GENE THERAPY PPT

1. Dr. Prabhjot kaur
DEPTT OF BIOCHEMISTRY
DMC LUDHIANA

2. Gene Therapy
“A promising future to
Diseases Treatment”

3. GENE:
 Genes are specific sequence of bases on the chromosome, which are expressed
in form of proteins
 Majority of cellular structures
 Variations in the DNA sequence
called mutations.
 Mutations are often harmless
but sometimes lead to serious disease.

4. Gene therapy: FOR GENETIC DISEASES
 Insertion of genes into a cell to treat a disease in which a defective mutant allele
is replaced with a functional one
 Some common approaches:
Replacing an abnormal gene with a healthy copy of the gene.
Inactivating or knocking out an abnormal gene that is functioning improperly.
Introducing a new gene into the body to help fight a disease.

5. How It Works?
 Vector delivers the therapeutic gene into a patient’s target cell
 The target cells become infected with the viral vector
 Functional proteins are created from the therapeutic gene causing the cell to
return to a normal state

7. History:
 1990:
 On September 1990, the first approved gene therapy
clinical trial took place when Ashanthi DeSilva,
a 4-year-old girl with Adenosine Deaminase
Deficient which causes Severe Combined
Immunodeficiency disease treated by gene Therapy.

9. TYPES OF GENE THERAPY:
SOMATIC CELL GENE
THERAPY
GERM -LINE GENE
THERAPY
 Therapeutic genes transferred into
the somatic cells. Eg. Introduction of
genes into bone marrow cells, blood
cells, skin cells etc.
 Will not be inherited later
generations.
At present all researches directed to
correct genetic defects in somatic
cells.
 Therapeutic genes transferred into
the germ cells. Eg. Genes introduced
into eggs and sperms.
 It is heritable and passed on to
later generations.
 For safety, ethical and technical
reasons, it is not being attempted at
present.

10. Types of somatic gene therapy:

11. 1. EX VIVO GENE THERAPY:
Transplant the modified cells to the patient.
Select genetically corrected cells.
Introduce the therapeutic genes .
Grow the cells in culture
Isolate cells with genetic defect from a patient

12. EXAMPLE OF EX VIVO GENE THERAPY:
 1st gene therapy clinical trial took place when Ashanthi DeSilva, a 4year old
girl with Severe Combined Immunodeficiency disease
 In patient with SCID, there is deficiency of Adenosine Deaminase due to a
gene defect.
 Deoxy adenosine accumulate and destroys T lymphocytes, disrupts immunity,
suffer from infectious diseases and death at young age.

14. 2. IN VIVO GENE THERAPY:
 Direct delivery of therapeutic gene into target cell into patients body
 Carried out by viral or non viral vector
systems.
 It can be the only possible option in
patients where individual cells
cannot be cultured in-vitro in
sufficient numbers (e.g. brain cells).
 In-vivo gene transfer is necessary when cultured cells cannot be re-implanted
in patients effectively.

15. Example of invivo gene therapy:
Therapy for cystic fibrosis:
 In patients with cystic fibrosis, a protein called cystic fibrosis
transmembrane regulator (CFTR) is absent due to a gene defect.
 In the absence of CFTR chloride ions concentrate within the cells and it
draws water from surrounding.
 This leads to the accumulation of sticky mucous in respiratory tract and
lungs.
 Treated by in-vivo replacement of defective gene by adenovirus vector.

17. 1. VIRAL VECTOR
2. NON VIRAL VECTOR

18. VECTORS:
 DNA is a large, long, fragile molecule easily degraded by serum nucleases
and intracellular nucleases. Therefore needs to be compacted and protected.
 So delivery is aided by a vector which safely delivers to target cells.
 Vectors are the vehicles used to carry the desired gene to the target cells
 They may be:
 Viral vectors
 Non viral vectors.

20. 1. VIRAL VECTOR:
 Virus bind to their hosts and introduce their genetic material into the
host cell.
 Viruses used as vectors are altered.
 Viruses bind to the cell surface receptors of cell membrane and deliver its
genetic contents.

21. Desirable characteristics of virus:
Some important characters of virus to be vector
in gene therapy are:
 Safety: by deleting the viral genome critical for viral replication.
 Low toxicity.
 Should efficiently deliver genes
 should not induce an allergic reaction or inflammation
 Tissue tropism (cell type specificity): should be able to target specific cell
types.
 should not elicit immune response in the host.

22. Types of viruses used:
 To deliver the desired gene clinical trials of Gene Therapy
mostly rely on:
 Retroviruses
 Adenoviruses
 Other viruses used as vectors include adeno-associated viruses,
lentiviruses, pox viruses, alphaviruses, and herpes viruses.

