Gene Therapy

What is Gene Therapy?

Gene therapy is a technique for

introducing the genetic material of a gene
in a patient that lacks that gene because
of a mutation. (= Gene transfer)

It represents the Application of nucleic

acids for treatment of diseases

Gene therapy targets

In principle, any disease can be targeted for gene therapy. In

practice, we need to know the following
(1) Does the disease arise from a mutation in one (or a few)
genes?
(2) Has the mutant gene been identified ?
(3) Has the corrected gene been cloned?
(4) Where is the gene expressed (tissues)?
(5)Will a normal gene solve the problem (dominant negative
mutations)?
(6) Can we deliver a normal gene to the affected tissue?

Gene Delivery:
Gene delivery is the Key to successful gene therapy
Successful gene delivery involves:
(1) Targeting: repaired gene must specifically enter the
correct cells
(2) Activation: repaired gene needs to enter nucleus and
be successfully transcribed in response to regulatory cues
(3) Integration: for long term protection, the repaired gene
may need to integrate into the genome and be replicated
(4) No side effects: the introduction of any foreign
biological material runs the risk of being toxic, damaging
the cells or stimulating an immune response

Barriers for introducing Nucleic acid into cells/tissues

1- Crossing plasma membrane:
A- Cell membrane has a negative charge & would
block attachment of DNA/RNA to cell membrane.
B- Also DNA/RNA are hydrophilic & would block
direct fusion with lipid plasma membrane.
2- Release from phagosomes (Phagocytosis)
3- Dissociation of nucleic acid from the chemical
vehicle/carrier
4- Transportation to the nucleus (can be improved by
adding ORI sequence from SV40 virus to the nucleic
acid or adding to the chemical vehicle a protein with
nuclear localization signal,NLS,e.g.SV40

Vectors
The way you insert the normal gene in the
patients cell is by vectors.
Vector: A carrier for therapeutic nucleic acid
for its delivery to target tissues/ cells
Vehicle: A chemical substance, which
improves delivery of nucleic acid to the cells
The most common vectors that are used in
gene therapy are virus vectors

Transformation: is Introduction of foreign genes into prokaryotic cells

such as bacteria

Transfection: Introduction of foreign genes to eukaryotic cells by

means of plasmid or vectors
Transduction: Introduction of genes by means of viral vectors

Qualities of a suitable vector for gene therapy

1) An optimal vector should be one that can be made available in
a highly intense form using an ideal and reproducible production
plan.
2) The vector must protect the genetic material from degradation
by host.
3) Those vectors that can accomplish site-specific integration are
more preferred and desirable in case of insertional mutagenesis.
4) A perfect vector should be harmless and should not cause any
noxious effect on human health.

Why Viruses?

Viruses through the time of evolution have

evolved to infect the cells with great specificity
Viruses tend to be very efficient at transfecting
their own DNA into the host cell genome.
This allows them to produce new viral particles
at the period of synthesis of the cell

Types Of Viruses
Retrovirus
Adenovirus
Adeno associated virus
Lentiviruses
Poxviruses
and Herpes Viruses

Which Virus to Use?

Depends
how well they transfer the genes to cells
Mainly on which cells they can recognize and infect
and whether they alter the cells DNA permanently
(integration) or temporarily (Transient)
Whether cells are dividing or not?

Viral vectors have natural host cell populations that they infect most
efficiently. Retroviruses have limited natural host cell ranges. Adenovirus
and adeno-associated virus are able to infect a relatively broader range
of cells efficiently. And are the most popular vectors for use in gene
therapy trials.

Advantage

Disadvantage

Retrovirus

-Stable integration into chromosome

- Moderate transduction efficiency
- (ss RNA virus with Reverse transcriptase &
integrase enzyme)

- Only infect actively dividing cells;

especially useful for tumor therapy
- Limited size of introduced gene
about 8 kb
- Insertional mutagenesis possibility

Adenovirus
(ds DNA
virus)

-Higher transduction efficiency

- Broad range of target cells
- Does not require dividing cells
- Low risk for insertional mutagenesis
- Most successful vector with 30 kb capacity

-Transient expression
- Immuno genecity Respiratory tract
infection)

Adenoassociated
virus (AAV)
(ss RNA)

-Does not require cell division

- Site specific integration
-Not immunogenic
-Infect both dividing and non-dividing cells

- limited size of introduced gene

about 4.5 kb

Herpes
simplex
(ds DNA)

- Large size of introduced gene about 30 kb

-Can infect a wide range of cells, including
neurons

- Cytotoxic

Non viral

-No limitation for size

- Easy to produce
- Not immunogenic

- Low efficiency

Non viral vectors are biocompatible moieties which can directly deliver genetic material
(DNA/RNA) into a specific cell with minimal toxicity. DNA is encapsulated with liposomes
(cationic lipids mixed with nucleic acids) or nanoparticles and then injected into a
specific cell. They can be produced in large scale, easy to manipulate and are easy for
cell or tissue targeting. The only disadvantage of non-viral vector is its reduced
transfection efficiency in cells or tissues. Non-viral delivery systems utilize different
genetic materials such as antisense oligonucleotide ( AON ), plasmid DNA, small
interfering RNA ( siRNA ), short hairpin RNA ( shRNA ), and micro RNA (miRNA) that
works on electrostatic interaction. On the other hand cationic vectors such as lipid and
polymers help in the formation of lipoplexes and polyplexes.

