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 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 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

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

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 zGeiLk8http:// zGeiLk8 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 zGeiLk8http:// zGeiLk8 Lived for 1 1/2 years...died in March of pneumonia

RNAi 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 Macular degeneration is the leading cause of adult blindness in the developed world.

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.