23. 1. Retrovirus:
Retroviruses have
a single stranded
RNA genome

24. Retrovirus:
Advantages:
 Good at targeting and entering cells
 Can infect only dividing cells .
 Can target specific cells through modification of surface proteins
 Can be modified to not replicate within host cells
Disadvantages:
 Can trigger an immune response within the host
 Can't be used to carry larger genes
 No long-term benefits.
 The random insertion of genes can disrupt other genes.

25. 2. Adenoviruses:
Adenovirus is double-stranded DNA virus.
vector has all viral DNA
eliminated to improve
safety and
immunogenicity

27. Adenovirus:
Advantages:
 Easy production of virus in high titres
 Efficient gene transfer
 Ability to be expressed in both proliferating and non-proliferating cells
Disadvantages:
 Inflammatory response elicited by their injection
 Immune response developed due to the inflammation
 Reduced expression so need frequent doses

28. 3. Adeno-associated virus:
 AAVs are small viruses from the Parvovirus family
 Simple, Non-pathogenic
 Single stranded DNA virus
 Dependent on the helper virus (usually adenovirus) to replicate.
Advantages:
 Persistent expression
 No insertional mutagenesis
 Infect both dividing and nondividing cells
 Safe
Disadvantages:
 Small genome limits size of foreign DNA.
 Low level of gene expression

29. 4. Herpes Simplex Virus:
• Have natural tendency to infect a particular type of cell.
• Infect and persist in nervous cells.
Advantages:
• Large insert size
• Could provide long- term CNS gene expression
Disadvantages:
• Current vectors provide transient expression

30. 5. LENTIVIRUS
 Genus of slow viruses of retroviridae family.
 Characterized by long incubation period.
 Can deliver significant amount of genetic information
into the DNA of host cell eg.HIV

31. HUMAN ARTIFICIAL CHROMOSOME-
Synthetic chromosome that can replicate with other
chromosomes
BONE MARROW CELLS-
Contain totipotent embryonic stem cells
Cells are capable of dividing and differentiating into various
cell types

33. 2. Nonviral methods:
 Methods of non-viral gene delivery include:
A. Physical approaches
B. Chemical approaches

34. A. Physical Methods:
 Physical approaches, including
 Nacked DNA injection
 Electroporation
 Gene gun
 Ultrasound
 Hydrodynamic pressure
employ a physical force that permeates the cell membrane and facilitates
intracellular gene transfer

35. 1) Nacked DNA injection:
 The DNA of a gene inserted into a plasmid is injected in the cells.
 There was no need for complex vectors.
 Direct injection of plasmid DNA into certain
cells has been shown to produce
comparatively low levels of
gene expression.

36. 2) Electroporation:
 Use high voltage electric field to carry DNA across the cell membrane.
 High voltage results in temporary breakdown and formation of pores in the
cell membrane, allowing
DNA molecules to pass
through.

37. 3) Gene Gun:
 Delivery with gene gun method also termed ballistic DNA delivery or
DNA-coated particle bombardment
 was first used for gene transfer to plants in 1987.
 Involve coating macromolecule (DNA, RNA) onto micro-carrier particles
such as gold and tungsten
 The particles achieve sufficient speed due to a pressurized inert gas
(generally helium)
 Momentum allows penetration of these particles at a certain speed to a few
millimeters of the tissue and then cellular DNA release
 Due to small size(1µm), the particles easily penetrate the cell
membrane and transport DNA into the cell.

39. 4) Sonoporation:
 Ultrasound applied to the cell which increase the permeability of cell
membrane to macromolecules such as plasmid DNA. therapeutic DNA then
can effectively be transferred into the cell
 Ultrasound creates membrane pores and facilitates intracellular gene
transfer through the membrane pores
 It could become an ideal method for noninvasive gene transfer into cells of
the internal organs.

40. 5) Hydrodynamic pressure:
 Involve rapid injection of a large volume of physiological solution to
increase the permeability of cell membranes of the cell.
 Hydrodynamic pressure created by the injection of the large volume of
DNA solution with blood pressure inside veins increases the permeability of
the capillary endothelium and pores form in the plasma membrane of
parenchyma cells.
 DNA or other related molecules can reach the cell from these pores.

42. NANOENGINERED SUBSTANCES
 Nonviral substances such as ormosil used as DNA
vectors
 Deliver DNA loads to specifically targeted cells in
living animals[organically modified silica]

43. B. Chemical Methods:
1. Lipoplexes:-
 DNA is covered with lipids .
 3 types of lipids:
• anionic
• neutral
• cationic

44. 2. Polyplexes:
 Complexes of polymers with DNA are called polyplexes
 consist of cationic polymers and their production is regulated by ionic
interaction.

