In this slide, You will get to learn abut Gene Therapy and different types of gene therapy. Various method of Gene Therapy and Advantage & Disadvantage and Recent advances in Gene Therapy.

1. GENE THERAPY
Presented by:-
Shaikh Nisar Ali (M.pharm)
Dept. of Pharmacology
Guided by:- Mrs. Arati Malpani. (Ph.D)
(Associate Professor)
29 January 2021

2. Advances in Biochemistry and Molecular biology have helped to
understand the genetic basis of inherited disease. It was a dream
of the researchers to replace the defective genes with new one and
cure genetic disorders.
DEFINATION:- Gene therapy can be broadly defined as the
transfer of genetic material to cure the disease or at least to
improve the clinical status of the patient.
The gene used in gene therapy are called GENE DRUG.

3. Principle :-
• Gene therapy is the process of inserting genes into cells to treat
diseases.
• The newly introduced genes will encode proteins and correct
deficiencies that occour in genetic disease.
• It involves genetic manipulation in animals or humans to
correct disease and keep organisms in good health.
• Initial experiment is carried out first on animals than on
Humans.

6. Approaches for Gene therapy
1) Somatic Cell Gene Therapy
2) Germ Cell Gene Therapy

7. Somatic Cell Gene therapy:-
• The Non-Reproductive cells ( Non-sex cells ) of an organism are
referred to as Somatic Cells. These are cells of organs other
than sperm or egg cells.
• Example- Bone marrow cells , Blood cells , skin cells, intestinal
cells.
• Somatic cell gene therapy involves insertion of an expressible
gene into Somatic cells and at present all research on gene
therapy is directed to correct genetic defects in Somatic Cells.
• Here Gene therapy is given to somatic cells that’s why Its called
Somatic Cells Gene therapy.

8. Germ Cell Gene Therapy:-
• The Reproductive cells ( Sex cells ) of an organism are referred
to as Germ Cells.
• Here Gene Therapy is given to germ cells that’s why its is called
Germ cell gene therapy.
• Gene therapy involving the introduction of DNA into germ cells
is passed to Successive Generation.
• For safety , Ethical and technical reason , germ cell gene therapy
is not being attempted at present.

9. There are 2 Types of gene therapy:-
1) EX-VIVO
2) IN-VIVO

10. EX-VIVO
• Here cells are modified
outside the body and then
transfer back again.
• It involves patient own cells
for culture and genetic
correction and then return
back to patient.
• Called as EX-Vivo because
cells are treated outside the
body.
IN-VIVO
• Invivo Gene therapy involves
direct delivery of therapeutic
gene (DNA) in to target cells
of a particular tissue of the
patient.
• Genes are changed in cell
when the cells are still in the
body.
• Called IN-Vivo because the
gene is transferred into the
cell inside patient’s body.

14. Steps involved in Ex Vivo gene therapy:-
First patients own cells are isolated with genetic defects from patient.
Grow cells in culture.
Introduce the therapeutic gene to correct gene defects.
Select the genetically corrected cells(stable Transformants) and
grow.
Transplant the modified cells in to the patient.

15. Ex-Vivo Gene Therapy

16. Vectors in gene Therapy:-
VECTORS :-
The carrier particles or molecules that are used to deliver genes
to Somatic Cells are called Vectors.
Types of vectors:-
1) Viruses.
2) Human artificial Chromosomes.
3) Bone Marrow Cells.

17. Viruses:-
• Viruses such as Reterovirus, adenovirus, Murine leukemia Virus
etc. are used as vectors.
• The vectors frequently used in gene therapy are virus
particularly reterovirus.
• RNA is a genetic material in reterovirus.
• As RNA enters host cells , it synthesize DNA by reverse
transcription.
• The formed viral DNA (Provirus) gets incorporated into DNA of
host cell.
• IT is normally harmless , but some reterovirus convert Normal
cells to cancer cell.

