GENE / DNA THERAPY

• By A.Arputha Selvaraj

WHAT IS GENE THERAPY ?• Definiton: an experimental technique for

correcting defective genes that are responsible for disease development

• The most common form of gene therapy involves inserting a normal gene to replace an abnormal gene

• Other approaches used: Replacing a mutated gene that causes

disease 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.

• Researchers are studying gene therapy for a number of diseases, such as Severe combined immuno-

deficiencies (SCID) Hemophilia Parkinson's disease Cancer HIV

HISTORY AND DEVELOPMENT OF GENE THERAPY

• 1960: The concepts of Gene Therapy was introduced

• 1970: Friedmann and Roblin author of a paper in Science titled "Gene therapy for human genetic disease?” cite the first attempt to perform gene therapy

• 1990: The first approved gene therapy case at the National Institute of

Health, U.K. It was performed on a four year old girl named Ashanti DaSilva. It was a treatment for a genetic defect that left her with an immune system deficiency

New gene therapy approach repairs errors in messenger RNA derived from defective genes. This technique has the potential to treat the blood disorder Thalassaemia, Cystic fibrosis, and some cancers

Sickle cell disease is successfully treated in mice

• 1992: Doctor Claudio Bordignon working at the Vita-Salute San Raffaele University, Milan, Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases

• 1999: Death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant setback to gene therapy research in the United States

• 2006: Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients. This study constitutes one of the first demonstrations that gene therapy can be effective in treating cancer.

• 2007- 2011: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases.

Retinal disease Colour blindnessAdrenoleukodystrophy

• 2011: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has cured a man from HIV using gene therapy

TYPES OF

GENE THERAP

Y

GERM LINE GENE

THERAPY

SOMATIC GENE

THERAPY

GERM LINE GENE THERAPY

Result in permanent changes. Potential for offering a permanent therapeutic

effect for all who inherit the target gene. Possibility of eliminating some diseases from a

particular family. Also raises controversy: Some people view this type of therapy as

unnatural, and liken it to "playing God”. Others have concerns about the technical

aspects.

SOMATIC GENE THERAPY

Affects only the targeted cells in the patient, and is not passed to future generations.

Short-lived because the cells of most tissues ultimately die and are replaced by new cells.

Transporting the gene to the target cells or tissue is also problematic.

Appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases.

Types of somatic gene therapy

Ex vivocells are modified outside the body

and then transplanted back

in again

called ex vivo because the cells

are treated outside the body

In vivogenes are changed in cells when the

cells are still in the body

called in vivo because the gene is transferred to cells inside the patient’s body

BASIC PROCESS OF GENE THERAPY

1. VIRAL VECTOR 2. NON VIRAL VECTOR

GT utilizes the delivery of DNA into cells, which can be accomplished by a number of methods.

The two major classes of methods :recombinant viruses – VIRAL VECTORnaked DNA or DNA complexes – NONVIRAL

VECTOR

VIRAL VECTOR

Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to harness this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones .

VIRUS

Virus bind to their hosts and introduce their genetic material into the host cell.

Plausible strategy for gene therapy, by removing the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA.

The viruses used are altered to make them safe, although some risks still exist with gene therapy.

TYPES OF VIRUS

Many GT clinical trials rely on retroviruses or adenoviruses to deliver the desired gene.

Other viruses used as vectors include adeno-associated viruses, lentiviruses, pox viruses, alphaviruses, and herpes viruses.

Differ in how well they transfer genes to the cells they recognize and are able to infect, and whether they alter the cell’s DNA permanently or temporarily

VIRAL VECTOR

Are a tool commonly used by molecular biologists to deliver genetic material into cells.

Can be performed in vivo or in vitro.

Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect.

Delivery of genes by a virus is termed transduction and the infected cells are described as transduced.

NON VIRAL VECTOR

Methods of non-viral gene delivery have also been explored using physical (carrier-free gene delivery) and chemical approaches (synthetic vector-based gene delivery).

