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1. SANA SAIFI
M.PHARM
Topic:- Gene Therapy:Introduction,clinical
application and recent advances in gene
therapy, priciples of RNA and DNA estimation.

2. Content
• Introduction
• Types of gene therapy
• Clinical application
• Recent advances in gene therapy
• Principles of RNA ana DNA estimation.

3. GENE THERAPY
Introduction
 Gene therapy is to transfer a gene from one DNA molecule to another
DNA molecule.
 It is the insertion of genes into the individual's cell and tissues to treat
a disease.
 The directed desirable gene transfer from one organism to another and
the subsequent stable integration and expression of foreign gene into
the genome is referred as genetic transformation.
Definition:
A novel approach to treat, cure or prevent a disease
by changing the expression of a person's genes.

4.  Unstable transformation occur when DNA is not
integrated into host genome.
 Stable transformation occur when DNA is integrated into
host genome and is inherited in subsequent generations.
 The transferred gene is known as transgene and the
organism that develop after a successful gene transfer is
known as transgenic organism.

5. Steps involved in gene therapy
 Identification of the defective gene
 Cloning of normal healthy gene
 Identification of target cell / tissue/organ
 Insertion of the normal functional gene into the host DNA.

6. Approaches:
1. Gene Replacement/Correction
- Replacing a mutated gene with a healthy copy.
Ex.) Severe Combined Immunodeficiency.
2. Gene Silencing/Gene Interference
- Inactivating or "knocking out" a mutated gene.
Ex. Sickle Cell Disease.
3. Gene Augmentation/Gene Addition
- Introducing a new gene to help fight disease
Ex. Parkinson's Disease.
4. "Suicide Gene“
- Can cause a cell to kill itself through apoptosis
- Makes cancer cells more vulnerable and sensitive to anticancer drugs
Ex. Solid Tumors.

7. Types of Gene Therapy
 Germ Line Therapy
 Somatic Cell Therapy
1.Ex Vivo
2.In Vivo

8. Germ Line Therapy
• Altering a gene of an egg or a sperm cell.
or
• Altering the genetic composition of a blastomere during an
early stage of its division.
• Any zygote produced as a result of this germ cell will have
a correct version of the defective gene and will continue
passing it on to their offspring
• Considered unethical.

9. Somatic Cell Therapy
• Involves altering the genetic code of a person's somatic
cells.
• Inserting therapeutic genes into somatic cells like:
 Fibroblasts
 Myoblasts
 Epithelial cells
 Nerve cells
 Glial cells
• It is mostly performed in fully grown organisms.
• Less controversial.

10. Ex Vivo Approach
• Commonest method of the two
• Called as "Ex Vivo" because the cells
treated outside body.
In Vivo Approach
• Called "In Vivo" because events take
place within patient's body
• Uses vectors.

11. Vectors for Gene Therapy
Different carrier systems are used for gene
delivery;
1. Viral Vectors
2. Non-Viral Vectors

12. 1.Viral vectors
Viral vectors are mostly derivatives of viruses that infects animais.
 Viruses have evolved a way of encapsulating and delivering their
genes to human cells to remove disease-causing genes and insert
therapeutic ones.
 Virus bind to their hosts and introduce their genetic material into the
host cell.
1.Free replicating viruses- that multiply within the cell, but do not
integrate into the genome of the host.
2.Integrateing viruses-that can integrates into the host genome. They
enters the cell, copy their RNA genome into DNA.

13. Non viral vector system
Non-viral vectors depend on physical or chemical methods
of delivering genetic material into a cell.
A..Physical methods-
1.Electroporation
2.Microinjection
3. Gene gun or a biolistic particle delivery system
4. Magnetofection
B.Chemical methods
-1.DNA transfer by calcium phosphate method
2.Liposome medicated transfer

14. Applications of Gene Therapy
1.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.

15. 2.Chronic Granulomatus Disorder
 CGD is a genetic disease in the immune system that leads to the
patients' inability to fight off bacterial and fungal infections that can be
fatal.
 Investigators in Germany treated two patients with this disease some of
the blood-making cells are taken from the patient.
 The normal gene is placed into the cells using special viruses called
retroviruses. The cells are then able to produce the normal protein.
3.Hemophilia
 Patients are not able to induce blood clots and suffer from external and
internal bleeding that can be life threatening.
 In a clinical trial 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.

