Gene Therapy and Stem cells

1. Gene Therapy and
Stem cells
DR.GANESH
CHALISE
2ND YEAR,RESIDENT
ORL AND
HNS,NAMS

2. What is gene Therapy ?
 Means of delivering exogenous genetic material
for therapeutic purposes into the host cell target
using vectors.
 Basic Principle: Intrinsic expressions of certain
genes in the body tissues can be modified to
treat disease.

3. Done by ..
 Replacing the defective gene with a functional version
Introduction of functioning ADA gene into the bone marrow of children with SCID
using a viral vector - reconstituting the patient’s immune system
 Enhancing the baseline expression level of a gene
Gene for TNF Introduced into TILs ex-vivo and when transfused into patient
preferentially migrates to residual tumor providing the therapeutic TNF.
 Suppressing the expression of genes that may contribute to the
pathologic process.
Introduction of RNAi by any means into a patient selectively turn off the gene
in disease(Cancer),blocking the mRNA translation.

4. HISTORY..
 1960’s : The concepts of Gene Therapy was introduced.
 1972 : Friedman and Roblin authored a paper in Science titled "Gene
therapy for human genetic disease.”
 1984: A retrovirus vector system was designed that could efficiently insert
foreign genes into mammalian chromosomes.
 1990: The first FDA approved gene therapy in the US took place on four
year old Ashanti DeSilva for a genetic defect that left her with ADA-SCID, a
severe immune system deficiency.

5.  1992: Doctor Claudio Bordignon performed the first procedure of gene
therapy using hematopoietic stem cells as vectors to deliver genes
intended to correct hereditary diseases.
 1999: Death of Jesse Gelsinger in a gene-therapy experiment
 2003 : a research team inserted genes into the brain for the first time
using liposomes.
 2006 : Successful use of gene therapy to treat two adult patients for X-
linked chronic granulomatous disease.
 2007: first gene therapy trial for inherited retinal disease

6.  2010 : an 18 year old male patient in France with beta-thalassemia major had been
successfully treated.
 2011: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has
cured a man from HIV using gene therapy (repeated haematopoietic stem cell
transplantation)
 July 2012, European commission approved alipogene tiparvovec(Glybera) ,an AAV
viral vector harbouring human lipoprotein lipase for familial lipoprotein lipase
deficiency disease.First commercial available gene therapy technology in Europe.
 Research is still ongoing and the number of diseases that has been treated
successfully by gene therapy increases.
 Most notable being gene therapy for ADA-SCID,B-Thalassemia.Sickle cell
disease,CFTR

7. As of 2012, some 1800 human clinical gene therapy trials (including phase I to
IV) exist world wide.The disease process involved can be broadely divided into
four groups (descending order)
 Cancer
 Monogenetic disease(CF,Haemophilia B)
 Cardiovascular disease
 Infectious disease (HIV)

8. Delivery
In the field of gene therapy,development of vehicle for the introduction of genetic
material into selected target cells has been a major focus.
Major barriers
 Targeting: only cells that require gene
 Binding and internalization (Transfection)
 Cellular trafficking to the nucleus(Transduction):Need to avoid endosomal
degradation
 Nuclear expression: Quantity and stability of the gene expression need to be
determined.
Different methods varies in ability in overcoming these barriers.

9. Methods
Vehicles of gene delivery are known as vectors
Ideal vector
 TARGET the right cells
 INTEGRATE the gene in the cells.
 ACTIVATE the gene.
 AVOID harmful side effects.
But, no universal vector exists.

10. Two general gene delivery methods(vectors) can be mentioned.
 Viral and
 Non-viral vectors

11. Non-viral vector
Most extensively studied method.
Includes
1) Plasmid mediated transfer of genes
 Gene can be effectively introduced into muscle and thyroid simply by injecting
DNA into tissues.
 Lipofection and gene gun methods- enhanced uptake of DNA in other tissues.
Advantages: Simplicity, Safe (do not alter cell’s genome),minimal immune response
Obstacle: Efficiency

12. 2 ) Direct delivery of RNA for gene silencing
IV administration of siRNA targeting VEGF gene has shown to reduce
significant tumor volume and intratumoral VEGF levels.
Lipofection: Liposomes significantly improves both the duration and level of
gene expression compared with non vector delivery methods.

