GENE THERAPY

ITS APPLICATIONS &

LIMITATIONS.

BY
AFTAB ALAM
JAMIA HAMDARD
 GENES
 Are carried on a chromosome.

 The basic unit of heredity.

 Encode how to make a protein.

 DNARNA proteins

 Proteins carry out most of life’s function.

 When altered, causes dysfunction of a protein.

 When there is a mutation in the gene, then it will change the codon
which will change the amino acid which will change the conformation
of the protein and subsequently will change the function of the
protein. (Genetic disorders result from mutations in the genome.)
 What is Gene Therapy
It is a technique for correcting defective genes that are responsible for disease
development.

There are four approaches:

1. A normal gene may be inserted into a
nonspecific location within the genome to
replace a nonfunctional gene.

4. An abnormal gene could be 2. The abnormal gene could

swapped for a normal gene be repaired through selective
through homologous reverse mutation, which returns
recombination.. the gene to its normal function.

3.The regulation (the degree to which a

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

AAV
Nucleus

Adenovirus

Therapeutic
Retrovirus/Lentivirus Protein

Target
Naked DNA
Cell
 The First Case
 The first gene therapy was performed on September 14th,
1990
 Ashanti DeSilva was treated for SCID

 Severe combined immunodeficiency

 Doctors removed her white blood cells, inserted the

missing gene into the WBC, and then put them back into
her blood stream.
 This strengthened her immune system.

 BY - W. French Anderson and

colleagues.
 GENE CORRECTIVE GENE
AUGMENTATION THERAPY

Random insertion of healthy Directing insertion of healthy

counterpart of defective gene gene at specific site to displace
somewhere in genome. defective gene is required.

No recombinational event Insertion at specific site would

required and non specific require some form of induced
insertion will work. recombinational event.
Suitable for recessive disorders Possible for dominant
and for single gene mutation. disorders.

Approach is feasible today. Extensive study is on.

 Types of gene therapy
 SOMATIC GENE THERAPY - therapeutic
genes are transferred into the somatic cells of a
patient(blood cells, liver cells,skin cells,lung cells)

 GERMLINE GENE THERAPY - germ cells,

i.e., sperms or eggs, (embryo) are modified by the
introduction of functional genes.
 SOMATIC GENE THERAPY V/s
GERMLINE GENE THERAPY
Changes confined to Changes will be passed to the
recipient. future generation.

No such problems associated. Technical and ethical

problems associated.

Any time of detection. Early diagnosis and therapy

required. (early embryo
stage)
Less as compared to germ cell High frequency of insertional
therapy. mutation.
 Strategies for gene delivery

Ex Vivo In Vivo
Cells removed from body Normal gene transferred
into the body directly.

Normal gene delivered

in cells cultured

Cells returned to the body

IDEAL GENE DELIVERY SYSTEM

 Biocompatible
 Non-immunogenic
 Stable in blood stream
 Protect DNA during transport
 Small enough to extravagate
 Cell and tissue specific
 TRANSFECTION TECHNOLOGY
 1) calcium phosphate
 2) electroporation
 3) microinjection
 4) liposome
 Retroviruses
Creates double stranded DNA copies from RNA genome

 The retrovirus goes through reverse transcription using reverse

transcriptase and RNA

 the double stranded viral genome integrates into the human

genome using integrase.
 integrase inserts the gene anywhere because it has no
specific site
 May cause insertional mutagenesis

 vectors used are derived from the human immunodeficiency

virus (HIV) and are being evaluated for safety ( also lentivirus)
 Picture 

http://encarta.msn.com/media_461561269/Gene_Therapy.html
 Adenoviruses
 Are double stranded DNA genome that cause
respiratory, intestinal, and eye infections in humans
 The inserted DNA is not incorporated into the
genome. 
 Has to be reinserted when more cells divide.
 Adeno-associated Viruses
 Adeno-associated Virus- small, single stranded DNA that
insert genetic material at a specific point on chromosome 19
 - causes no known disease and doesn't trigger patient
immune response. .

 hemophilia treatments, for example, a gene-carrying vector

could be injected into a muscle, prompting the muscle cells to
produce Factor IX and thus prevent bleeding.