1- Liposomes in gene therapy: (hollow sphere surrounded by a lipid bilayer)

lipos meaning fat and soma meaning body. A liposome is a tiny bubble (vehicle)
made out of the same material as a cell membrane. Liposomes can be filled with DNA
and can be used to deliver for cancer and other diseases. Membranes are usually made
of phospholipids which are molecules that have a head group and a tail group. Head is
hydrophillic and tail is long hydrocarbon chain and is hydrophobic. Usually phospholipids
are bilayer stable membranes. During packaging of the polynucleotide material in
liposome, a number of structures are formed. Each structure formed is the most
energetically favorable conformation based upon characteristics of specific lipids used.

2- Lipoplexes and polyplexes in gene therapy:

To improve the delivery of DNA into the cell, it must be protected from damage and its
entry into the cell must be facilitated. Plasmid DNAs are covered by lipids in an
organized structure like a micelle or liposome. When the organized structure is
complexed with DNA, it is called lipoplex. There are three types of lipids: cationic,
anionic and neutral. Cationic lipids are most preferred for gene delivery because these
are positively charged and thus complex well with negatively charged DNA and interacts
with the cell membrane, endocytosed by the cell and finally release the DNA.
Can be used to transfect cells in lung in cystic fibrosis treatments
Poor targeting and Inactivated in the blood
3- Naked DNA in gene therapy:
The simplest non-viral gene delivery system uses naked DNA as a vector. Direct
injection of free DNA into certain tissues specially muscles produce high levels of gene
expression. It is particularly applied to cancer tissues where the DNA can be injected
either directly into the tumor or can be injected into muscle in order to express tumor
antigens that might function as a cancer vaccine. But the expression is usually non
uniform and fails to correct the underlying histological and functional abnormalities of the
disease. Though it leads to gene expression but level of expression is much lower than
with either viral or liposomal vectors. It is unsuitable for systemic administration due to
the presence of serum nuclease. Therefore its application is limited
to muscle cells and skin.

4- Immunotherapy comprises of majority of the gene therapy trials for cancer. It has
been well established that cancer cells are weakly immunogenic because they are self
and by virtue of many ways to down regulate host immune response. Most of the studies
related to cancer gene therapy involve manipulation of tumor cells ex vivo to enhance
the production of interleukins, interferon gamma and tumor necrosis factor
Currently, antigen presenting cells such as dendritic cells isolated from a patient are
modulated to enhance the immune response against cancer.
5- Virosome is a drug or vaccine delivery system. It is a virus-like particle that acts as a
vaccine carrier and adjuvant thereby acting as an immune enhancing system. Vaccines
that are manufactured using virosome technology show high efficacy with high purity
and hence it is a safe and an effective way to vaccinate infants and adults. Virosomes
are reconstituted viral envelopes including membrane lipids and viral spike glycoproteins
but devoid of viral genome. They are highly effective as vaccine antigens and adjuvants
because they stimulate humoral immune response because of the presence of viral
glycoproteins. The main advantage of virosomes over other drug delivery system like
liposomal and proteoliposomal carrier system is that the virosomes protect
pharmaceutically active substances from proteolytic degradation and low pH within
endosomes. This helps the contents to be in intact form till it reaches cytoplasm. . It is
target specific and can stimulate both cellular and antibody immune response to
maximize protection against the targeted disease. In addition to this, they are completely
biodegradable.

Strategies for Transgene Delivery

Ex Vivo
Cells removed from body

Transgene delivered
Cells cultured

Cells returned to the body

In Vivo
Transgene delivered
directly into host

Target site for somatic gene therapy

There are different target sites for the gene therapy
Endothelium: The advantage of using endothelium as a target site for gene therapy
includes formation of new blood vessels and ease to get the gene of interest directly into
the blood stream. Eg- Hemophilia
Muscle: It is easily accessible to blood stream and muscle specific promoter is well
studied. Eg- Duchenne Muscular Dystrophy.
Liver: It has a capacity to regenerate and involve in a variety of functions. Thyroid
binding promoter is well studied for liver specific gene transfer. Eg- Familial
hypercholesterolemia.
Skin: Grafting of skin is possible and small piece can be grown to a large.
Brain: Important for nervous system related illnesses.
Lung: Airway epithelium is easily accessible organ. Eg- gene therapy for cystic fibrosis.

