Gene TherapyCan 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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Adenovirus• Nonenveloped (naked)• DNA genome• Responsible for 5-10% of upper
respiratory infections• 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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RISKS??• Viruses can usually infect more than one type of cell– Healthy and mutated
• Transferred genes could be over-expressed– 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 improvedThree 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
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“There is plasticity still in the brain and it is possible to treat cone defects with gene therapy” - A. Smith
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• 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=rCbuzGeiLk8
• Lived for 1 1/2 years...died in March of pneumonia
• 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=rCbuzGeiLk8
• 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– 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
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Macular degeneration is the leading cause of adult blindness in the developed world.
Hepatitis C• Hepatitis C
– 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
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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• 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
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A brain devastated by Huntington's disease, a genetic disorder for which there is now no effective treatment or cure
HIV• 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
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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.
Respiratory Infections
• 2005: RNAi molecule to shut down various respiratory syncytial virus (RSV) genes
• Inhaled RNAi• Trials began in 2006
in mice
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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.