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Gene Therapy
Can it save us??
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What is it?
• Replacing a mutated gene
with a healthy copy of the
gene
• Inactivating, or “knocking
out”, a mutated gene that is
functioning improperly
• Introducing a new gene into
the body to help fight a
disease
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Therapy for Hemophilia
How does it work?
• Vector is used to carry in the gene
• Viruses: altered to be made safe
– Retroviruses
– Adenoviruses
Let’s look at these viruses ..........
Retrovirus
• Introduces its RNA with
reverse transcriptase
and integrase into the
cell
• Needs to make a copy
of DNA
• Reverse transcription
• DNA is free to move
into the host nucleus
and incorporated into
the genome by
integrase
• RNA
• Surrounded by lipid envelope
• Ex: HIV
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• Nonenveloped (naked)
• DNA genome
• Responsible for 5-10% of upper
respiratory infections
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Adenovirus
• DNA is not
incorporated into host
cell’s DNA
• Left free in nucleus
• Instructions are
transcribed
• Not replicated when
the cell replicates
• Re-administered
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• Injected intravenously into a specific tissue or
cells can be removed and exposed to the
vector and replaced into the patient
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• Viruses can usually
infect more than one
type of cell
RISKS??
– Healthy and mutated
• Transferred genes
could be overexpressed
– Create so much
protein that it is
harmful
– Immune reaction
• Virus could be
inserted in the wrong
location
– Possible cause more
mutations/cancer
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Trials receive approval
• Must be approved by at least
two review boards at the
scientists’ institution
• Approved by the US FDA
• Trials funded by the National
Institute of Health must be
registered with the NIH rDNA
Advisory Committee
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1st Disease Approved
• Adenosine deaminase
deficiency (ADA)
• Essential to the body’s immune
system..makes wbc
• Patients do not have normal
ADA genes and do not make
the functional protein
• Prone to repeated serious
infections (SCID)
• WBC were taken and the
normal genes for making ADA
were inserted into them and
injected back into the patient
– Sept 14, 1990
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wbc
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•
Color Blindness
http://www.youtube.com/watch?v=0IBT-jGja28
• Used to restore color vision in two adult squirrel monkeys
• Unable to distinguish red and green
• Missing one version of the opsin gene (carried on X
chromosome)
-Sept 16, 2009, NATURE
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• Injected human form of red-detecting opsin gene into virus behind the retina
of 2 squirrel monkeys
• Assessed ability to find colored patches of dots on a background of gray
dots by training them to touch colored patches on a screen and then
rewarding them with grape juice
• After 20 weeks, color skills improved
Three human trials are under way for loss of sight due to degeneration of the
retina
• Different genes
• No serious adverse effects more than a year after
• Some with marked improvement in vision
“There is plasticity still in the
brain and it is possible to treat
cone defects with gene
therapy” - A. Smith
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Sickle Cell Disease in Mice
• Fatal, genetic mutation
in the hemoglobin gene
that causes rbc to
become crescent-shape
and sticky
• 2 bad copies of gene
leads to clumping of rbs
• December 14th,
SCIENCE
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• Removed bone marrow from
mice with disease, isolated
stem cells, and inserted the
new anti-sickling gene
• Cells transplanted back and
they started to produce health
round rbc
• Used a modified version of
HIV as the vector
• 10 months after therapy, 99%
of the rbc in the mice contain
the anti-sickling gene
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Treatment of Cancer
• Replace missing or altered genes with healthy
genes
• Used to stimulate the body’s natural ability to
attach cancer cells
– Insert gene to make T-cell receptor
– Transferred into wbc and put back in patient
– WBC produce TCR which recognize molecules on
tumor cells
– TCRs activate wbc to attack and kill
• Introducing “suicide genes” into cancer cells
– Pro-drug is given which leads to destruction of cancer
cells
Batman: Cancer
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U of M College of Veterinary Medicine
10-yr old German Shepard mixed breed
Brain cancer
Used surgery to remove the tumor
gene therapy at the surgical site to
attract immune cells to destroy
remaining tumor cells
made an anti-cancer vaccine from the
dog’s own cancer cells to prevent tumor
recurrence
http://www.youtube.com/watch?v=rCbu
zGeiLk8
Lived for 1 1/2 years...died in March of
pneumonia
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RNAi
• http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
• RNA interference
• Helps to control which genes are active and how active
they are
• dsRNA
• Highly specific
• Remarkably potent
– Only a few molecules/cell required for effective interference
• Interfering activity can cause interference in cells and
tissues far removed from the site of introduction
Muscular Degeneration
• Macular Degeneration
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Macular degeneration is the leading cause
of adult blindness in the developed world.
– RNAi injected into the
eye
– Shuts down genes that
make VEGF (blood
vessels)
– 2004, 24 people in the
trial, 25% had
significantly clearer
vision, other patients’
vision had stabilized
Hepatitis C
• Hepatitis C
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– 2002, RNAi controlled
the virus in laboratory
mice
– Injected “naked” RNA
into the tail veins of
mice
– Trying to find ways to
use viruses as vectors
To test their RNAi treatment, Stanford researchers
used mice infected with a specially crafted, "glowing"
version of a hepatitis C gene (left). The treatment
effectively turned off the glowing gene (right).
Huntington’s Disease
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A brain devastated by
Huntington's disease, a genetic
disorder for which there is now
no effective treatment or cure
• 2004: used virus vector to
transport RNA-making
molecules
• Treated mice with
spinocerebellar ataxia,
neurological disorder similar
to Huntington’s
• Gene was turned off
• Treated mice with
Huntington’s as well
• Turned off the harmful gene,
but also the healthy version
• Still optimistic to tweak the
design of the RNAi drug
HIV
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As with the best current HIV drug
regimes, new RNAi therapies must
attack the virus on multiple fronts at
once to counter the problem of drug
resistance.
• 2002: able to interrupt
various steps in the HIV
life cycle with RNAi in cell
cultures
• Engineered an RNAi
therapy aiming at multiple
HIV genes
• Used in combination with
2 other RNA technologies
to block HIV’s replication
and invasion of the
immune system
• Extract stem cells, alter
with RNA therapy and
transfuse them back
Respiratory Infections
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A child's lungs, infected with RSV.
The virus prompts as many as
125,000 pediatric hospitalizations
in the U.S. each year.
• 2005: RNAi
molecule to shut
down various
respiratory syncytial
virus (RSV) genes
• Inhaled RNAi
• Trials began in 2006
in mice