19. Human Artificial Chromosomes(HAC)
• The Human Artificial Chromosomes are the synthetic
chromosomes that can replicate with other chromosomes ,
besides encoding human protein.
• As the use of reterovirus in gene therapy is associated with
heavy risk such as cancer , This problem can be overcome if
human artificial chromosomes is used.
• HAC was developed in 1997 and it possesses the characteristics
of Human Chromosomes.

20. Bone Marrow cells:-
• Bone marrow cells contain totipotent embryonic stems cells.
• These cells are capable of dividing and differentiating into
various cell types. Ex- RBC , Platelets , B & T Lymphocytes.
• Bone Marrow cells are most widely used in gene therapy for
disease like SCID , Sickle Cell Anaemia.

21. Stratagies OF Gene Therapy:-
1 ) Gene Augmentation therapy :-
• Used to treat disease caused by mutation that stops a gene
from producing a functioning product such as protein.
• Thus therapy Adds DNA containing a functional version of lost
gene back in to the cells.
• The new gene produce a functioning product at sufficient levels
to replace the protein that was originally missing.

23. 2) Gene Inhibition Therapy:-
• It is suitable for treatment of infectious disease , cancer , and
inherited disease caused bt inappropriate gene activity.
• This therapy inhibits or eliminate the activity of gene that
encourages the groeth of Disease related cells.
• Aim is to introduce a gene whose product either-
Inhibits the expression of another gene.
Interferes with the activity of product of another gene.
EX- Cancer is sometimes the result of over activation of an
ONCOGENE ( Gene which stimulate cell growth).
So by inhibiting or eliminating the activity of that
Oncogenethrough gene inhibitiob therapy it is possible to prevent
further cell growth and stops cancer in its track.

25. 3) Killing of specific cells :-
• Its is suitable for disease such as cancer that can be treated by
destroying certain group of cells.
• Aim is to insert DNA into disease cell that causes thet cell to die.
• Achived by one of 2 ways :-
Insert DNA that contain ”Suicide Gene” that produces a highly toxic
product which kills the diseases cells.
Insert DNA that causes expression of a protein that marks the cell so
that the diseases cells are attacked by body’s natural Immune
System.
• It is essential with this method that the inserted DNA is targeted
appropriately to avoid the death of cells hat are functioning
Normally.

27. Various types of Gene Transfer Technique
Natural Methods-
• Congugation
• Agrobacterium Mediated Transfer
• Reteroviral Transduction
Physical Methods-
• Microinjection
• Biolistics Method

28. Chemical Methods-
• DNA transfer by Polyethylene glycol (PEG)
• Liposome Mediated transport
Electrical Methods-
• Electroporation
• Electrofusion

29. Electroporation:-
• It is an efficient process to transfer DNA into cells.
• Microscopic pores are induced in biological membrane by
applicaation of electric field.
• These pores are known as electropores which allows the
molecule , ions , water to pass from one side of membrane to
another.
• Or in simple terms , here electric field is applied on the cells –
because of this there is a disturbance on the phospholipid
bilayer of the cells – because of this polar molecules enters in to
the cells.

30. Electroporation

32. Electroporation

33. Advantages :-
• Fast Method
• Less Costly
• Simultaneously , large number of cells can be treated.
Disadvantages:-
• Cell damages because of disturbances.

34. Microinjection:-
• Here DNA is directly injected into cells . Specifically in to
nucleus or cytoplasm.
• Fine tipped ( 0.5 – 1.0 Micrometer ) diameterglass needle or
pipette is used.
• Microinjection can be done by only skilled person.
• Computerised control of holding pipette needle , Microscopic
stage and video technology has improved the efficiency of
technique.

35. Micro Injection

37. • Limitation:-
1) Coslty
2) Skilled personal Required
3) More useful for Animal cells

38. Biolistics :-
• Simplest methodof direct introduction of therapeutic DNA into
target cells.
• Looks like a pistol but works more like a Shotgun Golden
Pellets.
• The Gene gun fires DNA into the cells
• The DNA to be transformed into the cells is coated on to
microscopic beada of either gold or tungsten.
• The coated beads are then attachedto the end of plastic bullets.

