GENETHERAPY

GENE THERAPY

Guided By:
Mrs.Arulmozhi.s

Co Guide:
Dr.S.L
Bodhankar
Prof. & head of
Dept of pharmacology Presented By:
Jyoti Arora
M.Pharm 1st year
 What genes can do?

• Genes which are carried on chromosomes

are the basic physical and functional units of
heredity.
• Genes are specific sequences of bases that
encodes instructions on how to make
proteins.

12 APRIL 2011 PCP,PUNE 2

 Why genetic disorders?

• When genes are altered so that encoded

proteins are unable to carry out their
normal functions,gentic disorders can
result.

12 APRIL 2011 PCP,PUNE 3

 Gene therapy
• Gene therapy is the insertion, alteration, or
removal of genes within an individual's cells
and biological tissues to treat disease.

• Gene therapy is a technique for correcting

defective genes responsible for disease
development.

12 APRIL 2011 PCP,PUNE 4

 Approaches for correcting faulty genes
• A normal gene may be inserted into a nonspecific
location within the genome to replace a nonfunctional
gene. This approach is most common.

• An abnormal gene could be swapped for a normal

gene through homologous recombination.

12 APRIL 2011 PCP,PUNE 5

 • The abnormal gene could be repaired
through selective reverse mutation, which
returns the gene to its normal function.

• The regulation (the degree to which a

gene is turned on or off) of a particular gene
could be altered.

12 APRIL 2011 PCP,PUNE 6

 TYPES

Germ line gene therapy- change due to

therapy would be heritable and would be
passed on to later generations.

Somatic gene therapy- effects will be

restricted to the individual patient

12 APRIL 2011 PCP,PUNE 7

12 APRIL 2011 PCP,PUNE 8
 What is vector?
• A vector is a carrier molecule which is used
to deliver the therapeutic gene to the
patient's target cells.

• Currently, the most common vector is a virus

that has been genetically altered to carry
normal human DNA.

12 APRIL 2011 PCP,PUNE 9

 Vectors used in gene therapy
 Viral vectors –
1. Adenovirus
2. Retrovirus
3. Adeno associated virus
4. Herpes virus
 Non viral vectors –
1. Liposome
2. Microsphere
3. Plasmid DNA

12 APRIL 2011 PCP,PUNE 10

 Retrovirus
 Enveloped viruses – single stranded RNA as genome
 Viral genome contains 3 genes –
• - gag - coding for core protein
• - pol - coding for reverse transcriptase
• - env - coding for viral envelop protein
 Viral vector – replace viral genes with transgene
 Infect only dividing cells – not able to cross nucleaer
membrane
 The retroviral envelop interacts with a specific cellular
protein to determine the target cell. By
altering ‘env’ gene, transfer of therapeutic gene to
targeted cell is possible.
12 APRIL 2011 PCP,PUNE 11
 Adenovirus
 Non enveloped viruses containing double
stranded DNA as genome.
 Viral genes – E1, E2 , E3, E4
 Infect dividing & non-dividing cells.
 Produce only temporary effect.

 Disadvantage –
• - Host immune response
• - Short lived expression

12 APRIL 2011 PCP,PUNE 12

12 APRIL 2011 PCP,PUNE 13
 Adeno-associated virus
 Non pathogenic virus containing single
stranded DNA as genome.
 Viral genome contains 2 genes –
• - Rep - controls viral replication
• - Cap - codes for capsid structural
protein
 Infect dividing & non-dividing cells.
 Gene is always ‘on’ hence protein is
continuously expressed even if it is not
needed.

12 APRIL 2011 PCP,PUNE 14

 Vector Advantages Disadvantages

Adenovirus Very high transfection efficiency ex Repeat dosing ineffective owing to

vivo and in vivo. Transfects strong immune response.
proliferating and non-proliferating Insert size limit of 7.5 kb.
cells. Sub-stantial clinical experience Manufacture, storage, QC are
acquired. Efficient retargeted moderately difficult. Short duration
transfection demonstrated. of expression.

