it is a topic related gene mutation , disease undertaking ,classification ,curable or not main application.

1. GENE THERAPY
PRESENTED BY ,
Pooja .g. anil
M-PHARM
pharmaceutical chemistry
Amrita school of pharmacy ,kochi

6. EX VIVO GENE THERAPY
► STEPS:
1. Isolate cells with genetic defects from a
patient
2. Grow the cells in culture
3. Introduce the therapeutic genes
4. Select genetically corrected cells and
grow.
5. Transplant the modified cells to the
patient.

7. EXAMPLE OF EX VIVO GENE THERAPY
o 1st gene therapy: to correct deficiency of
enzyme, Adenosine deaminase(ADA)
o Performed on a 4 year old girl
o She was st1ffering from SCID- Severe
combined immunodeficiency
o Caused due to defect in gene coding forADA

8. IN VIVOGENE THERAPY
► Direct delivery of the therapeutic gene
into target cell into patients body.
► Carried out by viral and non viral vector
systems
► It can be only possible option in patients
where individual cells cannot be
cultured in vitro in sufficient
numbers.(eg-brain cells.)

9. EXAMPLE OF IN VIVO GENE THERAPY
► Therapy for cystic fibrosis -
► In patients with cystic fibrosis, a protein called cystic
fibrosis transmembrane regulator(CFTR) is absent.
► In the absence of CFTR chloride ions concentrate
within thecells and it draws water from
surrounding.
► This leads to the accumulation of sticky mucous ii1
respiratory tract and lungs.
► Treated by in vivo replacement of defective gene
by adenovirus vector.

10. VECTORS FOR GENE THERAPY
•To transfer the desired gene into a
target cell, a carrier is required.
Such vehicles of gene delivery are
known as vectors.
•Two main classes
•Viral vectors
•Non viral vectors

11. VIRAL VECTORS
► 1) retrovirus vector system
► the recombinant retrovirus have the ability to
integrate into the host genome is a stable fashion.
► Target cell-dividing.
► 2) adeno virus vector system-
► Adeno virus with a DNA genome – good vector
► Target- non dividing human cell.

12. ► 3) Adeno associated virus vector-It is a
single stranded, non pathogenic small DNA
virus.
► AAV enters host cell, becomes double stranded
and gets integrated into chromosome.
► 4)Herpex simplex virus vector-
► Viruses which have natural tendency to
infect a particular type of cell.

13. NON VIRAL VECTORS
► 1)PURE DNACONSTRUCT
► Direct introduction of pure DNA construct
into target tissue.
► Efficiency of DNA uptake by cells and
expression rather low.
► Consequently Large quantities of DNA
have to be injected periodically.
► 2) LIPOPLEXES
► Liquid DNA con1plexes;DNAconstruct
surrounded by artificial lipid layer.

14. ► 3) DNA MOLECULAR CONJUGATES
► Commonly used synthetic conjugates is poly-
L- lysine bound to specific target cell
receptor.
► Therapeutic DNA is then made to combine
with the conjugate to form a complex.
► It avoids lisosomal breakdown of DNA.
► 4) HUMAN ARTIFICIAL
CHROMOSOME:
► Can carry a large DNA ie, with one or more
tlherapeutic genes with regulatory elements.

15. METHODS OF GENE DELIVERY
PHYSICAL METHODS
► GENEGUN
► Employs a high- pressure delivery system to shoot
tissue with gold or tungsten particles that are coated
with DNA
► MICRO INJECTION
► Process of using a glass micropipette to insert
microscopic substances into a single living cell.
► Normally performed under a specialized optical
microscope setup called a micromanipulator

16. CHEMICAL METHODS
► USING DETERGENT MIXTURES
► Certain charged chemical compounds
like calcium phosphates are mixed with
functional CDNA of desired function .
► The mixture is introduced near the vicinity
of recipient cells
► The chemical disturbs the cell membrane,
widens the pore size and allows the
CDNA to pass through the cell.

17. ► LIPOFECTION
► It is a technique used to inject genetic
materials into a cell by means of liposomes.
► Liposomes are artificial phospholipid vesicles
used to deliver a variety of molecules
including DNA into the cells.

