gene therapy

1. GENE THERAPY
- Dr. Chandini
Moderator: Dr. Vinitha
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2. OVERVIEW
o Introduction
o History
o Types of gene therapy
o Techniques
o Approaches
o Methods of gene
delivery (vectors)
o Applications
o Pros & Cons
o Success stories in
the recent past
o Recent advances
o Conclusion
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3. INTRODUCTION
• Genes, the basic functional unit of heredity.
• Variations in the DNA sequence or code of a gene
= mutations.
• Each human being carries normal as well as some
defective genes.
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4. Why Gene therapy ??
• Faulty gene  defective protein 
DISEASE.
• Genetic disorder = Disease caused by a
"variation or "mutation of a gene.
• Passed on to family members
• > 4,000 genetic disorders identified,
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5. TYPES OF GENETIC DISEASES
1. Single gene disorders
Eg :- Sickle cell anemia, Cystic fibrosis, Marfan’s
syndrome
2 . Chromosome disorders
Aneuploidy, Deletion, Inversion, Translocation,
Mosacism
Eg. Down’s, syndrome, Turner’s syndrome
3 . Multifactorial inheritance disorders
Eg. CVDs, Alzheimer's disease, Diabetes mellitus,
AIDS,
Most cancers 5

6. WHY IS GENE THERAPY
SO ATTRACTIVE?
Radical cure for single gene diseases
Common acquired conditions –large genetic
component
Conventional treatment- far from ideal,
Ability to control gene expression
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7. WHAT IS GENE THERAPY?
• Technique in which a functional gene replaces
defective gene so that the body can make the
functional protein & therefore eliminate the root
cause of the disease,
or
transfer genetic material into cells or tissues to
prevent or cure a disease.
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8. HISTORY..
o 1971: key scientific experiment (galactosemia)
- DNA could be injected into human cells could fix the
biological problem
o 1972: formal proposal to use gene therapy
as a means to treat human genetic disease
1st put forth.
o late 1980s: 1st attempt to use gene therapy
to treat live humans in clinical trials began
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9. FIRST SUCCESSFUL CASE ..
• Sep 14th 1990: Ashanthi DeSilva,
ADA-deficient Severe
Combined
Immunodeficiency,
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10. TYPES
 Somatic Gene therapy:
• Transfer of genes into body cells other than
germ cells ie somatic cells
• Not passed on to offspring
• affects only targeted cells
• More conservative & safer
approach
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11.  Germline gene therapy –
• Functional genes introduced into germ cells (sperm or egg).
 Changes are permanent
 Passed on to future generations
• Theoretically highly effective.
 Offer the possibility of removing an inherited disorder from
a family line forever
 Controversial
Existing gene therapy treatments &
experiments are all somatic.
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12. TECHNIQUES IN GENE THERAPY
 Ex vivo technique
 In vivo technique
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13. 13

14. 1. Ex vivo gene therapy :
a)Isolate cells with genetic defect from
patient
b) Grow the cells in culture
c) Introduce the therapeutic gene to correct
gene defect
d) Select the genetically corrected cells &
grow
e) Transplant the modified cells to the patient
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15. 2. In vivo gene therapy:
• The direct delivery of the therapeutic gene into
the target cells of a particular tissue.
• Many tissues are the potential candidates for
this approach.
For eg. liver, muscle, skin, spleen, lung, brain and
blood cells etc.
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16. 16