PHYSICAL METHOD

Physical approaches, including Needle injectionElectroporationGene gunUltrasoundHydrodynamic deliveryemploy a physical force that permeates the cell membrane and facilitates intracellular gene transfer

I. NAKED DNA

The simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection.

This success, however, does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

CHEMICAL METHODS THAT ENHANCE THE

DELIVERY OF GENE THERAPY-lipoplexes & polyplexes-

LIPOPLEXES

DNA must be protected from damage & its entry into the cell must be facilitated

Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposomecomplexed with DNA it is called a lipoplex

3 types of lipids:anionic (negatively charged) neutralcationic (positively charged)

Initially, anionic and neutral lipids :-were used for the construction of lipoplexes for

synthetic vectors.-but,there is little toxicity associated with them,-they are compatible with body fluids-there was a possibility of adapting them to be tissue

specific-they are complicated turned to the cationic

versions.

Cationic lipids, due to their positive charge, -naturally complex with the negatively charged DNA.-their charge they interact with the cell membrane-endocytosis of the lipoplex occurs -DNA is released into the cytoplasm. -The cationic lipids also protect against degradation of

the DNA by the cell.

Common used of lipoplexes

In gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell

decrease the activity of oncogenes. useful in transfecting respiratory epithelial cells,

so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis.

POLYPLEXES

Complexes of polymers with DNA are called polyplexes

consist of cationic polymers and their production is regulated by ionic interactions.

large difference compared to lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm,

End= co-transfection with endosome-lytic agents such as inactivated adenovirus must occur (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell)

SUCCESS CASES OF GENE THERAPHY

PARKINSON’S DISEASE

BLINDNESS

GENE THERAPY CURES BLINDNESS

Cure blindness of inherited condition Leber’s conginetal amaurosis - inherited disease caused by an abnormality in a gene called RPE65. - The condition appears at birth or in the first few months of life and causes progressive worse and loss of vision. 

HOW IT WORKS?? used harmless viruses enable access to the cells beneath the

retinas of patients By using a very fine needle -safe in an extremely fragile tissue and can improve vision in a condition previously considered wholly untreatable.

• http://www.youtube.com/watch?v=d_YJZn-ft_Q

SUCCESS CASES OF GENE THERAPHY

PARKINSON’S DISEASE

BLINDNESS

GENE THERAPY REDUCES PARKINSON’S DISEASE SYMPTOMS

it significantly improved the weakness of the symptoms such as tremors, motor skill problems, and rigidity

Main- overactive brain region: the subthalamic nucleus should be introduced with gene

that would produce GABA—an inhibitory chemical—then they could potentially quiet that brain region and alleviate tremors.

HOW IT WORKS?? Done with local anesthesia, used a harmless,

inactive virus [AAV-2 GAD] Deliver the GAD gene into patient’s

subthalamic nucleus The gene instructs cells to begin making

GABA neurotransmitters to re-establish the normal chemical balance that becomes dysfunctional as the disease progresses

• Give a chance of a normal life to baby born with genetic disease.

• Give hope of healthy life to cancer patient.

• For certain disease that do not have any cure except gene therapy, it could save many lives

ADVANTAGES OF GENE THERAPY

• The genetic testing, screening and research in finding the availability of certain gene is very controversy.

• May increase rate of abortion if prenatal test regarding baby with genetic disease is done.

• The cost is very high and the patient might need an insurance to cover the treatment.

• Cosmetic industry may monopolized this gene therapy if it is used in enhancing beauty and in vanishing the aging effect, rather than used for treatment of a disease.

DISADVANTAGES OF GENE THERAPY

ETHICAL QUESTIONS SURROUNDING GENE THERAPY

• How can “good” and “bad” uses of gene therapy be distinguished?

• Who decides which traits are normal and which constitute a disability or disorder?

• Will the therapy only benefit the wealthy due to its high cost?

• Could the widespread use of gene therapy make the society less accepting of people who are different?

• Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

THANK YOU

EMAIL ME : arputhaselvaraj@gmail.com