13. Viral mediated gene transfer:
 Till date majority of the research has been focussed on developing
methods of using viruses as vectors.
 Viruses introduce their genetic material into the host cell as part of their
replication cycle
Involves construction of synthetic virus particles bearing the properties:
1.Lack pathogenic function
2.Incapable of replication
3.Contain a therapeutic gene within the viral genome and
4.Can deliver the genes to the cells by the process of infection.

14. Viral mediated gene transfer:
Advantages:
 Target specific types of cells. So, they're very good at targeting and entering cells.
 Can be modified so that they can't replicate and destroy cells.
 Certain virus can permanently integrate their genes into chrosomes of the
cell-Permanent gene therapy
 Can be introduced for both the dividing and non dividing cells
Disadvantage:
 Immune reaction,Toxicity or may cause disease inside.

15. Common viruses that have been used for human gene
therapy includes:
 Adenovirus
 Adeno-associated virus and
 Retrovirus/lentivirus

16. Retrovirus:
Original prototypes
 Useful properties:
Permanent gene therapy
Modifications can be made – Enhanced safety features
 Limitations:
Only integrate into actively dividing cells and efficiency is relatively low
Solution: lentiviral vectors
Most serious: Difficulty in achieving stable,regulated gene expression despite
permanent genomic integration.

17. Complications:
 Serious complications have been reported in clinical trials for X-linked SCID
 Cases of T-cell leukemia have been reported
Solution: Suicide gene inclusion in vector genome that will initiate cell death in
overproliferating cells.

18. Adeno Virus:
Powerful and effective vehicle for gene transfer.
Features:
 Unlike retrovirus the virus remain episomal, so the virus do not integrate
genes into target cells chromosome
Advantage
 Infecting both dividing and non dividing cells
 Both in- vivo and ex vivo with high level of efficiency.

19. Adeno-Associated virus:
Permanently integrates into target cell chromosome but unlike retrovirus can stably
infect both dividing and nondividing cells for prolonged periods.
Safe options:
 Apparent lack of pathogenecity
 Do not induce insertional mutagenesis or an innate immune response.
Disadvantage:
 Purified from the potentially cytotoxic,wild type helper virus.
 Limited capacity(4.8kb)

20. Strategies for administering gene
therapy:
Ex-vivo
 Cells isolated from surgical biopsy are
grown in culture,modified(genes inserted
using typically retroviral vector) and the
cells are reimplanted in the body via
autologous transplantation
 Creates a population of cells within the
body that permanently express a
therapeutic function
 Cells are treated outside.
 E.g.1st Gene Therapy done in a SCID
patient.
In-vivo
 DNA or viral vectors ,predominantly
adenoviral vectors are administrated directly
to patients.
 Intent can be single treatment for certain
tumors,administered intermittently in
response to acute disease or given long term
to establish steady state level of therapeutic
gene product.
 E.g. Adenovirus mediated replacement of
defective CFTR gene in CF.

21. Ex-vivo In-vivo

22. Gene Therapy: Types
1) Somatic vs germ cell gene therapy based on possible target cells and
1) Permanent vs Temporary gene therapy.

23. Somatic cell Gene Therapy
 Genes transferred to somatic cells
(Bonemarrow cells,Blood cells,Skin cells)
 Not inherited to later generations
 At present all researches directed to
correct genetic defects in somatic cells.
Embryonic cell Gene Therapy
 Genes transferred to germ cells
( Eggs and sperms)
 Inherited to future generation
 For safety, ethical and technical
reasons, it is not being attempted at
present
 Genetic manipulation of germ cells prohibited under
existing recombinant DNA guidelines.

24. Permanent Gene Therapy
 More desirable when the therapy involves
significant surgical procedures or substantial
risks( like organ resection)
 Requires tight control and regulation of
transferred genetic material and
 Not feasible with present techniques.
(growth hormone deficiency,juvenile diabetes)
Temporary Gene Therapy
 Beneficial for diseases like cancer, arthritis, CF
and disorders that require surgery.
 Demand is limited, one time gene expression or
repeat gene transfer needed over period of
time could prove effective
 Noninvasive routes like IM.IV or even aerosol
administration allow steady-state gene product
levels

25. Why Gene Therapy??
New Therapeutic approach:
 Novel approach to disease that are not satisfactorily managed using
conventional pharmacologic or surgical intervention.
E.g. Gives alternative to allogenic transplantation of bone marrow,solid
organs,or individual cells.
Site specific gene expression:
 Therapeutic products can be released from specific cell types in precise
locations within the body.