16.  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.
4. Gene therapy trial 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.

17. 5.Gene therapy for acquired disease
Cancer
 Multiple gene therapy strategies have been developed to treat a
wide variety of cancers, including suicide gene therapy, oncolytic
and therapeutic gene vaccines.
 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.

18. 6.Other acquired diseases
 The same gene therapeutic techniques have been applied to treat
other acquired disorders such as viral infections (e.g. influenza, HIV,
hepatitis), heart disease and diabetes, among others.
 Some of these have entered, or will soon be entering, into early
phase clinical trials.
Uses of gene therapy
 Clinical gene transfer applications.
 Vaccine development.
 Production of transgenic animals.
 Treatment of cancer and AIDS.
 Gene discovery.
 Gene therapy.
 Enhancing the resistance of plant.
 Genetically modified organism.

19. Recent Advancing on Gene
Therapy
 It depends on delivery of correct genes, viral vectors are tools use
by molecular biologist to deliver genetic material into the cell.
 This process can be performed inside living organism (in vivo) or in
cell culture(in vitro).
 Viruses have evolved specialized molecular mechanism to effective
transport their genomes inside the cell they infected.
 Viruses use as vector to introduce the genetic material inside the
bodies, this viruses are inactivated hence do not reproduce.
E.g. Adenoviruses, Adeno-associated virus, Herpes virus (DNA tumor
virus), Retrovirus (RNA tumor virus), Lenti virus, pox virus and measles
virus.

20. 1.Non viral vector:This include the use of liposomes (less immunogenic)
and use of nanotechnology( nanometer size).
2. 'Mending broken hearts' by using gene therapy Novel techniques to
"mend broken hearts" using gene therapy and stem cells represent a
major new frontier in the treatment of heart disease.
• It was achieved by the researchers at Gladstone Institute of
Cardiovascular Disease in California.
• They were able to re-programmed scar-forming cells into heart
muscle cells, some of which were capable of transmitting the kind of
electrical signals that make the heart beat,.
• They performed on a live mice, transforming scar-forming cells, called
fibroblasts, into beating heart muscle cells.
• They injected three genes (cocktail of genes) into the heart of live
mice that had been damaged by heart attack, fibroblasts could be
turned into working heart cells.
• Researchers said that the "cocktail of genes" used to regenerate cells
could one day be replaced with “small drug-like molecules”that would
offer safer and easier delivery.

21. 3.Mucopolysaccharidosis Type IIIA potential gene therapy.
• Mucopolysaccharidosis Type IIIA (MPSIIIA) is a metabolic disorder in
which the body is missing an enzyme required to break down long
chains of sugars known as glycosaminoglycans.
• The glycosaminoglycans accumulate in body and cause damage,
particularly in the brain if not broken.
• Fatima Bosch at university of Barcelona in Spain developed a form of
gene therapy to replace the missing enzyme in MPSIIIA.
• They injected the replacement gene into the cerebrospinal fluid that
surrounds the brain and spinal cord.
• This study demonstrates that gene therapy can be delivered to the
brain through the cerebrospinal fluid.
4. Stem cell gene therapy gives hope to prevent inherited neurological
disease.
• Scientists from The University of Manchester have used stem cell
gene therapy to treat a fatal genetic brain disease.
• Install NowIt was used to treat Sanfilippo - a fatal inherited condition
which causes progressive dementia in children.

