GENES• Are carried on a chromosome

• The basic unit of heredity

• Encode how to make a protein

• DNA RNA proteins

• Proteins carry out most of life’s function.

• When altered causes dysfunction of a protein.

WHAT IS GENE THERAPY?

• It is a technique for correcting defective genes that are responsible for disease development.

• There are four approaches:

1. A normal gene inserted to compensate for a nonfunctional gene.

2. An abnormal gene traded for a normal gene.

3. An abnormal gene repaired through selective reverse mutation.

4. Change the regulation of gene pairs.

THE BEGINNING…

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

• They would insert human genes into a bacteria cell.

• Then the bacteria cell would transcribe and translate the

information into a protein.

• Then they would introduce the protein into human cells.

THE FIRST CASE…

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

• Ashanti DeSilva was treated for SCID.

• Sever combined immunodeficiency.

• 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

GENE THERAPY STRATEGIES

• Ex-Vivo:-

Outside the Body.

• In-Vivo:-

Inside the Body.

TYPES OF GENE THERAPY

• Somatic Gene Therapy:-

Genes are transferred into the somatic cells or the body of the

patient.

• Germ Line Gene Therapy:-

Germ cells are modified by the introduction of functional genes that

are integrated into the genome.

VECTORS FOR GENE DELIVERY

• Two types of vectors:-

1. Viral Vectors;

Uses recombinant Viruses.

2. Non Viral Vectors;

Uses naked DNA or DNA complexes.

• It is recognized as powerful

strategy for gene therapy.

• Some of the important viral

vectors includes,

1. Retrovirus.

2. Adenovirus.

3. Adeno-associated Virus.

4. Herpes Simplex Virus.

VIRAL VECTORS

RETROVIRUS

• Creates double stranded DNA copies from RNA genome.

• The retrovirus goes through reverse transcription using reverse

transcriptase and RNA.

• The double stranded viral genome integrates into the human

genome using integrase.

• Integrase inserts the gene anywhere because it has no specific

site.

• Human immunodeficiency virus (HIV) is a common example of

Adenovirus.

ADENOVIRUS

• Non-enveloped viruses.

• Contains a linear double stranded DNA genome.

• The inserted DNA is not incorporate into genome.

• Has to be reinserted when more cells divide.

• Ex. Common cold.

ADENO-ASSOCIATED VIRUSES

• Small, single stranded DNA that insert genetic material at a

specific point on chromosome 19.

• From parvovirus family- causes no known disease and doesn't

trigger patient immune response.

• Low information capacity.

• Gene is always "on" so the protein is always being expressed,

possibly even in instances when it isn't needed.

• Hemophilia treatments, for example, a gene-carrying vector

could be injected into a muscle, prompting the muscle cells to

produce Factor IX and thus prevent bleeding.

HERPES SIMPLEX VIRUSES

• Double stranded DNA viruses that infect neurons.

• Ex. Herpes Simplex Virus Type 1.

Adenovirus

+ Infects many cell types.

- Does not integrate into host genome and can be lost.

Retrovirus

+ Integrates into host genome and cannot be lost

- Integrates into host genome and can cause cancer.

Adeno-Associated Virus (AAV)

+ Integrates into host genome and cannot be lost.

- Difficult to work with.

Herpes Simplex Virus (HSV)

+ DNA stays in nucleus without integrating into host

genome.

- Only infects cells of the nervous system.

NON-VIRAL VECTORS

• Used to overcome the disadvantages of viral vectors.

• Some of the most common non-viral vectors include,

1. Naked DNA.

2. Oligonucleotides.

3. Liposomes.

4. Microinjection.

5. Gene Gun.

6. Chemical Methods.

7. Hybrid Methods

NAKED DNA

• Simplest method of non-viral transfection.

• Different methods like Electroporation or Sonoporation can be

used for direct injection of Naked DNA.

• Expression rate is very low as compared to other methods.

• Only possible for certain tissues.

• Requires large amount of DNA.

INJECTION OF NAKED DNA

OLIGONUCLEOTIDES

• To inactivate genes involved in disease process.

• Different approaches are used for Oligonucleotides based Gene

Therapy

• One strategy utilizes antisense specific to the target gene which

disrupts the transcription of faulty genes.

• Another method uses siRNA to signal the cell to cleave specific

sequences in the mRNA transcript of the faulty genes which

results in disruption of translation.

• It is an artificially prepared spherical

vesicle made up of lipid bilayer.

• DNA is encapsulated within liposome.

• Can carry any size of DNA fragment.

• Liposome easily enters the cell

membrane and delivers the genes to

the target cell.

• It does not cause an immune response.

• This method is less efficient

LIPOSOMES

• Refers to the process of using

a glass micropipette to inject

the desired gene.

• DNA is injected directly into the

nucleus of the target cell.

• The whole process is viewed

under powerful microscope.

• This method is mostly used to

produce transgenic animals.

MICROINJECTIONS

• A specially designed gun

used for injecting the genes

into the nucleus of the target

cell.

• A special metallic sphere is

coated with DNA.

• The sphere is fired into the

cell from the gun.

• The method is used for

producing transgenic plants.

GENE GUN(BIOLISTICS)

CHEMICAL METHODS

• Chemical methods involves the use of certain chemicals which

increases cellular permeability.

• One example is CaPO4 that increases cellular permeability by

creating pores.

• The desired genes are then inserted into the target cell through

these pores.

HYBRID METHODS

• Involves the combination of two or more techniques.

• An example is Virosome.

• It is the combination of liposome with inactivated HIV or

Influenza virus.

• Results in more efficient gene transfer.

RECENT ADVANCES

• Introduction of artificial chromosome.

• Becomes the 47th chromosome.

• Exists alongside the other 46.

• Could carry a lot of information.

• Only problem being how to introduce such a big molecule

through cell membrane?

PROBLEMS WITH GENE

THERAPY

Short Lived

Hard to rapidly integrate therapeutic DNA into genome and rapidly

dividing nature of cells prevent gene therapy from long time.

Needs multiple rounds of therapy.

Immune Response

New things introduced leads to immune response.

Increased response when a repeat offender enters.

Viral Vectors

Patient could have toxic, immune, inflammatory response.

Also may cause disease once inside.

Multigene Disorders

Heart disease, high blood pressure, Alzheimer’s, arthritis and

diabetes are hard to treat because you need to introduce more than

one gene.

GENE THERAPY

DISAPPOINTMENTS

• In 1999 a boy died due to an immune response to an

adenovirus gene therapy vector.

• Four children have developed cancer due to a retrovirus gene

therapy vector.

ETHICAL ISSUES

• Who are we to play God?

• Should we interfere with nature?

• Where does gene therapy stops?

• Who gets gene therapy treatment?

• What type of gene therapies are acceptable?

• What about reproductive issues?