Gene Therapy Can it save us??. What is it? Replacing a mutated gene with a healthy copy of the gene Inactivating, or “knocking out”, a mutated gene that

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



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



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





• Injected intravenously into a specific tissue or cells can be removed and exposed to the vector and replaced into the patient



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



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





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



wbc



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



http://www.youtube.com/watch?v=0IBT-jGja28

• 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



“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



• 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



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



http://www.youtube.com/watch?v=rCbuzGeiLk8




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

http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html

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



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



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



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



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



A child's lungs, infected with RSV. The virus prompts as many as 125,000 pediatric hospitalizations in the U.S. each year.

Documents

Gene Therapy Can it save us??. What is it? Replacing a mutated gene with a healthy copy of the gene Inactivating, or “knocking out”, a mutated gene that