Gene therapy

A power point presentation on gene therapy for medical dental pharmacology and biotech students to facilitate easy learning .

1. Gene Therapy
Dr. Rohini C Sane

2. Gene Therapy
•Gene Therapy:
1.is the process of inserting genes into cells to treat diseases.
2.is a means of replacing defective genes with good ones and cure
the genetic disorders.
3.involves genetic manipulations in human to correct diseases and to
improve their health. Thus ,there is intracellular delivery of genes to
generate therapeutic effect by correcting an existing abnormality.
•Gene augmentation therapy: a DNA is inserted into the genome
to replace the missing gene product.
•Gene inhibition therapy :the anti-sense gene inhibits the
expression of the dominant gene.

3. Vectors in gene therapy
•Vectors used in gene therapy:
1.Viruses
2.Human artificial chromosomes
3.Bone marrow cells

4. Gene delivery by viruses
•Viruses used as vectors(carriers) for gene therapy :
1.Retroviruses
2.Adenovirus
3.Adeno- associated viruses
4.Herpes simplex virus
Limitations of viral vectors in gene therapy : Viral vectors
induce inflammatory responses in the host.

5. Viruses as vectors in gene therapy
Most frequently used viruses in gene therapy: retroviruses
1.Entry of genetic material of retrovirus in the host cell.
2.synthesis of DNA (provirus)from RNA by reverse
transcription.
3.Multiplication of provirus( normally harmless)
Risk of the technique: some retroviruses can convert normal
cells into cancerous ones. Such thing should be avoided.

6. Retroviruses as vectors
1.Retroviruses are RNA viruses that replicate through a DNA intermediate.
2.Moloney murine leukemia virus(MMLV) is commonly used.
3.The gag, pol ,env genes are deleted from wild type retrovirus,rendering it
incapable of replication inside human body.
4.human gene is inserted into virus.
5.This is introduced into a culture containing packaging cells having gag, pol
and env genes.
6.These cells provide the necessary protein to pack the virus.
7.The replication-deficient ,but infective ,retrovirus vector carrying the human
gene, now comes out of cultured cells.
8.These cultured cells are introduced into the patient.
9.The virus enters into the target cell via specific receptor.
10.In the cytoplasm of the human cells ,the reverse transcriptase carried by the
vector converts the RNA to proviral DNA, which is integrated into the target
cell DNA. The normal human gene can now express.

7. Gene transfer by retroviral vector
Retrovirus (wild type) RNA
pol LTRgagLTR env
LTR LTR
Delete gag ,pol ,env genes. In that site add cassette containing human gene
Virus particle with protein coat .
These are infective (can enter
into human cells ),but cannot
replicate or produce disease.
Receptor mediated entry of
retrovirus into human host cell.
Human gene
Human gene
Human cell
cytoplasm
Human gene
integrates with host DNA inside nucleus
Reverse transcriptase of virus RNA to virus DNA
Nucleus
Naked viral RNA has to get protein coat to enable them to enter into human host
cell.So defective virus is put into packaging cells, which provide the viral coat.

8. Advantages of retroviruses as vectors
•virus is modified and replication deficient.
•So infection with viral particle is limited to one cycle
and is very safe .
•They can infect wide variety of human cells .
•This strategy is very suitable for treatment of all
diseases produced by single gene mutation.

9. Disadvantages of retroviruses as vectors
1.Retrovirus requires dividing cells as the targets.
2.It allows only low titres of virus to be generated.
3.the length of the gene inserted should be less than 25kbp.
4.Illegitimate combination (the gene getting inserted at
undesirable sites) and insertional mutagenesis are
possibilities.

10. Adenoviruses
•Adenoviruses :
1.are DNA viruses
2.functions as a carrier of the human gene reaches nucleus of
target cells.
3.is not integrated ,but remains as epichromosomal (episomal)
Advantages of Adenovirus as a vector: they can carry longer genes
of higher titres.
Disadvantages of Adenovirus as a vector: the expression is
transient , the useful effect varying from a few weeks to months only.

11. Gene delivery by non-viral system
•Pure DNA constructs : that can be directly introduced
into target tissues.
•Lipoplexes (liposomes): lipid -DNA complexes that have
DNA surrounded by lipid layers.
•Plasmids:
•Human artificial chromosomes: which can carry large
DNA(one or more genes).