17. APPROACHES FOR GENE THERAPY
1. Gene augmentation therapy.
2. Gene replacement therapy.
3. Gene inhibition therapy.
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18. 1. GENE AUGMENTATION THERAPY
• Corrects metabolic deficiencies caused by a
missing or defective gene.
• Does not substitute the flawed / absent gene.
• Eg. Cystic fibrosis
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19. 2. GENE REPLACEMENT THERAPY
• Replacement of mutant copy with a correctly
functioning copy in situ.
Eg. oncogenes
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20. 3. GENE INHIBITION THERAPY
• Aim:
• Inhibition of expression of faulty gene
• Inhibit activity of product of another gene.
• Eg. oncogene
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21. METHODS OF GENE DELIVERY
VECTORS:
• The carrier particles or molecules used to deliver
genes.
 Viral vectors
 Non-viral vectors
 Physical methods
 Chemical methods
Ideal vector
- accommodate
foreign genes of
sufficient size
-deliver gene to a
specific cell type,
-non-immunogenic
and safe
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22. VIRAL VECTORS
• Most promising system of gene delivery
1. Gene transfer - more efficient & specific
2 . Multiple & repeated doses not required.
- single dose is sufficient.
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23. VIRUSES USED IN GENE
THERAPY
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24. 24

25. 25

26. NON-VIRAL VECTOR SYSTEMS
PHYSICAL METHODS
• Microinjection
• Electrophoration
• Gene guns
• Sonoporation
• Magnetofection
CHEMICAL
METHODS
• Liposomes
• Oligonucleotides
• Dendrimers
• Amino acid
polymers
• Nanoparticles
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27. Gene guns-
• DNA particle bombardment by
gene gun
• Gold or tungsten spherical particles (1–3 μm
diameter) coated with plasmid DNA,
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28. DNA microinjection
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29. Electroporation-
• Disadvantage:
A high rate of cell death following electroporation
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30. Sonoporation-
• Ultrasonic frequencies  nanomeric pores in
membrane
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31. Magnetofection-
• Magnetic fields used to
concentrate particles
containing nucleic acid
into the target cells.
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32. CHEMICAL METHODS OF GENE
DELIVERY
• More common than physical methods
• Nanomeric complexes –
compaction of negatively charged nucleic acid
by polycationic nanomeric particles
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33. Liposomes (lipoplexes) –
• The nanomeric complex between a cationic liposome
and nucleic acids = lipoplex
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34. Oligonucleotides-
• Aim in gene therapy: to inactivate the genes involved
in the disease.
• Approaches:
o Antisense specific to target gene- to disrupt the
transcription of the faulty gene.
o Short interfering RNA (Si RNAs)  cleave specific
sequences in the mRNA transcript of the faulty
gene,  disrupting translation of the faulty mRNA.
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35. GENE THERAPY
APPLICATIONS
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36. DISORDERS FOR WHICH GENE
THERAPY HAS BEEN TRIED OR
CONSIDERED
Genetic diseases
1. Adenosine deaminase deficiency
2 . Cystic fibrosis
3 . Familial hypercholesterolemia
4 . Storage disorders - eg. Gaucher disease
5 . Coagulopathies - eg. Haemophilias A , B
6 . Haemoglobinopathies - Β thalassaemia , sickle cell
disease 36

37. Acquired diseases
1 . Cancer - eg. Melanoma , brain & renal tumors
2 . AIDS
3 . Vascular disease
4 . Neurological disorders - Parkinson disease
Alzheimer disease
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38. GENE THERAPY IN CANCER
Strategies –
1. Immunogene therapy:
Mking cancer cells immunogenic.
2. Suicide Gene therapy –
• converts prodrugs of cytotoxic drugs to active
compounds  lethal to tumor cells.
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39. 4. Gene therapy with Antisense oligonucleotide
• Antisense RNA hybridizes with sense RNA.
5. Gene therapy with chemoprotection Genes
• Transfer of drug resistance genes into
hematopoietic stem cells
6. Gene therapy with tumor suppressor gene
• Reintroduction & expression of wild-type p53 into
p53 altered tumor cells 39