22. • Sanfilippo, is currently untreatable mucopolysaccharide (MPS)
disease.
• It is caused by the lack of SGSH(N-sulfoglucosamine sulfohydrolase)
enzyme in the body which helps to breakdown and recycle long
chain sugars, such as heparan sulphate (HS).
• Children with the condition build up and store excess heparan
sulphate (HS) throughout their body from birth which affects their
brain and results in progressive dementia and hyperactivity, followed
by losing the ability to walk and swallow.
5. UCLA researchers combine cellular and gene therapies to develop
treatment for breast cancer.
• Carol Kruse, a professor of neurosurgery and member of the
Jonsson Cancer Center and the UCLA Brain Research Institute led
the research on breast cancer.
• Breast cancer is the most common form of cancer in women, and
metastasis is a major cause of health deterioration and death from
the disease.
• Cellular therapy and gene therapy were used together to treat breast
cancer.

23. • Cellular therapy is a type of immunotherapy that uses T cells, the foot
soldiers of the immune system, that have been sensitized in the
laboratory to kill breast cancer cells.
• These sensitized T cells are injected into the parts of the brain to which
cancer has spread.
• The research shows that the T cells can move through tissue and
recognize and directly kill the tumor cells.
6.First Real-Time MRI-Guided Gene Therapy for Brain Cancer.
• Neurosurgeons at the University of California, San Diego School of
Medicine and UC Sar Diego Moores Cancer Center are among the first
in the world to utilize real-time magnetic resonance imaging (MRI)
guidance for delivery of gene therapy as a potential treatment for brain
tumors.
• Using MRI navigational technology, neurosurgeons can inject Toca 511
(vocimagene amiretrorepvec), a novel investigational gene therapy,
directly into a brain malignancy.
• The new approach offers a precise way to deliver a therapeutic virus
designed to make the tumor susceptible to cancer-killing drugs.

24. • Toca 511 is a retrovirus engineered to selectively replicate in cancer
cells, such as glioblastomas.
• Toca 511 produces an enzyme that converts an anti-fungal drug,
flucytosine (5-FC), into the anti- cancer drug 5-fluorouracil (5-FU).
• Cancer cell killing takes place when 5-FC comes into contact with
cells infected with Toca 511.

25. DNA
Principle:
• This reaction is generally given by deoxypentose, the 2-deoxyribose
of DNA, in presence of acid is converted to ẞ-hydroxilevulinic
aldehyde which then react with diphenylamine to form blue colored
complex which is read at 595nm.
• The intensity of blue color is directly proportional to concentration of
DNA.

26. Procedure:
• Dissolve 1.5g diphenylamine in 100ml of glacial acetic acid.
• Add 1.5ml of concentrated sulphuric acid store the solution in dark
glass bottle.
• On the day of use prepare a fresh solution of ethanal (1ml) in 50ml
water.
• Add 0.5ml of this solution to each 100ml of diphenylamine solution.
ASSAY:
• Prepare series of dilutions of standard DNA (0.25mg/ml) in saline
citrate buffer to give concentration of 50-500µg/ml.
• Prepare all samples in triplicate.
• To 2ml of each dilution of blank, standard and unknown add 4ml of
diphenylamine reagent and mix.
• Warm, cool and read absorbance at 595nm against the blank.
• Construct the standard curve and calculate the concentration of
unknown DNA sample dissolve in saline citrate solution.

27. RNA
Principle:
• This is a general reaction of pentose and depends on the
formation of furfural when pentose is heated with concentrated
hydrochloric acid, orcinol react with furfural in presence of ferric
chloride as catalyst to give blue- green complex which is
measured at 665nm.
• The absorbance is directly proportional to concentration of RNA.

28. Procedure:
• Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1ml of working standard into
series of labeled test tubes.
• Pipette out 1ml of given sample into other test tubes.
• Makes up the volume to 1ml in all test tubes, test tube with 1ml act
as blank.
• Add 2ml of orcinol reagent to all test tube including blank.
• Mix by shaking and heat on boiling water for 20 minutes.
• Cool the content and record the absorbance at 665nm against blank
.
• Plot standard curve by taking concentration of RNA on X-axis and
absorbance at 665nm on Y-axis.
• From this standard curve calculate the concentration of RNA in given
sample (µg RNA/ml).