12. Plasmid liposome complex
1.is a non viral vector system.
2.Liposomes are artificial lipid bilayers which could be
incorporated with plasmids carrying the normal human DNA.
3.These complexes enter the target cells by fusing with the
plasma membrane.
4.Cationic liposomes (positively charged) can form complexes
spontaneously with DNA(negatively charged).
•Advantages : the vector can carry human gene of big size ,do not
replicate and evoke only very weak immune responses.
•Disadvantages: the most complexes are destroyed inside the
host cell, and so the efficiency of gene transfer is less.

13. Gene Transfer by plasmid liposome
Plasmid carrying normal human gene is inserted into liposome
Plasmid -liposome complex enters the host cell by fusion of membranes .
It will not enter into nucleus,but remains as epichromosome.
Expression of normal gene

14. Gene Gun method
•Tungsten particles are coated with plasmid DNA and
accelerated by helium pressure discharge.
•This enables particles to penetrate the target tissues.
•It is quick and could be used in almost all tissues.
•Cellular damage and transient gene expression are the
drawbacks.

15. Combining stem cells and gene therapy
•Patients with chronic liver cirrhosis (HCV or HBV
infection) conventionally treated by a liver transplant or
transplantation of mature hepatocytes.
•However, the transplanted organ or cells are likely to
become reinfected by the hepatitis virus.
•To avoid this, gene transfer of a vector encoding short
hairpin RNAs(shRNAs) directed against virus would
make transplanted cells resistant or immune to
reinfection.
•The resistant cells can repopulate the liver over time and
restore normal liver function.

16. Summary of the procedure of gene therapy
1.Isolate the health gene along with the sequence
controlling expression.
2.Incorporate this gene into a carrier or vector as an
expression cassette.
3.finally deliver the vector to target cells.

17. Approaches for gene therapy
Somatic cells : The non-reproductive (non-sex) cells e.g.
sperm or egg cells ,bone marrow cells, blood cells ,skin cells,
intestinal cells
Two approaches for gene therapy:
1.Somatic cell gene therapy: involves the insertion of a new fully
functional and expressible gene into a target somatic cell of the
patient under trial to correct a genetic disease permanently.
2.Germ cell gene therapy: the reproductive (sex) cells of an
organism constitute germ cell line.The introduction of DNA by
this therapy into germ cells is passed to the successive
generations. It is consider as unethical.

18. Defective gene
Two major approaches of gene Therapy
Gene augmentation therapy Gene inhibition therapy
functional gene
Antisense gene
Dominant functional gene
inhibitory action

19. Human gene therapy
Disease Gene therapy
severe combined immunodeficiency (SCID) Adenosine deaminase
Cystic fibrosis(CF)
Cystic fibrosis transmembrane
regulator (CFTR)
Familial hypercholesterolemia Low density lipoprotein (LDL)receptor
Emphysema alpha1-Antitrypsin
Haemophilia FactorVIII and IX
Thalassemia Alpha or Beta Globin
Sickle cell anaemia Beta -globin
Lesch Nyhan syndrome Hypoxanthine-guanine
phosphoribosyl transferase (HGPRT)
Gaucher’s disease Glucocerebrosides
Peripheral artery disease Vascular endothelial growth factor
(VEGF)
Falconi anemia Falconi anemia C
Melanoma Tumor necrosis factor (TNF)
Leber’s hereditary optic neuropathy isomerohydrolase

20. Human gene therapy
Disease Gene therapy
Melanoma ,renal cancer Interleukin -2(IL-2)
Glioblastoma (brain tumour ),AIDS ,
ovarian cancer
Thymidine kinase( Herpes simplex
virus)
Head and neck cancer p
53
(tumor suppressor gene)
Breast cancer multi drug resistance I
AIDS rev and env
Colorectal cancer, melanoma ,renal
cancer
Histocompatibility locus antigen-B7
(HLA-B7)
Duchenne muscular dystrophy Dystrophin
Short stature Growth hormone
Diabetes
Glucose transporter -2, (GLUT-2),
Glucokinase
Phenylketonuria Phenylalanine hydroxylase
Citrulllnemia Arginosuccinate synthesise
mostly confined to animal experiments

21. Types of gene therapies
•Ex vivo gene therapy: this involves the transfer of
genes in patient’s cultured cells (e.g. bone marrow
cells) in the laboratory.The new genes are infused into
the cells . The modified cells are then reintroduced /
administered into the patients.
•In vivo gene therapy : The direct delivery of genes in
the form of vector into the cells of particular tissue.
e.g. cystic fibrosis gene to respiratory tract cells.
•In situ therapy : when the expression cassette is
injected to the patient either intravenously or directly
to the tissue.