26. Hurdles..
 Short lived nature: need multiple treatment
 Immune response:
 Viral vectors: Toxicity,gene control and targeting tissues
 Multigene disorders: Like Heart disease,Diabetes. Complicates gene therapy
 Insertional Mutagenesis:T cell leukemia during trial for X-SCID.
 Cost: Alipogene tiparvovec or glybera,famed as “million dollar drug” cost 31 million
Euro as of 2015 per trearment.
Deaths: Three deaths have been reported
 1st Jesse Gelsinger 1999 because of immune rejection response.
 2nd X-SCID patient died of leukemia in 2003.
 3rd in 2007, a rheumatoid arthritis patient died from an infection

27. Why Gene Therapy??
Improved efficacy and safety:
 Establishes the expression of normal human proteins in a directed
therapeutic fashion within the body maintaining efficacy and safety.
Improved route of administration and compliance:
 Provides continuous endogenous expression of natural protein products
and requires less frequent administration (of gene) ranging from one time
to weekly or monthy treatment depending upon disease target and
transfer strategy.

28. Why Gene Therapy??
Preventive medicine and reduction in health care costs:
 Gene Therapy can establish continuous release of a therapeutic product
with one time treatment or infrequent dosing
 Its use for prevention is more practical and affordable than conventional
therapies.
Atherosclerosis,cancer,diabetes,infections or degenerative diorders.

29. Application of Gene Therapy in
OTOLARYNGOLOGY
 Relatively new.
 Inherited Disease:
Includes sinus disease in CF, hearing loss in Usher,Pendred or Alport syndrome and
goiter in congenital hypothyroidism.
First clinical trial for CF involved introduction of normal CFTR gene into nasal mucosa
using adenoviral vectors that provided foundation for the studies in which the CFTR
gene would be replaced throughout the respiratoty tract using viral or DNA vectors.

30. Application of Gene Therapy in
OTOLARYNGOLOGY
Head and Neck ONCOLOGY
 Melanoma,Squamous cell carcinoma
Approaches:
Immune Modulation Approaches:
1.Genetic modification of Tumor-Infiltrating Lymphocytes
 Gene therapy involves transfer of the gene for IL-2 directly into a patients tumor
resulting in local formation of tumor specific cytolytic TIL cells.Il-2 increases the
immunogenesity of cancer cells and suppress the tumor growth.

31. 2. Direct In Vivo stimulation of a Antitumor Immune Response:
 Introduction of genes for various cytokines directly into tumor cells to increase the
natural immune response to tumor specific antigens.
In head and neck cancer animal model,use of adenoviral mediated delivery of the
cytokine IL-2 in combination with a cytotoxic gene –synergistic effect on tumor
regression.
Note:In a phase I clinical trial patients received TNFerade(2nd generation adenoviral vector with
TNFa gene inserted) in addition to hysroxyurea,5-FU,and reirradiation-response achieved 83% of
patients,although survival remained very poor in study cohort.

32. Suicide gene Therapy:
 Infecting tumor cells by direct injection of a gene with retrovirus that encodes
enzymes like thymidine kinase - making them vulnerable and susceptible to
chemotherapy.

33. Gene augmentation strategies:
Modifying oncogenes and tumor suppressor genes:
 TP53 and CDKN2A mutation - Head and NECK cancer.
Growing evidence from phase I and II trials, significant benefit from
mediated TP53 gene therapy
 Use of novel dominant negative Adenovirus vector that encodes a mutant RAD50
gene, causes downregulation of MRN expression leading to sensitization of
SCC cells to cisplatin with increased apoptosis.

34. Gene suppression strategies:
Gene silencing:
 Selective turning off or silencing genes to prevent development or progression of disease.
 Study says,silencing of EGFR gene (HNSCC) using antisense(plasmid mediated) technique
causes greater cytotoxic effect than conventional therapy.
 shRNA use in silencing cyclin D1 gene markedly sensitizes (HNSCC) cells to cisplatin in vitro
 0n Targeting E6 and E7 oncogenes(upregulation of P53 and PRb) – causes cytotoxic effect
HPV related oropharyngeal carcinoma.

