Gene Therapy

All of these diseases are caused by a defect at a single gene locus. (The inheritance is recessive so both the maternal and paternal copies of the gene must be defective.) Is there any hope of introducing functioning genes into these patients to correct their disorder? Probably.

Other diseases, also have a genetic basis, but it appears that several genes must act in concert to produce the disease phenotype. The prospects of gene therapy in these cases seems far more remote.

Case study: severe combined immunodeficiency (SCID)

• cell-mediated immune responses nor
• is able to make antibodies.

It is a disease of young children because, until recently, the absence of an immune system left them prey to infections that ultimately killed them.

Treatment Options:

• Raise the child in a strictly germfree environment: all food, water, and air to be sterilized. David, the "bubble boy" from Houston, survived this way until he was 12 years old.
• Give the child a transplant of bone marrow from a normal, histocompatible, donor. Ideally, this would give the child a continuous source of ADA⁺ T and B cells. However,
  • even though the child cannot reject the transplant (the child has no immune system), T cells in the transplant (unless the donor was an identical twin) can attack the cells of the child producing graft-versus-host disease.
  • the donor cells may be infected with a virus which could overwhelm the recipient before his or her immune system was restored. (David received a bone marrow transplant from his sister, but she, like many people, had been infected earlier with the Epstein-Barr virus (the cause of "mono")). The virus was still present in the cells she donated, and killed her brother.
• Give injections of ADA (the enzyme is currently extracted from cows). When conjugated with polyethylene glycol (PEG) to delay its breakdown in the blood, ADA-PEG injections have kept SCID patients reasonably healthy. But just like the insulin injections of a diabetic, they must be repeated at frequent intervals. So,
• what about giving the patient functioning ADA genes; that is, gene therapy?

Gene Therapy: requirements

• The gene must be identified and cloned.This has been done for the ADA gene.
• It must be inserted in cells that can take up longterm residence in the patient. So far, this means removing the patient's own cells, treating them in tissue culture, and then returning them to the patient.
• It must be inserted in the DNA so that it will be expressed adequately; that is, transcribed and translated with sufficient efficiency that worthwhile amounts of the enzyme are produced.

• Their envelope protein enables the virus to infect human cells.
• RNA copies of the human ADA gene can be incorporated into the retroviral genome using a packaging cell.

• an RNA copy of the human ADA gene along with
  • a packaging signal (P) needed for the assembly of fresh virus particles
  • inverted repeats ("R") at each end; to aid insertion of the DNA copies into the DNA of the target cell.
  
• an RNA copy of the retroviral gag, pol, and env genes but with no packaging signal (so these genes cannot be incorporated in fresh viral particles).

• the envelope protein needed to infect the human target cells
• an RNA copy of the human ADA gene, complete with R sequences at each end
• reverse transcriptase, needed to make a DNA copy of the ADA gene that can be inserted into the DNA of the target cell
• none of the genes (gag, pol, env) that would enable the virus to replicate in its new host.

Once the virus has infected the target cells, this RNA is reverse transcribed into DNA and inserted into the chromosomal DNA of the host.

What to use for target cells?

T cells

The first attempts at gene therapy for SCID children (in 1990), used their own T cells (produced following ADA-PEG therapy) as the target cells.

• placed in tissue culture
• stimulated to proliferate (by treating them with the lymphokine, Interleukin 2 (IL-2)
• infected with the retroviral vector
• returned, in a series of treatments, to the child

• the injections had to be repeated because T cells live for only 6-12 months in the blood

  But someday there may be a way to make genetically-engineered T cells live indefinitely by getting them to express telomerase. See the discussion.

• the children also continued to receive ADA-PEG so the actual benefit of the gene therapy was unclear

Stem cells

In 1993, ADA gene therapy was attempted with blood stem cells removed from the umbilical cords of three newborn babies who were known to be homozygous for ADA deficiency.

• produce (by mitosis) all the types of blood cells, including T and B lymphocytes
• produce (by mitosis) more stem cells, thus ensuring an inexhaustible supply

Blood stem cells are present in everyone's bone marrow, but represent only a tiny fraction of the total cells present there. Umbilical cord blood has a much higher proportion of stem cells in it.

At last report, some of the circulating T cells in these children carried the ADA gene. But because the children continue to receive ADA-PEG, it is unclear how much gene therapy has contributed to their good health.