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Gene therapy
Goal: Treat a genetic disease (a defect in some gene)
by replacing the defective gene.
Why is gene therapy appealing?
Consider hemophilia, a bleeding disease caused by a defect in
a blood protein, a clotting factor, required to activate clotting.
There are two forms of hemophilia,
one due to a mutation in clotting factor VIII, hemophilia A,
one due to defect in clotting factor IX, hemophilia B.
Both are sex-linked diseases (genes on the X chromosome).
The disease affects ~ 30,000 American males.
The disease is characterized by frequent spontaneous
bleeding episodes, usually into soft tissues and joints.
Bleeding into closed spaces can be fatal.
Current treatment:
Infusion of clotting factor concentrates in response to bleeds.
Clotting factor isolated from human blood became available in
the 70s. Many patients getting this isolated factor became
infected with blood-born pathogens such as hepatitis and HIV.
Now a cloned clotting factor is available—it doesn’t come
from human blood—so young patients are not infected with
these viruses.
Nevertheless the current treatment has many disadvantages
-ongoing tissue damage occurs because bleeds are treated
rather than prevented.
-inconvenience. The clotting factor protein has to be
injected. It can not be taken orally as the digestive enzymes
in our intestine would destroy it.
-protein has short half life. Clotting is only enhanced for a
brief time after treatment.
-expense. World wide only 20 - 30% of hemophiliacs are
treated.
Kathy High
Dr. Kathy High at CHOP has been studying the possibility
of gene therapy for this disease.
Why is this a good disease to try gene therapy?
Precise regulation of gene expression is not required.
Raising blood levels of clotting factor even a few percent is
a big help; if as high as 100%, it is still ok.
Animal models exist (mice and dogs with hemophilia) so gene
therapy can be tested on them first.
Determination of efficacy is straight forward—measure
clotting time.
Tissue specific expression of gene is not required –only need
secretion of the protein into blood.
How does one get the cloned gene into cells?
In this case, the investigators chose to use a virus,
adeno-associated virus (AAV-2).
This is a non-pathogenic, replication-deficient virus—(most
of us have it).
The coding sequence of the virus was replaced with gene of
interest.
Using this virus, sustained expression of a target gene has
seen in skeletal muscle, liver and CNS.
Advantages of gene-based therapy
Continuous maintenance of clotting factor in the plasma.
Bleeds would be prevented rather than treated after
they occurred.
Risks of blood products avoided
Cheaper?
Long term?
•More convenient
Who is being treated by current attempts at gene therapy?
An individual? Or the progeny of an individual?
What determines whether the progeny or just the individual
will be affected by the therapy?
If the added gene is incorporated into somatic cells—liver, muscle,
(any cell except germs cells)-- only the treated individual will be
affected.
If the gene is incorporated into germ cells, i.e. eggs and sperm, then
the progeny will also be affected.
Gene therapy is only being used to treat an individual. The
government requires controls (separate studies) to demonstrate that
germ cells are not being affected.
Results:
Test on animals, both small animals (mice) and large animals
(dogs) were successful. Virus did not appear in the semen.
Initial tests on humans with hemophilia were approved by
the FDA.
The initial tests were positive:
The gene was expressed in the liver,
Clotting times were increased.
No virus was detected in semen
Leg muscle was injected with adeno-associated virus carrying a
corrective gene for hemophilia
Cells expressing clotting factor are green.
Trials of gene therapy of hemophilia were recently stopped
because the virus was found in the semen of one of the
individuals in the trial.
Currently the trial is on hold.