Retrovirus Fairly prolonged expression high Low transfection efficiency in vivo.

transfection efficiency ex vivo.
Substantial clinical experience ex
vivo.
Low immunogenicity.
Insert size limit of 8 kb. Transfects
only proliferating cells.
Safety concern of insertional
mutagenesis. Manufacture, storage
and quality control are extremely
difficult.

Lentivirus Transfects proliferating and non- Safety concerns from

proliferating cells. Transfects immunodeficiency virus origins.
hatematopoietic stem cells. Manufacturing storage QC are
extremely difficult. Insert size limit
of 8 kb. No clinical experience

Adeno-associated virus(AAV)
Efficiently transfects wide variety of
cells in vivo. Very prolonged
expression in vivo. Low
immunogenicity.
Insert size limit 4.5 kb. Manufacture,
QC is very difficult. Little clinical
experience. Safety concern of
insertional mutagenesis. Repeat
dosing affected by neutralizing
antibody.

12 APRIL 2011 PCP,PUNE 15

 Non-viral vectors
 Liposome –
• - Lipid bilayer structure
• - Gene is present in inside core
• - Low efficiency , sometime cytotoxic

 Plasmids –
• - Directly injected into target cell
• - Attached to gold particles
• - Non efficient , low level of expression

12 APRIL 2011 PCP,PUNE 16

  Microsphere –
• - Biodegradable microsphere made from
polyanhydride copolymer of Fumaric &
Sebacic acid loaded with plasmid DNA.
• - EX. A plasmid with β – galactosidase
activity formulated by this technique when
given to rat by oral route, show systemic
absorption & expression of enzyme in liver,
raising the possibility of oral gene therapy.

12 APRIL 2011 PCP,PUNE 17

 How gene therapy works?
A vector
The target
vector’s
delivers
cells
genetic
the
therapeutic
become
material infected
is inserted
genewith
into
a patient’s
the
intoviral
the target
vector.
target
cellcell

Fun
ctio
nal
pro
tein
s
are
cre
ate
d
fro
m
the
the
rap
euti
c
gen
e
cau
sing
the
cell
to
ret
urn
to a
nor
mal
stat
e

12 APRIL 2011 PCP,PUNE 18

12 APRIL 2011 PCP,PUNE 19
 Problems and ethics

Short-lived nature of gene therapy

Immune response

Problems with viral vectors

Multigene disorders

Chance of inducing a tumour (insertional mutagenesis)

Religious concerns

12 APRIL 2011 PCP,PUNE 20

 Applications

Gene therapy for genetic disorders-

Cystic Hemophili
fibrosis a

Muscular
dystrophy SCID

12 APRIL 2011 PCP,PUNE 21

 Gene therapy for acquired disorders-

Parkinson’
s disease Cancer
Cardiovascul Neurologic
ar disorders al disorders

12 APRIL 2011 AIDS

PCP,PUNE 22
Disease Target cells Transfected gene(s)

Hemophilia A liver, muscle, Factor VIII

Hemophilia B bone marrow cells, fibroblasts Factor IX

Familial liver LDL receptor

hypercholesterolaemia

Severe combined bone marrow cells, T cells Adenosine deaminase

Immunodeficiency
(ADA)
Hemoglobinopathies red blood precursor cells a-globin, b-globin

Cystic fibrosis lung airway cells CFTR

Gaucher disease bone marrow cellsglucocerebrosidase

macrophages

Cancer tumor cells p53, Rb,interleukins,,

apoptosis genes
12 APRIL 2011 PCP,PUNE 23
Haemophilia-

A virus containing the gene

that produces Factor VIII or
Factor IV (in case of
Hemophilia B) is injected into
a large group of cells in the
patient.

12 APRIL 2011 PCP,PUNE 24

 SCID-
• Severe Combined Immunodeficiency
Disease (SCID) is due to a defective gene for
Adenosine Deaminase (ADA).
• A retrovirus, which is capable of
transferring it's DNA into normal eukaryotic
cells (transfection), is engineered to
contain the normal human ADA gene

12 APRIL 2011 PCP,PUNE 25

12 APRIL 2011 PCP,PUNE 26
Retinal gene therapy-
The replacement
gene is injected
between the two
layers of cells which
make up the retina. It
is a faulty gene in the
pigment layer which
is preventing the
photoreceptor cells
from detecting light.