22. Technique of Ex vivo gene therapy
1.isolate cells with genetic defect from patient.
2.grow the cells in culture.
3.introduce therapeutic gene to correct gene
defect.
4.transplant the modified cells to the patients.

23. The technique for ex vivo gene therapy
Patient with a genetic defect
isolated lymphocytes
In vivo culture
Therapeutic gene construct
Genetically transformed cells selected
Transplantation

24. The technique for ex vivo gene therapy using retrovirus as a vector
Patient with a genetic defect
isolated cells from patient
cells with defective gene
Retrovirus carrying normal gene
Normal gene is introduced into patient’s cell by the virus
Patient’s cell now contains a normal gene
cells with normal gene is injected
back to patient

25. Ex vivo gene therapy
1.can be applied to only selected tissue(e.g. bone
marrow) whose cells can be cultured in the
laboratory.
2.is not associated with adverse immunological
responses after transplanting the cells.
3.is efficient only if the therapeutic gene (remedial
gene) is stably incorporated using vectors and
continuously expressed.

26. Human artificial chromosomes as a vector for gene therapy
•Human artificial chromosome (HAC): is a synthetic
chromosome that can replicate with other
chromosomes , besides encoding a human protein.
•Risk of converting normal cells into cancerous ones
( common when retroviruses are used as a vector)can be
avoided if HAC is used.

27. Bone marrow cells as a vector for gene therapy
Totipotent embryonic stem cells of bone marrow capable of
dividing and differentiating into various cell types (e.g. red
blood cells, platelets , macrophages. osteoblasts , B-
lymphocytes ,T- lymphocytes.
It is the most widely used technique for several genetic
disorders.

28. Gene therapy for the
enzyme Adenosine
deaminase deficiency
Dr.Rohini C Sane

29. Stem cells
•Stem cells : defined as cells with the capacity for self-renewal and having
potential to differentiate into progenitor of different lineages which
ultimately give rise to mature tissues.
•Mario Capecchi ,Sir Martin Evans and Oliver Smithies(Nobel 2007): for
introducing specific gene modifications in mice by the use of embryonic
stem cells
•Sources of stem cells : embryos, umbilical cord ,adult bone marrow
•Developmental plasticity : ability of stem cells to divide for an indefinite
period and give rise to a variety of specialised cell type.
•Plasticity : as the ability of stem cells from one germinal layer to give rise
to tissues of germinal layer and is more for embryonic cells.
•Applications of stem cell therapy: treatment of cancer and coronary artery
disease

30. Types of stem cells
Stem cells :
•have capacity to produced unaltered daughter cells (renewal).
•also to generate specialised cells (potency).
•totipotent : capable of producing all types of cells of organism .
•pleuripotent: able to generate cells of three germ layers .
•multipotent :able to produce only closely related cell types.
•unipotent : may produce only one cell type .
•embryonic type : capable to differentiate.
•adult type : limited capacity to differentiate.
•reside in a special microenvironment called niche .

31. Adenosine deaminase (ADA)deficiency
ADA gene is expressed in variety tissues but in cytoplasm of
human lymphocytes enzyme has the highest activity.
ADA deficiency
accumulation of adenosine
conversion of adenosine to its ribonucleotide or deoxyribonucleotide
increase in concentration of dATP
Inhibition of ribonucleotide reductase
Decrease in synthesis of dATP,dGTP,dCTP, dTTP
severe combined immunodeficiency (SCID)
cellular kinases

32. Severe combined immunodeficiency(SCID)
•Severe Combined immunodeficiency (SCID):
1.is rare inherited (autosomal recessive)immune disorder associated with T-
lymphocytes (to lesser extent B-lymphocytes dysfunction)and natural killer cells
dysfunction.
2.is due to genetic defect in the gene located on chromosome 20 and gene has
32,000 base pairs with 12 exons that code for ADA.
3. Deoxyadenosine and its metabolites primarily deoxyadenosine 5’ triphosphate
accumulate and destroy T-lymphocytes . Accumulation of dATP and adenosine
in ADA deficiency lead to developmental arrest and apoptosis of lymphocytes.
T-lymphocytes participate directly in body’s immune response and also promote
the function of B-lymphocytes to produce antibodies.
The patient with ADA deficiency suffer from infectious diseases and die in young
age(two years ). ADA deficiency is prevalent In USA (14% of total SCID cases).
Previously , the children suffering from SCID were treated with conjugated bovine
ADA ( enzyme replacement therapy - ERT)or bone marrow transplantation(BMT).

33. The first clinical trial of Gene therapy
for Adenosine deaminase deficiency
Gene therapy for Adenosine deaminase deficiency :
•the first gene therapy (14th September 1990) by a
team of workers led by Blaese and Anderson at
National Institute of health ,USA.
•Patient’s name : Ashanti (4years old then)

34. Technique of gene therapy for ADA deficiency
1.selection of plasmid vector bearing a proviral DNA.
2.replacement of a part of proviral DNA by ADA gene and a gene (G 418) coding for
antibiotic resistance and then cloned.
3.selection of desired clones with ADA gene with the help of antibiotic resistance
gene.
4.Removal of circulating lymphocytes from patient suffering from ADA deficiency.
5.exposure of lymphocytes to billions of retroviruses carrying ADA gene to facilitate
their trans-infection.
6.growth of genetically modified lymphocytes in culture.
7.confirm the expression of ADA gene.
8.return transfected lymphocyte cells to the patients.
These lymphocytes persist in the circulation and synthesise ADA. Consequently ,
ability of patients to synthesise antibodies is increased.Transfusions have to be
carried out frequently as lymphocytes have short life span (just live for few months).

35. Transfer of ADA gene into stem cells
•ADA gene was transferred into the stem cells obtained
from umbilical cord blood ,at the time of baby’s
delivery.
•Infant received these genetically modified stem cells
back four ays after birth.
•A permanent population of ADA gene producing cells
was established .(in 1995 for the first time)

36. Ex vivo gene therapy for the patient with severe
combined immunodeficiency (SCID) caused by
lack of synthesis of adenosine deaminase
1.separation of lymphocytes from other cells in blood of the patient with
SCID.
2.Infection of the lymphocytes carrying defective gene with a modified
retrovirus carrying the normal gene for adenosine deaminase.
3.integration of the normal gene into chromosome of patient lymphocyte
cell.
4.Expression of the normal gene to synthesise adenosine deaminase in
some of the patient’s lymphocytes.
5.transfer of modified lymphocytes to patient’s blood circulation .
6.Immune function of the patient regained.

37. Treatment of Adenosine deaminase
(ADA)deficient patient by ex vivo gene therapy
Schematic diagram
Child with SCID
(deficient in ADA gene)
Isolation of lymphocytes
Vector DNA
Retrovirus containing ADA+ gene
Human ADA+
gene
Vectors
Transfection
Lymphocytes with viral DNA and ADA+ gene
cell culture to verify expression of ADA+transgene
synthesis of ADA
Correction of SCID
Infuse
lymphocytes
with ADA+
gene
expression into
patients

38. Gene Therapy
Normal gene
Retrovirus
Lymphocyte from patient with a normal gene
reverse transcription
Infection of a lymphocyte with a modified retrovirus carrying a normal gene
Lymphocyte from patient
with the defective gene
defective
gene
Schematic diagram

39. SCID and PNP
Purine nucleoside phosphorylase (PNP) deficiency
results in less severe immunodeficiency involving
T- lymphocyte cells primarily.

40. In vivo gene therapy
In vivo gene therapy: direct delivery of therapeutic
(DNA) gene into target cells of particular tissue of a
patient .
Potential tissue for in vivo gene therapy: liver ,
muscles ,skin, spleen, lung ,brain, and blood cells
Gene delivery : viral or non-viral systems

41. Diagrammatic representation of in vivo gene therapy
(p-Promoter gene) specific for therapeutic gene
Schematic diagram
P
Therapeutic gene
Patient
Target tissue

42. Factors governing outcome of in vivo gene therapy
•The efficiency of the uptake of the remedial (therapeutic) gene
by target cells.
•Intracellular degradation of the gene and its uptake by nucleus.
•The expression capacity of the gene.

43. Gene therapy strategies for cancer
Cancer treatment strategies :
1.surgery
2.chemotherapy
3.radiation therapy
4.gene therapy( latest therapy)

44. Tumor necrosis factor(TNF) gene therapy
•Tumor necrosis factor (TNF):
1.protein produced by human macrophages
2.provides defence against cancer cells
3.enhances ability of tumor infiltrating lymphocytes (TILS), a special
type of immune cells.
4.highly toxic
5.gene (along with a neomycin resistant gene) and used for the
treatment of malignant melanoma( a cancer of melanin producing cells
usually occurs in skin).
No toxic effects were detected in the melanoma patients injected with
genetically altered TILS with TNF gene. Some improvement in the cancer
patients was observed.

45. Suicide gene therapy
The gene encoding the enzyme thymidine kinase:
1.referred as suicide gene.
2.phosphorylates nucleosides to form nucleotides which are
used for synthesis of DNA during cell division.
3.is used for treatment of certain cancers.

46. Prodrug activation gene therapy
The drug Ganciclovir (GCV) bears close structural resemblance
to certain nucleosides (thymidine).
Thymidine kinase phosphorylates Ganciclovir to form
triphosphate -GCV, a false and unstable nucleotide for DNA
synthesis . Triphosphate -GCV inhibits DNA polymerase.
Ganciclovir = prodrug
Application of Ganciclovir (prodrug activation gene therapy):
used for brain tumours (Glioblastoma, a cancer of glial cells
in brain) with a limited success.

47. Mechanism action of Ganciclovir
Inhibition of DNA polymerase by Triphosphate -GCV
The elongation of DNA polymerase abruptly stops at a point
containing the false nucleotide of Ganciclovir
Entry of triphosphate -GCV in cancer cells
Bystander effect (the neighbouring cancer cells killed)
Multiplication of cancer cells halted ,followed by their death by
Ganciclovir.

48. The action of Ganciclovir mediated by thymidine
kinase to inhibit the growth of cancer cells
DNA synthesis
Nucleoside Nucleotide Thymidine kinase
Ganciclovir
false nucleotide
DNA synthesis
blocked
Thymidine kinase
inhibits DNA
polymerase
Death of cancer cells
Phosphates

49. Gene replacement Therapy
p
53
:
1.a gene coding for a protein with molecular weight of 53 kilodaltons
(hence p
53
) .
2.is a tumor suppressor gene since the protein it encodes binds with
DNA and inhibits replication.
Mutated p
53
:
A.is

a

causative

factor in tumor development.
B.mutated form present in tumor cells of several tissue (breast , brain,
lung ,skin bladder ,colon bone) coding different proteins from the
original.

50. Antigene and Antisense therapy
•Cancer ,viral parasitic infections and inflammatory diseases result
from overproduction of certain normal proteins .
•Antigene therapy :
1.using a single-stranded nucleotide sequence (antigene
oligonucleotide) that hybridise with the specific gene thus,
blocking transcription.
2.is used to treat these diseases by blocking transcription.
•Antisense therapy :
A.using a single-stranded nucleotide (anti-sense oligonucleotide) to
inhibit of transcription and translation by blocking the transcription
factor responsible for the specific gene expression.

51. Antisense therapy for cancer
•Oncogenes :
1.are responsible for causation of cancer.
2.can be targeted in antisense technology by using antisense
transgenes or oligonucleotides.
Antisense oligonucleotides:
are used for the treatment of myeloid leukaemia(1991 for the
first time).
specifically bind to the target mRNA and block protein
biosynthesis(translation).

52. The cloned AS cDNA introduced into cells to
produce antisense RNA
•Antisense cDNA can be cloned and transfected into cells.
•Antisense mRNA is synthesised by transcription.
•This can readily bind with the specific mRNA and block translation.
•The mRNA is actually formed by gene containing exons and introns
through transcription ,followed by processing.

53. Inhibition of translation by antisense RNA
AS cDNA E1 E2I DNA
E2E1I
Transcription
Primary transcript
Processing
Antisense RNA
mRNA
E1E2
mRNA -antisense RNA complex
Transcription
No translation
Inhibition of translation by
antisense RNA:The cloned
AS c DNA introduced into
cells to produce antisense RNA.
AS cDNA =
Antisense complementary DNA
E1,E2 = Exons in a gene
I=Intron

54. Inhibition of translation by antisense RNA by
introduction into cells
The other way to block translation is directly introduce antisense
RNA into the cells . This hybridise with target mRNA and blocks
translation.

