Ex Vivo Gene Therapy Methods

  • In this Ex Vivo gene therapy methods post we have briefly explained about ex vivo gene therapy, Vectors, and examples.

Ex Vivo Gene Therapy Methods

  • Only some organs (e.g., bone marrow) whose cells can be cultivated in the laboratory can be treated using ex vivo gene therapy. The following are the steps involved in ex vivo gene therapy.
  • The treatment entails the cultivation and genetic repair of the patient’s own cells, followed by their return to the patient. As a result, this approach is not linked to negative immunological reactions after cell transplantation.
  • Only when the therapeutic gene (remedial gene) is stably integrated and constantly produced is ex vivo gene therapy effective. The usage of vectors can help with this.
Ex-Vivo Gene Therapy

The procedure for ex vivo gene therapy


  • The carrier particles or molecules used to deliver genes to somatic cells are referred to as vectors. The important vectors employed in ex vivo gene therapy are listed below.
  • Viruses
  • Human artificial chromosomes
  • Bone marrow cells


  • Viruses, particularly retroviruses, are commonly utilised as vectors in gene therapy. Retroviruses have RNA as their genetic material.
  • The retrovirus synthesises DNA from RNA as it enters the host cell (by reverse transcription). The viral DNA (referred to as provirus) is then incorporated into the host cell’s DNA.
  • In most cases, proviruses are harmless. However, because some retroviruses can transform healthy cells into malignant ones, there is a significant risk. As a result, it is critical to ensure that such an event does not occur.

Harmless Retroviruses

Ex-Vivo Gene Therapy

Large scale production of vector viruses by using helper viruses

  • Before utilising retroviruses as vectors, researchers use biochemical procedures to transform hazardous retroviruses to innocuous ones. For example, the retrovirus can be disabled and rendered harmless by deleting a gene that encodes for the viral envelope.
  • This is because retroviruses cannot enter the host cell without the envelope. Starting with a single envelope-defective retrovirus, a vast number (billions) of viral particles can be produced. This is accomplished by the employment of helper viruses that possess the usual gene for envelope construction.
  • The vector (with a faulty envelope gene) can enter the host cell with the helper virus, and both of them replicate. Hundreds of billions of vector and helper viruses are created by recurrent multiplication in host cells.
  • The vector viruses can be purified after being separated from the helper viruses. It is critical to isolate vector viruses that are completely free of helper viruses. The health of patients undergoing gene therapy is jeopardised by the presence of helper viruses.

Human artificial chromosome

  • A human artificial chromosome (HAC) is a synthetic chromosome that, in addition to encoding a human protein, may replicate with other chromosomes. As previously stated, using retroviruses as vectors in gene therapy carries a significant risk. If HAC is employed, this problem can be solved. In this direction, some progress has been made.

Bone Marrow Cells

  • Bone marrow contains totipotent embryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types (e.g., red blood cells, platelets, macro phages, osteoclasts, B- and T-lymphocytes).
  • For this reason, bone marrow transplantation is the most widely used technique for several genetic diseases. And there is every reason to believe that the genetic disorders that respond to bone marrow transplantation are likely to respond to ex vivo gene therapy also e.g. sickle cell anaemia, SCID, thalassemia.

Examples of Ex Vivo Gene Therapy Methods

Adenosine Deaminase Deficiency

  • The earliest and most widely published human gene therapy was performed to treat adenosine deaminase insufficiency (ADA). This was accomplished on September 14, 1990, at the National Institute of Health in the United States, by a group of workers led by Blaese and Anderson.
Ex-Vivo Gene Therapy

Treatment of adenosine deaminase (ADA) deficient patient by somatic ex vivo gene therapy (SCD-Severe combined immunodeficiency)

Severe Combined Immunodeficiency

  • This is an uncommon genetic immunological illness characterized by malfunctioning of T lymphocytes and (to a lesser extent) B-lymphocytes. A deficiency in the adenosine deaminase gene (located on chromosome 20, with 32,000 base pairs and 12 exons) affects about half of SCID patients.
  • Deoxyadenosine and its metabolites (mainly deoxyadenosine 5′-triphosphate) accumulate and kill T-lymphocytes in the absence of ADA. T-lymphocytes are vital to the body’s immune system. They boost the function of B-lymphocytes to create antibodies, in addition to engaging directly in the body’s defence.
  • As a result, SCID (ADA-deficient) patients are susceptible to infectious illnesses and die at a young age. Children with SCID were previously treated with either conjugated bovine ADA or bone marrow transplantation.

ADA deficiency

  • A plasmid vector containing proviral DNA is chosen. The ADA gene and a gene (G 418) coding for antibiotic resistance are cloned to replace a portion of the pro-viral DNA. With the ADA gene, the antibiotic resistance gene will aid in the selection of desired clones.
  • A patient with ADA deficiency has his or her circulating lymphocytes eliminated. The ADA gene is transfected into these cells by exposing them to billions of retroviruses containing the gene. The genetically modified lymphocytes are cultivated in cultures to ensure that the ADA gene is expressed before being reintroduced to the patient. These cells survive in the bloodstream and produce ADA.
  • As a result, the patient’s ability to generate antibodies improves. There is, however, a limitation. Because lymphocytes have a short life span (only a few months), transfusions are required on a regular basis.
  • In 1995, the ADA gene was implanted into stem cells derived from umbilical cord blood at the time of the baby’s birth. The infant received the changed cells four days after birth. A stable population of ADA gene-producing cells was produced in this manner.


  • The patients of familial hypercholesterolemia lack the low density lipoprotein (LDL) receptors on their liver cells. As a result, LDL cholesterol is not metabolised in liver. The accumulated LDL- cholesterol builds up in the circulation, leading to arterial blockage and heart diseases.
  • Attempts are being made by gene therapists to help the victims of familial hypercholesterolemia. In fact, there is some success also. In a woman, 15% of the liver was removed. The hepatocytes were transduced with retroviruses carrying genes for LDL receptors. These genetically modified hepatocytes were infused into the patient’s liver.
  • The hepatocytes established themselves in the liver and produced functional LDL-receptors. A significant improvement in the patient’s condition, as assessed by estimating the lipid parameters in blood, was observed. Further, there were no antibodies produced against the LDL-receptor molecules, clearly showing that the genetically modified liver cells were accepted.

Lesch-Nyhan Syndrome

  • Lesch-Nyhan syndrome is an inborn error in purine metabolism due to a defect in a gene that encodes for the enzyme hypoxanthine-guanine phosphoribosyl transferase (HCPRT). In the absence of HGPRT, purine metabolism is disturbed and uric acid level builds up, resulting in severe gout and kidney damage.
  • The victims of Lesch-Nyhan syndrome exhibit symptoms of mental retardation, besides an urge to bite lips and fingers, causing self-mutilation. By using retroviral vector system, HGPRT producing genes were successfully inserted into cultured human bone marrow cells.
  • The major problem in humans is the involvement of brain. Experiments conducted in animals are encouraging. However, it is doubtful whether good success can be achieved by gene therapy for Lesch-Nyhan syndrome in humans, in the near future.


  • Hemophilia is a genetic disease due lack of a gene that encodes for clotting factor IX. It is characterized by excessive bleeding.
  • By using a retroviral vector system, genes for the synthesis of factor IX were inserted into the liver cells of dogs. These dogs no longer displayed the symptoms of hemophilia.

Further Readings