40. CARDIOVASCULAR DISEASE
• Gene transfer into vascular cells (endothelial) &
smooth muscle
 Prevent restenosis
 Thrombolysis
 Hypertension
 Ischemic heart disease
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41. GENE THERAPY IN CNS
 Gene-based therapies,
 Neurotrophic factors (NTFs),
 Nervous-system growth factors,
 Stem cells,
 Novel vaccines,
• potential to prevent cell loss & degeneration in
brain,
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42. PROS AND CONS OF GENE
THERAPY
 Pros:
Gene therapy is a “medicine” for the future
- can wipe out genetic diseases before they can begin &
eliminate suffering.
 Cons:
1) Short-lived nature of gene therapy:-
have to undergo multiple rounds of gene therapy
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43. 2) Problems with viral vectors
3) Immune response
4) Multi-gene disorders
5) Insertional mutagenesis
6) Risk of procedure
7) Expensive. 43

44. 7) Ethical issues
• Recombinant Advisory
Committee (RAC)
• Misuse of gene therapy
- personality or physical enhancement
(Human genetic engineering)
• Religious issues ?
- Somatic cell therapy >> germline therapy 44

45. SUCCESS STORIES IN THE RECENT
PAST..
• world’s 1st gene therapy trial
for Leber’s Congenital
amaurosis, (mutation in
RPE65 gene.)
• Sub-retinal delivery of
recombinant AAV carrying
RPE65 gene - positive results
2007:
London’s
Institute of
Ophthal-
mology
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46. • Approved Glybera (Adipogene
tiparvovec) - lipoprotein lipase
deficiency
July
2012:
EMEA
• Gene Rx for Metachromatic
Leukodystrophy & Wiskott- Aldrich
Syndrome (7-32 months)
July
2013:
Italian San
Raffaele
Telethon
Institute
• 6 patients with choroidemia treated
with genetically engineered AAV
with a copy of REP1 gene –
improvement in sight
Jan
2014:
Universit
y
Of
Oxford
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47. BREAKTHROUGH 2017-2018!
 CAR – T immunotherapies: FDA-approved (2017)
- B-cell ALL (kids & young adults)
- Non-HL (adults)
• in those who don’t respond
to std Rx.
• Ongoing –
multiple myeloma,
glioblastoma
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48. • novel gene therapy treatment of Leber’s
congenital amaurosis.
• targets mutated RPE65 gene.
• subretinal injection
• 1st in vivo gene therapy approved by the FDA.
• not a cure. Improves vision.
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49. CRISPR-CAS9 COMING TO A HUMAN NEAR
YOU !
Clustered regularly interspaced
short palindromic repeats (CRISPR)
– Cas9
- used to edit genes within organisms
- goes into the nucleus and directly
cuts out faulty genes.
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50.  Trial to fix genetic defect in beta thalassemia
(CRISPR therapeutics Cambridge)
 Human trial for sickle cell anemia (Stanford
University) : FDA approved
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51. CONCLUSION
Gene therapy is the permanent solution for genetic
diseases.
• Both beneficial & harmful.
• Potential cure for deadly
diseases (AIDS, cancer etc)
• Ethical concerns –
human genetic
engineering
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52. Future prospects:
 Identify more efficient ways to deliver genes
 Develop more target-specific vectors,
(insert genes on the precise location)
 Ensure transplanted genes are precisely
controlled by the body’s normal physiologic signals
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53. REFERENCES
1) Najmul Hasan, Dr. Savita Saini, Gene therapy: Current status
and future perspectives / International Journal of Pharma Sciences
and Research (IJPSR)
2) Biju mammen, Ramakrishnan T, Uma sudhakar . Principles of
Gene therapy .Indian journal of pharmacology, 2010 ,18: 196-200 .
3) Yamamoto, M. & Tani, K. Current status and recent advances of gene
therapy in hematological diseases. Int J Hematol (2016) 104: 4
4) https://singularityhub.com/2018/01/09/gene-therapy-had-a-breakthrough-
2017-2018-may-be-even better/#sm.0001zyc2phb9crg10gp1gf7sm3x91
5) Rang & Dale’s Pharmacology, 8th edition
6) Goodman & Gilman’s The Pharmacological Basis of
Therapeutics, 12th edition
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