12 APRIL 2011 PCP,PUNE 27

 Parkinson-

12 APRIL 2011 PCP,PUNE 28

Cancer-

12 APRIL 2011 PCP,PUNE 29

Type of cancer Tissue Gene

Brain tumors Tumor cells HS-tk

Brain tumors Tumor cells Antisense IGF-1

Breast cancer Fibroblasts IL-4

Breast cancer HSC MDR-1

Malignant melanoma T-cells TNF

Malignant melanoma Tumor cells TNF or IL-2

Ovarian cancer HSC MDR-1

12 APRIL 2011 PCP,PUNE 30

 Approaches for cancer treatment
1. Enhance the immunogenicity of the tumor by inserting
genes for: cytokines; allogeneic surface antigens.
2. Genetically alter immune cells to increase antitumor
efficacy by the insertion of: immune-stimulatory factors
(e.g., IL-2).
3. Insert a sensitivity or “suicide” gene into the tumor:HS-
tk/GCV or genes that disrupt DNA repair (e.g., antisense
DNA polymerase).
4. Block oncogene expression: antisense oligonucleotides.
5. Insert tumor suppressor genes.
6. Genetically protect tissues from the systemic toxicities of
chemotherapy: insertion of a MDR-1 gene.
12 APRIL 2011 PCP,PUNE 31
12 APRIL 2011 PCP,PUNE 32
Hepatic fibrosis-

• Telomerase gene delivery inhibit and

protect mice against CC14-induced hepatic
fibrosis.
• NF-қB inhibition by means of adenoviral
gene delivery of Ib suppressor reduces
early alcohol induced liver injury in rats.
• Other approaches-
1. Use of Ad vectors containing MMP8 gene
as therapeutic agent
2. Using Urokinase plasminogen activator
gene as a therapeutic agent.

12 APRIL 2011 PCP,PUNE 33

 Recent developments -
 Nanotechnology + gene therapy yields treatment to
cancer. March, 2009. The School of Pharmacy in London is
testing a treatment in mice, which delivers genes wrapped
in nanoparticles to cancer cells to target and destroy hard-
to-reach cancer cells.
 Antisense Oligonucleotide – Oligonucleotide’s are
complementary to gene or it’s product.It blocks the gene by
forming triplex with it. Also form complex with m-RNA &
block it.
• Certain enzymes present in cell are responsible for
destruction of oligonucleotide.
- Methylphosphorate analogue
- Phosphothiorate analogue
12 APRIL 2011 PCP,PUNE 34
 Scientists suceeded in using induced stem cells for gene
therapy for the treatment of sickle cell anaemia in animal
model.

12 APRIL 2011 PCP,PUNE 35

 References

 C. Calvo , Gene therapy of human sever combined

immunodeficiency (SCID) disease, science , 288(5466):669-
672
 D.Stone & A.David, Viral vectors for gene delivery & gene
therapy within endocrine system , Journal of endocrinology ,
103-118 , 2000
 H.P.Rang & M.M. Dale , Rang & Dale’s pharmacology ,
sixth edition , Elsevier publication , 773-780 , 2007
 K.D.Tripathi , Essential of medical pharmacology , fifth
edition , Jaypee brother medical publisher , 790-792 , 2004

12 APRIL 2011 PCP,PUNE 36

  M.A.Kay & J.C.Glorioso , Viral vectors for gene therapy :
the art of turning infectious agents into vehicle of
therapeutics , Nature medicine , Vol – 1, No. 1, 2001
 Mc cormact & P.Matthew , Activation of Tcells oncogene
LMO2 after gene therapy for SCID , New England journal
of medicine , 346 : 1185-1193 , 2002
 S.C.Rastogi & N.Mendiratta , Bioinformetics cocept , skills
& applications , first edition , CBS publisher , 24-48 , 2003
 S.M.Scholl & S.Michaelis , Gene therapy application to
cancer treatment , Journal of Biomedicine & Biotechnology,
35-47 , 2003

12 APRIL 2011 PCP,PUNE 37

 Thank
Thank you
you

12 APRIL 2011 PCP,PUNE 38