 Study by Wilson and Kathy High (University of

Pennsylvania), patients have not needed Factor IX
injections for more than a year.
 Herpes Simplex Viruses
 Double stranded DNA viruses that infect
neurons
 Ex. Herpes simplex virus type 1
 NEW TECHNIQUES LIKE

 Hybrid method (virosomes)

 Dendrimers
 DNA nanoballs
 Trying to introduce a 47’th chromosome,
which would carry a lot of information. 
APPLICATIONS
 CANDIDATE DISEASE FOR GENE
THERAPY
DISEASE PATTERN OF MUTATED SYMPTOMS
INHERITANCE GENE
1) SCID X-linked ADA-gene loss of B and T
cells
2) CYSTIC Autosomal CFTR blocked airways of
FIBROSIS recessive lungs, pancreatic
insufficiency.

3)PHENYLKETO- Autosomal Phe-hydroxylase excess

NURIA recessive phenylalanine in
blood stream
causing mental
retardation.

4) HEMOPHILIA X-linked Factor-VIII defective blood

clotting.
5) DUCHENNE X-linked dystrophin progressive
MUSCULAR muscle wasting.
DYSTROPHY

6) SICKLE Autosomal β- globin chronic anemia,

CELL DISEASE recessive damage to spleen,
heart, kidney.

7) LESCH- Autosomal HGPRT-gene Gout, Kidney

NYHAM recessive disease
DISEASE

8) Autosomal Globin gene anemia

THALASSEMIA recessive

9) Huntington’s Autosomal huntingtin dementia

disease dominant
SCID – GENE THERAPY
Bubble boy, David, lived until removed from isolation, after a bone
marrow transplant to restore his immune system.
 Cystic Fibrosis
 An autosomal-recessive disorder in which thick, sticky
mucous covers the respiratory tract and digestive tract.
 Defective gene-CFTR on chromosome-7 which code for a
chloride channel.
 Skin is salty.

3. Gene structure (discovered in

1989)

Long arm of chromosome 7

Gene Therapy for Cystic Fibrosis
 GENE THERAPY FOR CANCER
Repair of cell cycle defects
 caused by loss of tumor suppressor gene

 e.g. p53 = DNA repair enzyme – “Guardian of the

genome”
 Mutations passed to progeny

 In most human cancers, the p53 gene appears

defective.
 Results from studies in experimental tumor models of

gene therapy for cancer and for certain viral

infections have been encouraging and suggest that this
may be a feasible and attractive alternative to
interferon protein therapy.
 GENE THERAPY FOR AIDS

 AIDS
 HIV patients  T lymphocytes treated ex vivo with rev
and env defective mutant strains of HIV
 Large numbers of cells obtained
 Injected back into patient
+
 Stimulated good CD8 cytotoxic T cell responses (Tcyt)

 By gene therapy we can also manipulate hematopoitic

stem cells with HIV resistant genes (RevM10 and

ribozyme gene against HIV ) by using retroviral
vectors system, to restrict viral growth and replication
GENE THERAPY IN ANIMALS
 Hemophilia A: clotting factor VIII has introduced in
Rat and Dog (Xu et al., 2005)
 Epilepsy: Type A receptors - In Rat
(Brooks et al, 2002 )
 Deafness: Atoh1 or Math1- Guinea pigs
(Izumikawa, 2003)
 Mastitis: lysostaphin gene in Jersey cattle
(Robert-J-Wall et al 2007)
 Blindness:GC1 gene in incubating egg
(Semple Rowland et al 2005 )
 Successful Gene Therapy
 SCID
 Successful One Year Gene Therapy Trial For Parkinson's
Disease
 RNA interference or gene silencing to treat Huntington’s
disease.
 Sickle cell successfully treated in mice
 To date 600 gene therapy clinical trials.
 Unsuccessful Gene therapy

 In 1999, 18-year-old Jesse Gelsinger died from multiple

organ failure 4 days after treatment for ornithine
transcarbamylase deficiency.
 Death was triggered by severe immune response to

adenovirus carrier.
 FDA hasn’t yet approved any

human gene therapy product

for sale due to this great
failure.
 LIMITATIONS
 Short-lived nature of gene therapy

 Immune response

 Insertional mutagenesis

 Multigene disorders

 viral vectors
 regulations associated with human exp.
 High costs associated with developing this novel
technology experimentation
 SOCIAL AND ETHICAL ISSUES

 Privacy & Confidentiality

 Psychological Impact

 Available to rich & powerful

 Playing with God’s creation

 Designer babies
 Accumulation of defective genes in future
generation.
 Probability of occurrence of new diseases
 GENE THERAPY – TWO STEPS
FORWARD OR TWO STEPS BACK ?

 PERHAPS WE SHOULD VIEW GENE THERAPY

AS WE HAVE ANTIBIOTICS, ORGAN
TRANSPLANT AND MAN FOR SPACE TRAVEL.
 REFERENCES
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http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg