GENE THERAPY

D of PG Studies

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Introduction Gene therapy and use Chromosome intro. Steps of gene therapy Types Problems Current status Conclusion References

Objectives-

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Gene - A segment of DNA found on a chromosome that codes for a particular protein.

Humans have approximately 100,000 genes.

Genome - The sum of all genes that code for a particular organism.

Gene & Genome-

Are carried on a chromosome

The basic unit of heredity

Encode how to make a protein◦ DNARNA proteins

Proteins carry out most of life’s function.

When altered causes dysfunction of a protein

Genes-

Gene therapy = Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product

GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder

A. What is gene therapy? Why is it used?

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Structure of a Chromosome

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Disease Genetic defect

hemophilia A absence of clotting factor VIII

hemophilia B absence of clotting factor IX

cystic fibrosis defective chloride channel protein

muscular dystrophy

defective muscle protein (dystrophin)

sickle-cell disease

defective beta globin

Many human diseases are caused by defective genes

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The Beginning…

In the 1980s, Scientists began to look into gene therapy.

They would insert human genes into a bacterial cell.

Then the bacterial cell would transcribe and translate the

information .

Then they would introduce the protein into human cells.

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What is Gene Therapy

It is a technique for correcting defective genes that are

responsible for disease development.

OR

The treatment of disease by either replacing damaged

or abnormal genes with normal ones, or by providing

new genetic instructions to help fight disease.

Modification of somatic cells by transferring desired gene sequences into the genome.

Somatic cells necessary to ensure that inserted genes are not carried over to the next generation.

How is Gene Therapy Carried Out?

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How It Works

A vector delivers the therapeutic gene into a patient’s target cell

The target cells become infected with the viral vector

The vector’s genetic material is inserted into the target cell

Functional proteins are created causing the cell to return to a

normal state

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How It Works…….

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Steps in Gene Therapy

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The First Case,

The first gene therapy was performed on September 14th, 1990.

Ashanti DeSilva was treated for SCID.

Doctors removed her white blood cells,inserted the missing gene into the

WBC, and then put them back into her blood stream. This strengthened her

immune system, Only worked for a few months.

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A gene is inserted into the DNA of the germline cells (egg or sperm) so that the offspring of the patient will have the inserted gene.

The egg is a very large cell, relatively easy to manipulate and inject with DNA.

Inherited by the next generation.

Approaches: Germline therapy

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Somatic cells include all the non-reproductive cells in the

human body. Not inherited by the next generation. e.g:- bone marrow cells, blood cells, skin cells etc.

Somatic Gene Therapy

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Technique of Somatic Therapy

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Gene transfer

Vectors - Gene therapy delivery vehicles, or carriers, that encapsulate therapeutic genes for delivery to cells.Examples

Biological (Viral vectors, mammalian chromosomes etc)Physical (Microinjection, Gene gun, naked ‘DNA’, Electroporation etc)Chemical (Liposomes, Oligonucleotides etc)

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Different Delivery Systems are Available

In vivo = delivery of genes takes

place in the body

Ex vivo = delivery takes place out of

the body, and then cells are placed back into the body

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In vivo GENE THERAPY

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

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Viruses

Replicate by inserting their DNA into a host cell. Gene therapy can use this to insert genes that encode for

a desired protein to create the desired trait. The virus is engineered so that it cannot reproduce. Four different types 1) Retroviruses 2) Adenoviruses 3) Adeno-associated Viruses 4) Herpes Simplex Viruses

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Can't "expand" to fit a piece of genetic material larger than it is naturally built to carry, some genes may be too big to fit into a certain type of virus.

Viruses can cause immune responses in patients, resulting in two potential outcomes:

Patients may get sick.A patient's immunity to a virus may limit treatments.

General drawbacks of Viral Vectors

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More efficient to deliver a gene using a non-viral vector, which has fewer size constraints and which won't generate an immune response

Typically circular DNA molecules- Plasmids Bacteria use plasmids to transfer genes from cell to cell Easily and efficiently store and replicate genes of interest

from any organism

Non-Viral Vectors

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No immune response.Could carry a lot of information i.e one or more therapeutic genes.

Chromosome

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General advantages of viral vectors They are very good at targeting and entering cells. Some viral vectors might be engineered to target specific types

of cells. They can be modified so that they can't replicate and destroy

the cell.

Viral Vectors

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Gene guns Isolate normal DNA fragment. Make very small spheres of a heavy metal like gold. Coat gold with DNA. Fire particles at cells at high speed so particles enter cells. May be used directly on tissues or organs in situ.

Other techniques… a) Electoporation b)Liposomes

Gene guns

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Problems with Gene Therapy

Short Lived ◦ Hard to rapidly integrate therapeutic DNA into genome

and rapidly dividing nature of cells prevent gene therapy for long time.

◦ Have multiple rounds of therapy.

Viral VectorsPatient could have toxic, immune, inflammatory response

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Multigene DisordersHard to treat because need to introduce more than one

gene. Immune Response

New things introduced leads to immune responseMay induce a tumor if integrated in a tumor suppressor

gene because insertional mutagenesis.

PROBLEMS CONT…..

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Current Status FDA hasn’t approved any human gene therapy product for

saleReasons: In 1999, 18-year-old Jesse Gelsinger died from multiple

organ failure 4 days after treatment for ornithine transcarboxylase deficiency.

January 2003, halt to using retrovirus vectors in blood stem cells because children developed leukemia-like condition after successful treatment for X-linked severe combined immunodeficiency disease.

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Successful Gene Therapy

Severe Combine Immunodeficiency

Infants with severe combined immunodeficiency are unable

to mount an adaptive immune response, because they have a

profound deficiency of lymphocytes.

Attempt of gene therapy for immunodeficiency was

successful in children with severe combined

immunodeficiency due to a deficiency of adenosine

deaminase.

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Multiple gene therapy strategies have been developed to treat a wide variety of cancers.

Phase III trial for head and neck cancer Phase III gene vaccine trials for prostate cancer and pancreas cancer.

Additionally, numerous Phase I and Phase II clinical trials for cancers in the brain, skin, liver, colon, breast and kidney are being conducted .

cancer

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Gene Therapy in Heart Failure

Gene therapy in HF must be aimed at correcting key molecular mechanisms in cardiac tissue.

This requires,introduction of DNA/RNA that targets specific cardiomyocyte processes that alter HF outcomes.

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Gene delivery and activation. Introducing changes into the germline. Immune response. Disrupting important genes in target cells. Success requires -efficient delivery, - the correct gene, -the correct cells, -the correct tissue,

Challenges in Gene Therapy

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Conclusion

The viral and non viral gene delivery strategies have been fairly

successful in cell culture systems and animal models

The therapeutic success of GT in human still remains

questionable.

With the explosive increase in the availability of information on

human genome, several genetic disorder have become

candidates for gene therapy.

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The field is still at its infancy, the need of the hour is to initiate

more studies in different systems on the various aspects.

In spite of various drawbacks, gene therapy is witnessing a

rapid growth and hope fully the progress would continue.

Conclusion….

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References

Vyas S. P. , Dixit V. K., Pharmaceutical Biotechnology 1st edition, CBS Publisher & distributors 2001, New Delhi,401-435

D’Souza J.I. , Killedar S. J., Biotechnology & Fermentation Process 3rd edition, Nirali Prakashan Pune. 2007, 7.1-7.32

K.Sambamurthy, Ashutosh Kar.“ Pharmaceutical Biotechnology”,2nd Edn.,New Age International Publishers123-135.

U.Satyanarayana Biotechnology 3rd edition uppala author publishers.

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