18. OTHER TYPE OF GENE THERAPY
GENEAUGMENTATION THERAPY
o Most common form of gene therapy.
o Foreign gene replaces missing or defective
gene.
o eg- replacement of defective p53 gene by a
normal one in liver cancer
o GENE INHIBITION THERAPY-
o Done to block the over production of some
proteins.
o 2 types- antigene and antisense therapy

19. ADVANTAGES
o Gene therapy has the potential to eliminate and
prevent hereditary disease such as cystic fibrosis,
ADA- SCID etc.
o It is a possible ct1re for heart disease, AIDS and
cancer.
o It gives someone born with a genetic disease a
chance to life.
o It can be used to eradicate diseases from the
future generations.

20. DISADVANTAGES
► Long lasting therapy is not achieved by gene therapy;
due to rapid dividing of cells benefits of gene therapy
is short lived.
► immune response to the transferred genestimulatesa
potentialrisktogenetherapy
► Viruses used as vectors for gene transfermay
cause toxicity, immune responses, and
inflammatory reactions in the host.
► Disorders caused by defects in multiple genes
cannot be treated effectively using gene therapy.

21. GENE THERAPY FOR GENETIC DISORDERS SEVERE COMBINED
IMMUNE DEFICIENCY ( ADA – SCID )
 Affected children are born without an effective immune
system and will succumb to infections .
 The disease is caused by a mutation in gene on
chromosome 20 .
 The gene codes for the enzyme adenosine deaminase
(ADA)
 The therapeutic gene called ADA was introduced into the
bone marrow cells of such patients in the laboratory ,
followed by transplantation of the genetically corrected
cells back to the same patients.
 The immune system was reconstituted.

22. HEMOPHILIA :
o Patients are not able to induce blood clots and suffer
from external and internal bleeding that can be life
threatening.
o In a clinical trail conducted in the United states ,the
therapeutic gene was introduced into the liver of
patients , who then acquired the ability to have normal
blood clotting time.
 The therapeutic effect was transient because the
genetically corrected liver cells were recognized as
foreign and rejected by the healthy immune system in
the patients and curative outcome by gene therapy .

23. GENE THERAPY TRAIL FOR INHERITED BLINDNESS :
 Choroideremia is a rare inherited cause of
blindness that affects around 1 in 50,000
people.
 There is currently no cure.
 It is caused by defects in the CHM gene on the
X chromosome.
 Without the protein produced by the CHM
gene, pigment cells in the retina of the eye
slowly stop working , then die off .

24. CANCER :
 Multiple gene therapy strategies have been developed to treat a
wide variety of cancers , including suicide gene therapy ,
oncolytic and therapeutic gene vaccines .
 & gt; Two-thirds of all gene therapy trials are for cancer and
many of these are entering the advanced stage, including a Phase
III trial.
 Additionally, numerous Phase I and Phase II clinical trials for
cancers in the brain, skin, liver, colon, breast and kidney among
others, are being conducted in academic medical centers and
biotechnology companies, using novel technologies and
therapeutics developed on-site.

38. What is CRISPR-Cas9?
• CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats).
• Genome editing system- Manipulate genes in different organisms.
• Major components- CRISPR (Small segment of RNA structure)Cas9
(Nuclease Protein)
• The complex cleaves target DNA at specific location.

39. Origin of CRISPR-Cas9 System
Concept –
 Bacterial defence against viral attack (acquired immunity)The
phage genome- CRISPR kind of sequences.
 Bacteriophages attack the bacteria and destroys it during the
growth of bacteriophage.
 When viral genome released for next time, the bacteria already
has viral geneticmaterial as a copy or memory.
 Bacterial cell produces RNA copy (crRNA) along with Cas9
protein.
 The CRISPR-Cas9 complex interacts with the viral genome and
cleaves the targetDNA.
 Scientists tried to replicate the process in in-vitro studies

40. Advantage of using CRISPR-Cas9 system:
1. The total DNA content can be changed.
2. The function of the gene can be analyzed.
3.If tagged with fluorescent dye, the CRISPR-Cas9 can mark specific region or
specific DNA in the whole genome of complicated organism.
E.g. HumansApplications:
1.Food industryImmunization of bacteria used in food production against
viruses (cheese/yogurt)
2. Medical TreatmentCRISPR therapy would allow silencing of defective genes
(Huntington Disease)
3. Agriculture Development of disease resistant transgenic plants,Increased
oil production in (Canola crop), etc.

41. REFERENCES
• Satyanarayana U, Biotechnology, 1st edition, Book and
allied (P) Ltd, Kolkata.
• http://www.rnedindia.net/articles/genetherapy_treat
men
t.Htm
• http://en.wikipedia.org/wiki/Gene_therapy