45. 3. Encapsulation:
 Encapsulation of DNA [Protected from enzymatic degradation]
Polymers are degraded and DNA is released

46. Gene therapy for hematopoietic
derived diseases
 All hematopoietic cells derive from– pleuripotent
hematopoietic stem cells (PHSCs)
 Stem cell gene therapy can be applied to a large variety of
congenital & acquired blood cell diseases.

47. Gene silencing
 Also called antisense technology
 Can inactivate a gene that may cause disease or is
defective
 Highly specific
 Gene knockdown & not gene knockout!!

48. Antisense technology

50. Potential approaches to dominant
negative
 Technique for repairing mutations: SMaRT
 Techniques to prevent the production of a mutated
protein: triple helix forming oligonucleotides
 Use of ribozyme technology

51. SMaRT
 Delivery of RNA that pairs specifically with intron next to
mutated segment of mRNA
 Simultaneous delivery of correct version of segment to
replace mutated piece in final mRNA product
 Translation of repaired mRNA to produce normal,
functional protein

52. Triple helix forming
oligonucleotides

53. Ribozyme technology

54. Creating new chromosome:
 Researchers are also experimenting with introducing a 47th Artificial
Chromosome into target cells.
 This chromosome would exist autonomously alongside the standard 46, not
affecting their workings or causing any mutation.
 It would be a large vector capable of carrying substantial amount of genetic
code and the body’s immune system would not attack it.
 A problem with this potential method is the difficulty in delivering such a
large molecule to the nucleus of the target cell.

55. Some Diseases for applying gene therapy:
Disease Defect Target cell
SCID Adenosine deaminase T-lymphocytes
Hemophilia Factor VIII, Factor IX
deficiency
Liver, muscle cells
Cystic fibrosis Loss of CFTR gene Airspaces in the lung
1-antitrypsin deficiency 1-antitrypsin Lung or liver cells
Cancer Many causes Many cell types
Neurological diseases Parkinson’s, Alzheimer's Direct injection in brain
Cardiovascular Arteriosclerosis etc Vascular endothelium
Infectious diseases AIDS, hepatitis B T cells, macrophages
Liver cirrhosis Fibrogenesis Hepatocyte growth factor
Autoimmune disease Lupus, diabetes MHC, 2-microglobulin

56. GENE THERAPY CURESBLINDNESS
 Cure blindness of inherited condition
 Leber’s conginetal amaurosis
- inherited disease caused by an abnormality in a gene
called RPE65.
- The condition appears at birth or in the first few
months of life and causes progressive worse and loss of
vision.

57. HOW IT WORKS??
 used harmless viruses
 enable access to the cells beneath the retinas of
patients
 By using a very fine needle
-safe in an extremely fragile tissue and can improve
vision in a condition previously considered wholly
untreatable.

58. GENETHERAPY REDUCESPARKINSON’S
DISEASESYMPTOMS
 it significantly improved the weakness of the
symptoms such as tremors, motor skill problems, and
rigidity
 Main- overactive brain region: the subthalamic
nucleus should be introduced with gene
 that would produce GABA—an inhibitory chemical—
then they could potentially quiet that brain region and
alleviate tremors.

59. HOW IT WORKS??
 Done with local anesthesia, used a harmless,
inactive virus [AAV-2 GAD]
 Deliver the GAD gene into patient’s subthalamic
nucleus
 The gene instructs cells to begin making GABA
neurotransmitters to re-establish the normal
chemical balance that becomes dysfunctional as the
disease progresses

61. GENE THERAPY USES AIDS VIRUS
TO FIGHT AIDS
 Immune cells are removed from patient
 Modified with disabled AIDS virus k/a LENTIVIRUS
then returned intravenously.
 Cells are genetically altered and prevent HIV from
producing

62.  Gene therapy to alleviate pain by introducing a
therapeutic protein into target structure

63. GENE THERAPY FOR CANCER
 TUMOR NECROSIS FACTOR GENE THERAPY
 Protein produced by macrophages
 Tumor infiltrating lymphocytes were transformed with a
TNF gene and used for the treatment of malignant
melanoma.

64. suicide gene therapy
 Involves transducing tumor cells with a gene encoding
an enzyme that can metabolize a nontoxic prodrug to
its toxic form
 Most commonly used technique in clinical trials for
brain tumors

65. HSV-1 Thymidine
Kinase/Ganciclovir
 HSVtk is an enzyme that metabolizes nontoxic
nucleoside analogues,such as ganciclovir, acyclovir, or
valacyclovir into a cytotoxic molecule= metabolite is
incorporated into DNA= cell death
 DNA polymerases inhibition

66. Bystander effect.
 The cytotoxic effect of transduced cells on adjacent
nontransduced cells is termed the bystander effect.

