What is Gene Therapy

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• What is Gene Therapy?

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The transfer a normal functional copy of a gene
into a somatic cell that has a mutation in its
copy of the given gene, with the intent to
correct disease.
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Why do we need gene therapy?
A treatment of this type promises to offer a
precise/ specific means of curing essentially any
disease with a genetic defect as the
root. Current therapies are inadequate and do not
CURE disease.
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Where did the idea of gene therapy come from?
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• 1944- Oswald Avery showed the heritable material
  was DNA.
• A little later- Hershey and Chase conduct
  different experiments and arrive at same
  conclusion.
• 1953- Watson and Crick put together model for
  structure of DNA.

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Edward Tatum and Joshua Ledenberg (1963) We
might anticipate the in vitro culture of germ
cells and such manipulations as the interchange
of chromosomes and DNA segments. The ultimate
application of molecular biology would be the
direct control of nucleotide sequences in human
chromosomes, coupled with recognition, selection
and integration of desired genesWe anticipate
that viruses will be used effectively for mans
benefit
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W. French Anderson (1968) Wrote article for New
England Journal of Medicine postulating how one
day gene therapy could be accomplished In order
to insert a correct gene into cells containing a
mutation, it will first be necessary to isolate
the desired gene from a normal chromosome. This
gene will probably have to be duplicated to
provide many copies. And, finally, it will be
necessary to incorporate the correct copy into
the genome of the defective cell. Paper said to
be fascinating and erudite, but far too
speculative to be published in a medical journal!
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Gordon Rattray Taylor (1968) Publishes The
Biological Time Bomb, a book describing the
potential for misuse of genetic engineering.
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Stanfield Rogers (1966) Published a paper in
Nature reporting that animals infected with the
Shope rabbit papilloma virus exhibited lowered
levels of arginine in their bloodstream, as did
workers in the labs handling this virus. No
other effects were observed.
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Lederberg (again) (1969) Publishes a paper in
Lancet describing two German sisters suffering
from a genetic defect that caused toxic levels of
the amino acid arginine to build up in their
bloodstream. As a result, the girls were
epileptic, spastic, grossly retarded, and
progressively becoming worse.
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Stanfield Rogers (1970) Conducts first human
gene therapy trial decides to infect the two
German sisters described by Lederberg with Shope
virus to lower their blood arginine.
Administered 1/20th of the dose of virus found to
be harmless in mice. Children had no benefit.
Rogers never conducted another human study.
Spent remainder of his career working with
genetic engineering of plants.
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Sir MacFarlane Burnet (1971) Publishes Genes
Dreams and Realities Begins optimistically If
rocks from the moon can be brought to Houston,
why should we not be able to apply molecular
biology to cure what is now incurable?
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Sir MacFarlane Burnet (1971), continued He then
goes on to say The next step would be the
crucial and probably impossible one, to
incorporate the gene into the genetic mechanism
of a suitable virus vehicle in such a fashion
that the virus in turn will transfer the gene it
is carrying to cells throughout the body and in
the process precisely replace the faulty gene
with the right one. I should be willing to state
in any company that the chance of doing this will
remain infinitely small to the last syllable of
recorded time.
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What kind of diseases would be treatable bygene
therapy?

• Diseases caused by a single faulty gene (over
  4000 known).
• Candidate diseases include
• Sickle cell anemia, Thalassemia, MPS,
• Gauchers, Hurlers

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Only about ¼ of all the ongoing gene therapy
trials are for this type of disease. WHY?
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Percent effort directed towards different gene
therapy trials.
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• Three strategies for somatic cell gene therapy
• Ex vivo cells removed from body, incubated with
  vector and gene-engineered cells returned to
  body.
• In situ vector is placed directly into the
  affected tissues.
• In vivo vector injected directly into the blood
  stream.

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Example of ex vivo somatic cell gene therapy

• Usually done with hematopoietic stem cells
  because they are easiest to remove and return
• Candidate diseases include
• Sickle cell anemia, Thalassemia, MPS,
• Gauchers, Hurlers

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Stem Cell Gene Therapy
Place gene in delivery vector
Harvest marrow

Expose cells to vector
Reinfuse corrected cells
Radiation/Chemotherapy
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Example of in situ somatic cell gene therapy

• Infusion of adenoviral vectors into the trachea
  and bronchi of cystic fibrosis patients.
• Injection of a tumor mass with a vector carrying
  the gene for a cytokine or toxin.
• Injection of a dystrophin gene directly into the
  muscle of muscular dystrophy patients.

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Example of in-vivo somatic cell gene therapy

• No clinical examples, but testing in animal
  models (dog).
• Safe in vivo injectable vectors must be developed.

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Basic Gene Transfer Vector
cDNA (protein coding sequence)
Transfer Vector

• Regulatory Region (promoter/enhancer)
• determines tissue-specificity
• determines level of expression
• allows cDNA to be regulated

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Types of vectors

• RNA viruses (Retroviruses)
• 1. Murine leukemia virus (MuLV)
• 2. Human immunodeficiency viruses (HIV)
• DNA viruses
• 1. Adenoviruses
• 2. Adeno-associated viruses (AAV)
• 3. Herpes simplex virus (HSV)
• 4. Foamy viruses
• Non-viral vectors
• 1. Liposomes
• 2. Naked DNA
• 3. Liposome-polycation complexes

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Does Gene Therapy Work?

• In mice - Yes
• In humans - ???