55. Inhibition of translation by antisense RNA
E1 E2I DNA
E2E1I
Transcription
Primary transcript
Processing
Antisense RNA
mRNA
E1E2
mRNA -antisense RNA complex
No translation
Inhibition of translation by
antisense RNA: Anti-sense RNA
directly introduced into cells .

56. Applications of Antisense mRNA therapy
•Applications of Antisense mRNA therapy: for treatment of
a brain tumour namely malignant glioma and cancer of
prostrate gland.
•Malignant glioma : overproduction of the protein insulin-like
growth factor I(IGF-I).
•Prostrate cancer : overproduction of the protein insulin-like
growth factor I receptor (IGF-IR).
•In both these cancers ,the respective antisense cDNAs can
be used to synthesise antisense mRNA molecules.

57. Peptide nucleic acid (PNA) therapy
•Peptide nucleic acids (PNA)s :
1.are artificial analogs of nucleic acid with a polypeptide
backbone.
2.possess standard nucleic acid bases attached to a
polypeptide in a place of sugar -phosphate backbone.
3.have been developed to inhibit translation of HIV viral
transcript ,gog -pol and to block translation of cancer
genes e.g. Has-ras and bcl-2.

58. Transgenic animals
•Transgenic animals can be produced by injecting a recombinant gene into
fertilised egg.
•If gene is permanently /successfully integrated into the chromosome of
fertilised egg , it will be present in the germline of the resulting animal .
This gene can be passed along from generation to generation .
•Super-mouse (a giant mouse) was produced by injecting a rat growth
hormone gene into fertilised mice egg.
•Transgenic cows and goats can be designed to produce human
proteins for therapeutic purpose( e.g. blood clotting factors in their milk).
•Knock -out mice : generated by inserting a non-functional version gene
into a mouse. If inserted gene expresses a mutated product / over /under
expressed product ,such genetically modified animals can serve as
models for the study of a corresponding human diseases.

59. Applications of Gene Therapy
1.Treatment of diseases : introducing a normal cloned DNA /gene in individual with a
defective gene e.g. sickle cell anaemia , thalassemia , adenosine deaminase
deficiency
2.Transgenic animals : introduction of cloned genes into the fertilised eggs to produce
transgenic animals that can develop normal offsprings. They can produce therapeutic
proteins(e.g.blood clotting factors) or can serve as a model for human diseases .
3.genetic concealing : is a device to prevent passing of defective genes to offsprings .
Recombinant DNA screening test is performed on the prospective parents prior to
conception and the foetus is tested for genetic defect. Treatment can be commenced
at an early stage even in utero if the foetus is defective .
4.Agriculture applications: plants that are resistant to disease , insects , herbicides ,
drought and temperature extremes or more efficient in fixing nitrogen are being
produced by gene therapy.
5.Industrial applications : production of enzymes ( to be used in detergents and
cheese).

60. Limitation of gene therapy
1.inconsistent results
2.lack of ideal vector
3.lack of targeting ability in non viral vectors
4.death of patient during the course of gene therapy for
Ornithine trans-carbamoylase deficiency(OTC)
5.Reactivation of retroviral vector due to illegitimate
combination of the inserted gene leading to leukaemia in
patient. This has posed setback in this field .

61. Gene therapy trial
A few patients treated by gene therapy for disease like
severe combined immunodeficiency (SCID-X1),
developed leukaemia and died. Presumably this is
because of activation of hematopoietic oncogene .
The gene introduced through a retroviral vector got
inserted at a site (illegitimate recombination) that lead to
activation of oncogenes that resulted in leukaemia.
At present trials are in progress ,but restricted with
stringent protocols and follow-up.

62. The future of gene therapy
•The Gene used for gene therapy :
1.should be harmless to patients.
2.is approximately expressed (too much or too little will not be good).
Gene therapy :
A.not simple but has several inbuilt complexities.
B.broadly involves isolation of a specific gene, making its copies,
inserting them into target tissue cells to make the desired protein.
C.is the body's immune system which reacts to the foreign proteins
produced by the new genes.
D.is the permanent solution for genetic diseases.
E.will revolutionise the practice of medicine!

63. Thank You