Gene Therapy I

Many human diseases are caused by defective genes.

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 long-term 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

• 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

In June of 2002, a team of Italian and Israeli doctors reported on two young SCID patients that were treated with their own blood stem cells that had been transformed in vitro with a retroviral vector carrying the ADA gene. After a year, both children had fully-functioning immune systems (T, B, and NK cells) and were able to live normal lives without any need for treatment with ADA-PEG or immune globulin (IG). The doctors attribute their success to first destroying some of the bone marrow cells of their patients to "make room" for the transformed cells.

Almost 8 years later (January 2009) these two patients are still thriving and have been joined by 8 other successfully-treated children.

Gene Therapy for X-linked SCID

Gene therapy has also succeeded for 15 French baby boys who suffered from another form of severe combined immunodeficiency, called X-linked SCID, because it is caused by a mutated X-linked gene encoding a subunit — called γc (gamma-c) — of the receptor for several interleukins, including interleukin-7 (IL-7).

IL-7 is essential for converting blood stem cells into the progenitors of T cells. [View]. Boys with X-linked SCID can make normal B cells, but because B cells need T-helper cells to function, these boys could make neither cell-mediated nor antibody-mediated immune responses and had to live in a sterile bubble before their treatment.

• isolated blood stem cells from the bone marrow of each boy;
• treated the cells with a retroviral vector containing the normal gene for the γc interleukin receptor subunit;
• returned the treated cells to each donor.

• are able to live normal lives at home instead of inside a sterile "bubble";
• have normal (with some exceptions*) numbers of T cells of both the CD4 and CD8 subsets;
• have responded to several childhood immunizations, including diphtheria, tetanus and polio by producing both T cells and antibodies specific for these agents.
• Antibody production is sufficiently good that they have no need for periodic infusions of immune globulin (IG).

Adenovirus Vectors

Adenoviruses are human pathogens responsble for some cases of the human "cold". Modified versions of two strains are currently being used as vectors in gene therapy trials.

• Unlike retroviral vectors, they do not integrate into the host genome and thus should not be able to disrupt host genes (thus avoiding the danger that resulted in the three little boys developing leukemia).
• They can infect nondividing cells with high efficiency.

• They elicit a powerful immune response, both by T cells and by B cells (antibodies) so repeated doses soon lose their effectiveness.
• Many people already have antibodies against the virus from earlier "colds", and these can inactivate the vaccine at the outset. A recent trial of an HIV vaccine using an adenovirus as the vector was halted when it was found not only not to be effective but, in people with preexisting high levels of anti-adenovirus antibodies, may have even increased their susceptibility to HIV.