GENE THERAPY &

GENE DELIVERY SYSTEM

BY
Amreen Samiullah
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

GENE THERAPY
• An approach of treating diseases by either modifying the expressions of an
individual’s genes or correction of abnormal genes

• This can be accomplished by:

➢ 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.

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 14th, 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

 Types of Gene therapy
SOMATIC GENE THERAPY

GERM LINE GENE THERAPY

 SOMATIC CELL GENE THERAPY & GERM LINE GENE THERAPY
1. Therapeutic genes transferred into
the somatic cells.
2. E.g.. Introduction of genes into bone
marrow cells, blood cells, skin cells
etc
3. Will not be inherited later
generations
4. At present all researches directed to
correct genetic defects in somatic
cells
1. Therapeutic genes transferred into
the germ cells
2. E.g.. Genes introduced into eggs and
sperms
3. It is heritable and passed on to later
generations
4. For safety, ethical and technical
reasons, it is not being attempted at
present
Types of somatic cell gene therapy
• Ex-Vivo:- Outside the Body
➢ cells are modified outside the body and then transplanted back
in again
➢ called ex vivo because the cells are treated outside the body
• In-Vivo:- Inside the Body
➢ genes 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
VECTORS FOR GENE DELIVERY
• Two types of vectors:-
➢ Viral Vectors
• Uses recombinant Viruses

➢ Non Viral Vectors

• Uses naked DNA or DNA complexes
VIRAL VECTORS
• 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
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

 ADVANTAGES &
DISADVANTAGES OF
VIRAL VECTORS
➢ 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

 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.
LIPOSOMES
➢ 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

MICROINJECTIONS
➢ 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.
 GENE GUN(BIOLISTICS)
➢ 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.

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
THANK YOU