Gene Therapy Principles

AAV

Nucleus

Adenovirus
Therapeutic
Protein

Retrovirus/Lentivirus

Naked DNA

Target
Cell

Adenovirus Cell Entry

Lesch-Nyhan Syndrome (LNS); is a rare, inherited disorder caused by a

deficiency of the enzyme hypoxanthine-guanine phosphoribosyl
transferase(HPRT). LNS is an X-linked recessive disease
The lack of HPRT causes a build-up of uric acid crystals in all body fluids, in
the joints, kidneys, central nervous system, leading to gout-like swelling in the
joints and severe kidney problems with poor muscle control With lip and finger
biting

Neurological symptoms include facial grimacing, involuntary writhing, and

repetitive movements of the arms.
Treatment for LNS is symptomatic. Gout can be treated with allopurinol to
control excessive amounts of uric acid. Kidney stones may be treated by using
shock waves or laser beams. There is no standard treatment for the
neurological symptoms of LNS.

HPRT deficiency- results in Lesch Nyhan syndrome

ADA deficiencyresults in severe immunodeficiency syndrome

The first gene therapy experiment was conducted on a 4-years-old American girl in
1990. She was suffering from a genetic disease having deficiency of adenosine
deaminase enzyme required for the proper metabolism of purine. Any malfunctioning of
adenosine deaminase leads to severe combined immunodeficiency in affected patients.
The white blood cells of the girl was taken out and corrected with a human gene using
retrovirus as a vector.

First partial success (~1990)

Young child with Severe Combined
Immuno Deficiency (SCID)
- point mutation in a single gene of white
blood cells
- withdrew blood; introduced normal gene;
transfused blood
Improved quality of life (not a cure) but
required the procedure be repeated
every 3-4 months
(Note: blood cells have life span of ~120
days)

The molecular basis of SCID (Severe combined immunodeficiency

disease)
ADA-SCID
Adenosine Deaminase defect. Adenosine metabolites build up in the
blood and kill T cells. Therefore, no T cell immunity.
First genetic disease targeted with gene therapy 1990; 2 girls. T cells
taken out, infected with ADA virus, re-infused Problem: Too low a
level of ADA expression Correction: LTR contained element that
suppressed transcription in stem cells--eliminated!
X-SCID
Kids lack the IL2 receptor gamma common (C) chain,
One of the components of the IL2, IL4, IL7, IL15 receptors.
T cells can not expand after stimulation (IL2, IL4).
B cell do not undergo class-switching (IL7, IL15). Have no natural
killer T cells. In 1998, the French study of 11 individuals was initiated
to replace gamma C in blood stem cells

Cancer gene therapy

1. Direct attack on tumor cells
a) transfer of tumor suppressor gene (p53)
b) Inhibition of oncogenes
-anti-sense therapy
-ribozymes (Degrading enzymes)
c) Suicide genes
d) Oncolytic viruses
2. Harnessing immuneresponse to tumor antigens
-overexpression of cytokines genes
-tumor vaccines
3. Anti-angiogenic therapy

Oncolytic viruses

Strategy of genetically modified tumor vaccines;

1.Isolate tumor cells from a patient=autologic cells

2. Alternativeculture other tumor cells-eg, cell line of

The same typei.e. allogeneic cell line (Dead cancer cell)
3. Transduce such cells with vectoreg. Retroviral vector
Harboring cytokine gene
4. Inject such modified cells into patients

5. Allogeneic tumor cells stimulate immune system, and also

cytokines enhance this response to kill cancerous tissues.

Disorder

SCID
Lesch-Nyhan
syndrome
Familial
hypercholesterolemia
(FH)
Phenylketonuria
(PKU)
-Thalassemia
Hemophilia B
Cystic fibrosis
Melanoma

Affected Gene

adenosine deaminase (ADA)

hypoxanthine-guanine
phosphoribosyltransferase (HGPRT)
LDL receptor
phenylalanine hydroxylase
-Globin
Clotting Factor IX
Cystic fibrosis transmembrane receptor
Tumour necrosis factor (TNF)

Gene therapy using germ cells result in permanent changes that are
subsequently passed on to next generation. Gene therapy using somatic
cells are not passed on to the next generation. Somatic cell gene therapy is
safer than the germ line gene therapy. Somatic cell gene therapy can be
done exterior (where cells are modulated in vitro and then transplanted
back) or interior (where genes are changed in vivo ).
ex vivo, which means exterior (where cells are modified outside the body
and then transplanted back in again). cells from the patients blood or bone
marrow are removed and grown in the laboratory. The cells are exposed to
the virus that is carrying the desired gene. The virus enters the cells and
inserts the desired gene into the cells DNA. The cells grow in the laboratory
and are then returned to the patient by injection into a vein. This type of
gene therapy is called ex vivo because the cells are treated outside the
body.
in vivo, which means interior (where genes are changed in cells still in the
body). This form of gene therapy is called in vivo, because the gene is
transferred to cells inside the patients body.

Today's Science Fiction

Gene therapy was viewed as Science
Fiction 40 years ago when the genes
were shown to be basic units of
heredity
Gene therapy is now a reality and there
are many successful treatments in that
field (still years to go ...)

So what can we look forward to (be

afraid of?) in another 40 years ....

Most diseases can be

(potentially) treated with
gene therapy, because every
disease has a genetic
background!