39. • An explosive force fires the bullet towards the target cells that
lie just beyond the end of the barrel.
• After the beads pass through the cell wall into cytoplasm of the
target cells.
• Then beads and DNA dissociate and cells become transformed.

40. Gene Gun

43. Gene Gun

44. Liposome Mediated Gene Transfer:-
• Liposomes are spheres of lipids which can be used to transport
genes or molecules in to the cell.
• These are artificial vesicles that can act as a delivery agent for
exogenous materials.
• They are considered as spheres of lipid bilayer surrounding the
molecule to be transported and promote transport after fusing
with cell membrane.

45. Liposome mediated gene transfer

46. Liposome mediated gene transfer

47. Clinical Application :-
• Severe Combined Immuno Defficiency ( SCID)
• Hemophilia
• Parkinson’s disease
• Cancer
• HIV
• Retinal Disease
• Colour Blindness
• Diabetes mallitus

49. First Gene therapy:-
• The first gene therapy was performed to treat the defect in the
gene which synthesizes Adenosine Deaminase(ADA).

50. • Bone marrow cells with the help of ADA enzyme produce B&T
Lymphocyte which are responsible for Immune response.
• Gene undergoes transcription and translation process to form
ADA enzyme which is a protein.
• Suppose If that gene is faulty then no B&T lymphocyte are
formed which weakens the Immune response.
• This disease is called SCID ( Severe Combine Immuno
Deficiency ).
• The first gene therapy was performed on 4 year girl in 1990 who
was suffeeing from SCID.
• What scientists did was.....

51. • They Isolate ADA synthesizing gene . This gene was introduced
in to lymphocyte from that girl using reterovirus.
• Because of this temporarily B&T cells starts synthesize. The
immune system becomes normal.
• But life of lympocyte is short thatswhy regular interval of
therapy is required.
• If given at early stage this correction can be permanent.
ADA
synthesizing
gene
Using Reterovirus
Lymphocyte
Bone
Marrow
Cells
Transformed
lymphocyte is
introduced in bone
marrow of girl.

52. Advantages of Gene therapy:-
• Gives a chance to live a normal life to a patient born with genetic
disease.
• Gives a hope of healthy life to Cancer patient , Alopecia patient etc.
Disadvantages of Gene therapy :-
• Costly
• Taking frequent gene therapy is dificult as it is very costly.
• New things introduced in body leads to immune response.
• As not much study done and not FDA approved , It is not not safe for
humans.

53. Current status...
• Till Right now USFDA has not approved any human gene
therapy for sale.
Reasons..?
• In 1999, A 18 year old Jesse Gelsingur died from multiple organ
failire afyer treatment for Ornithine Transcarbaylase (OTC) .
DEATH was triggered by severe immune response to
Adenovirus.
• Like this there are many reasons ...
• One problem with gene therapy is that one does not have
control over where the gene will be inserted into genome.

54. Recent Advances :-
• Genes get into brain using Liposomes coated in polymer called
polyethylene glycol found potential for treating Parkinson’s
disease.
• Sickle cell anaemia is successfully treated in Mice.
• Severe Combined Immuno Difficiency (SCID) treatment is
found successful by Gene therapy.
• Curing hemophilia B in mice is found Successful.
• Research is going on Animal level for treating Amyotropic
Lateral Sclerosis.

55. • Stem cell therapy gives hope to prevent Inherited neurological
disease where scientists from University of Manchester have
used stem cell gene therapy to treat a fatal genetic brain
disease.
• A very large variety of therapeutic genes are under investigation
such as Tumour supressor , Suicide antiangiogenesis , Micro-
RNA genes.
• Cancer gene therapy is not yet indicated in clinical practice
however basic and clinical advanceshave been reported and
gene therapy is promising. New therapeutic approach for
treatment of gastrointestinal tumours.

56. • Gene therapy holds promise for treating wide range of disease
such as Cancer , Cystic fibrosis , Diabetes , Hemophilia ,
Alopecia , AIDS etc.
• Researchers are still atudying how and when to use gene
therapy.
• Currently in US gene therapy is available only as a part of
clinical trail.

57. THANK YOU
29 January 2021