36. Inhibiting angiogenesis:
 Introduction of the gene that inhibit angiogenesis in vicinity of a tumor-
regression.
E10A-Adenoviral vector with gene that encodes endostatin - Inhibit
angiogenesis

37. Oncolytic viruses:
Early studies- viral vectors were replication incompetent
 Now,genetically engineered to selectively infect and replicate in targeted tumor
cells that have inherent genetic defects such as loss of TP53 gene expression
leading to tumor kill.
 Onyx-015(adenovirus): First replication selective viral vector on human trial.
Key-deletion of P53 binding protein gene that causes selective replication in
tumor cells lacking P53 expression causing lysis of target cells and spread to nearby
cells. More benefit on combination therapy .

38. Plastic and Reconstructive Surgery
Principle:
 Expression of growth regulating factors to enhance repair or regeneration of
damaged tissues and to enhance local proliferation to fill surgical defects.
Reconstrcutive tissue flaps and wound healing
 Gene encoding angiogenesis factor(bFGF),cytokines and growth factors(TGF-a,IGF-
1-2,PDGF) promotes tissue repair and regeneration.

39. Plastic and Reconstructive Surgery
Skin grafting :
 Feasibility of cultivating epidermal cells ex-vivo and subsequently engrafting the cells
Nerve Injury:
 viral vectors(Adenovirus,lentivirus,herpes simplex) can be used to prevent death of
motoneurons and enhance axonal regeneration
Optimizes bone growth:
 In case of removal of bone and soft tissue loss in extensive HNSCC
Repair and regeneration of irradiated tissues by enhancing viability and vascularity

40. Laryngology
 IGF-1 with its neutropic and myotropic effects holds potential for
optimizing function after acute laryngeal injury or for augmenting surgical
re innervation of the paralysed larynx.

41. Otology AND Neurotology
Experimental stage.
 Studies have demonstrated that adenoviral delivery of gene Atoh1 gene to
inner ear of guinea pigs result in production and innervation of inner ear
cells.

42. Stem Cells

43. Stem cells
 Unspecialized & undifferentiated cells that are able to develop into any
specialized type of cells.
Features:
 Self renewel
 Potency

44. Types:
 Embryonic Stem cells
 Somatic or adult tissue specific Stem cells
 Induced Pluripotent stem cells

45. Adult tissue specific Stem cells:
 Immature cell that is capable of dividing indefinitely both to renew itself and
produce cells maturing for tissue function.
Found in most normal and malignancy tissues.

46. Multipotent:
 Produce many types of cells that in one family e.g. Hematoid stem cells (make
types of the blood & lymph cells
Oligopotent:
 Can produce Few types of cells•e.g. lymphoid stem cells (make many types of
lymphoid cells like T, B, Nkcells
Unipotent:
 can produce one type of cells e.g. Muscle stem cell

47. Multipotent Stem cells

48. Embryonic Stem Cells
Totipotent stem cells(a.k.a.
omnipotent)
 Stem cells can differentiate into
embryonic and extraembryonic cell
types,like the Amnion, Yolk sac, Chorion,
placenta, Umbilical Cord
 The Zygote & the cells produced by the
first few divisions of the fertilized egg
are also totipotent (Zygote divides into
2, 4, 8, 16 cells ”morula”)
Toti: means ‘whole’as they make the whole
organism
Pluripotent Stem cells
 Stem cells are the descendants of totipotent
cells and can differentiate into nearly all
cells (except the extraembryonic cell )
 The inner cell mass, Bilamenlar &
Trilamenlar germ disc
 The differentiate into more than 200 types
of cells
Pluri: means ‘Several’

49. Induced Pluripotent stem cells (iPSCs)
 Induced expression of stem cell genes such as Oct4,Sox2,cMyc and Klf4 in
differentiated cells results in acquisition of stem cell properties similar to
those of Embryonic Stem cells.
 Potential clinical use for replacement of tissues that are genetically
defective or have been lost to damage or disease.

50. Application of stem cells:
Otolaryngology
The ear and nose:
 Engineered cartilage grafts generated from cartilage stem cells in biopsies
from nasal septum can be used for reconstruction.
Cochlea damage or degeneration:
 Loss of inner hair cells is currently irreversible but certain stem cells
procedure has been reported to generate hair cells and associated nerve.

51. Trachea:
 Small defect-Autologous cartilage graft
 Extensive tissue loss(malignancy):Tissue engineering can be used to generate
tracheal tissues from autologous stem cells.
Larynx and vocal cords:
 Regenerative medicine has the potential to enhance VF recovery beyond that
associated with conventional treatment.
Other tissues:
Muscle derived Multipotent Stem Cells have been shown to improve outcomes
facial and RLN injuries.

52. Thank you ……….