67.  Transfer of toxic phosphorylated forms of ganciclovir
to nontransduced cells presumably via gap junctions
 Targeting of mitotically active endothelial cells in
tumor vessels
 An immune-associated response against a nonhuman
protein,such as tk, leading to diffuse cell death that
affects neighboring nontransduced cells

68. Cytosine Deaminase/5-
Fluorocytosine
 Cytosine Deaminase= 5-Fluorocytosin= 5-fluorouracil
 The toxic effects of 5-FU are mediated by its
intracellular metabolites, which cause DNA strand
breakage leading to cell death

69. Cytochrome P-450
2B1/Cyclophosphamide
 The rat cytochrome P-450 2B1 (CYP2B1) activates CPA
with high efficiency
 HSV-1 vector (rRp450) that can kill tumor cells
through two modes:
 (1) using viral oncolysis
 2) rendering the infected cell sensitive to CPA

70. Tumor Suppressor Gene
 Transfer of functional gene controling cell cycle in
order to restore normal cycle functioning

71. Positive aspects:
 Gene therapy has the potential to eliminate and prevent hereditary diseases
such as cystic fibrosis, ADA- SCID etc.
 It is a possible cure for heart disease, AIDS and cancer and give hope of
healthy life to the patient.
 It gives someone born with a genetic disease a chance of normal life
 It can be used to eradicate diseases from the future generations.
 For certain disease that do not have any cure except gene therapy, it could
save many lives
 Gene therapy have a number of advantages over drug therapy such as
providing a cure rather than easing the symptoms

72. Negative aspects:
 Short Lived
• Long lasting therapy is not achieved by gene therapy
• Due to rapid dividing of cells benefits of gene therapy is short lived.
 Immune Response
• new things introduced leads to immune response
• Immune response to the transferred gene stimulates a potential risk to gene therapy.
 Viral Vectors
• Viruses used as vectors for gene transfer may cause toxicity, immune responses, and
inflammatory reactions in the host.

73.  Multigene Disorders
• Disorders caused by defects in multiple genes cannot be treated effectively using
gene therapy.
• Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to
treat because we need to introduce more than one gene
 Insertional mutagenesis
• The viruses may target the wrong cell.
• If the DNA is integrated in the wrong place in the genome, for example in a tumor
suppressor gene, it could induce a tumor
 Costly:
The treatment is very expensive for the patients. It is not accessible to most people
due to its high cost

74. Recent developments
 In Jan 2012, University of Florida, researchers found Rx for
a common form of blindness (X linked retinitis
pigmentosa) that strikes both youngsters & adults.
 GT called NLX-P101 dramatically reduces movement
impairment in parkinson’s patient’s according to results of
a phase II study published on march 2011 in the journal
lancet neurology

75. '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-programme 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

76. First Real-Time MRI-Guided Gene Therapyfor BrainCancer
Neurosurgeons at the University of California, San Diego School of Medicine and UC San
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

77. 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).
After the injection of Toca 511, the patients
are treated with an investigational extended-
release oral formulation of 5-FC called Toca
FC.
Cancer cell killing takes place when 5-FC
comes into contact with cells infected with
Toca 511.

78. UCLA researchers combine cellularandgene 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
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

79. stemcell genetherapy gives hopeto prevent inherited
neurological disease
Scientists from The University of Manchester have used stem cell gene
therapy to treat a fatal genetic brain disease
It was used to treat Sanfilippo – a fatal inherited condition which causes
progressive dementia in children
Sanfilippo, is currently untreatable mucopolysaccharide (MPS) disease
It is caused by the lack of SGSH 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 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

80. Mucopolysaccharidosis Type IIIApotential gene therapy
Mucopolysaccharidosis Type IIIA (MPSIIIA) is a metabolic disorder in which the body
is missing an enzyme that is required to break down long chains of sugars known as
glycosaminoglycans
The glycosaminoglycans collect in the body and cause damage, particularly in the
brain if not broken
Fàtima Bosch and colleagues at Universitat Autònoma de Barcelona in Spain
developed a form of gene therapy to replace the enzyme that is missing 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 and suggests that this approach could potentially be used as a therapy
for MPSIIIA

81. Future aspects and CONCLUSION:
 Theoretically, gene therapy is the permanent solution for genetic diseases
but has several complexities.
 A breakthrough may come anytime and a day may come when almost every
disease will have a gene therapy
 Current uses of gene therapy focus on treating or curing existing conditions
but in future focus could shift to prevention
 As more of the human genome is understood. With this knowledge in hand,
Gene therapy could be used to head off problems before they occur.

83. “THANK
YOU”