptor are complementary, i.e. they fit together like two pieces of a puzzle. After the binding of CAR and the epitope, a molecular signal is sent that leads to the activation of the immune response. When the CAR is designed to recognize an epitope specific to cells of a particular cancer type, its activation leads to an attack on those cancer cells. Because the altered cells come directly from the patient, there is little chance that the therapy will not be accepted. However, an important prerequisite is that the patient's type of cancer cells is known and that the right CAR is available. There are several drugs on the market in Europe that use the CAR-T system. They are aimed at treating certain types of leukaemia and lymphoma when conventional therapy has failed or the cancer has returned shortly after the treatment. The shortcoming at present is the small number of known pairs of CARs and epitopes specific for different types of cancer, as well as the observed toxicity of activated T lymphocytes, which slows down their use in practice.