Gene therapy

Principle, types, methods, vectors, techniques and applications

1. GENE THERAPY
Arushe Tickoo
B. Tech Biotech

2. GENE THERAPY
•The term gene therapy describes any procedure intended
to treat or alleviate disease by genetically modifying the
cells of a patient. It encompasses many different
strategies and the material transferred into patient cells
may be genes, gene segments or oligonucleotides.
•The genetic material may be transferred directly into
cells within a patient (in vivo gene therapy), or cells may
be removed from the patient and the genetic material
inserted into them in vitro, prior to transplanting the
modified cells back into the patient (ex vivo gene
therapy).

3. Gene therapy was first conceptualized in 1972.
The first approved gene therapy case in the United States
took place on 14 September 1990, at the National
Institute of Health, under the direction of Professor
William French Anderson. It was performed on a four
year old girl named Ashanti DeSilva. It was a treatment
for a genetic defect that left her with ADA-SCID, a
severe immune system deficiency. The effects were only
temporary, but successful.

4. In January 2014, researchers at the University of Oxford
reported that six people suffering from choroideremia had
been treated with a genetically engineered adeno-
associated virus with a copy of a gene REP1. Over a six
month to two year period all had improved their sight.
Choroideremia is an inherited genetic eye disease for
which in the past there has been no treatment and patients
eventually go blind

5. TYPES OF GENE THERAPY
1.Somatic Gene Therapy

In somatic gene therapy, the therapeutic
genes are transferred into the somatic cells (non sex-
cells), or body, of a patient. Any modifications and
effects will be restricted to the individual patient only,
and will not be inherited by the patient's offspring or
later generations. Somatic gene therapy represents the
mainstream line of current basic and clinical research,
where the therapeutic DNA transgene (either
integrated in the genome or as an external episome or
plasmid) is used to treat a disease in an individual

6. Most of these trials focus on treating severe genetic
disorders, including immunodeficiency, haemophilia,
thalassemia, and cystic fibrosis. These disorders are
good candidates for somatic cell therapy because they
are caused by single gene defects. The replacement of
multiple genes in somatic cells is not yet possible.

7. Germ cells will combine to form a zygote which will divide
to produce all the other cells in an organism and therefore if
a germ cell is genetically modified then all the cells in the
organism will contain the modified gene. This would allow
the therapy to be heritable and passed on to later
generations
2. Germline gene therapy
In germline gene therapy, germ cells (sperm or eggs) are
modified by the introduction of functional genes, which are
integrated into their genomes.

8. CLASSES OF DISEASES
•Because the molecular basis of diseases can vary widely, some
gene therapy strategies are particularly suited to certain types
of disorder, and some to others. Major disease classes include:
•Infectious diseases (as a result of infection by a virus or
bacterial pathogen);
•cancers (inappropriate continuation of cell division and cell
proliferation as a result of activation of an oncogene or
inactivation of a tumor suppressor gene or an apoptosis gene
•Inherited disorders (genetic deficiency of an individual gene
product or genetically determined inappropriate expression of
a gene);
•Immune system disorders (includes allergies, inflammations
and also autoimmune diseases, in which body cells are
inappropriately destroyed by immune system cells).

9. CLASSICAL GENE THERAPY
•An essential component of classical gene therapy is that
cloned genes have to be introduced and expressed in the
cells of a patient in order to overcome the disease.
Practically, this usually involves targeting the unaffected
cells of diseased tissues.
•Immune system-mediated cell killing In many gene
therapies the target cells are healthy immune system
cells, and the idea is to enhance immune responses to
cancer cells or infectious agents.
•Two major approaches for gene therapy: transfer of
genes into patient cells outside of the body (ex vivo) or
inside the body (in vivo).

10. IN-VIVO AND EX-VIVO GENE
TRANSFER
In vivo gene therapy (blue arrow) entails the genetic modification of the
cells of a patient in situ. Ex vivo gene therapy (black arrows) means that
cells are modified outside the body before being implanted into the patient.

11. EX VIVO GENE TRANSFER
•Transfer of cloned genes into cells grown in culture.
•Those cells which have been transformed successfully are
selected, expanded by cell culture in vitro, then introduced into
the patient.
• To avoid immune system rejection of the introduced cells,
autologous cells are normally used: the cells are collected
initially from the patient to be treated and grown in culture
before being reintroduced into the same individual.
•Clearly, this approach is only applicable to tissues that can be
removed from the body, altered genetically and returned to the
patient where they will engraft and survive for a long period of
time (e.g. cells of the hematopoietic system and skin cells).
•This type of gene therapy involves transplantation of autologous
genetically modified cells and so can be considered a modified
form of cell therapy.

13. IN VIVO GENE TRANSFER
•The cloned genes are transferred directly into the tissues of the
patient.
•This may be the only possible option in tissues where individual
cells cannot be cultured in vitro in sufficient numbers (e.g. brain
cells) and/or where cultured cells cannot be re-implanted
efficiently in patients.
•Liposomes and certain viral vectors are increasingly being
employed for this purpose.
•It is often convenient to implant vector-producing cells (VPCs),
cultured cells which have been infected by the recombinant
retrovirus in vitro: in this case the VPCs transfer the gene to
surrounding disease cells.
•As there is no way of selecting and amplifying cells that have
taken up and expressed the foreign gene, the success of this
approach is crucially dependent on the general efficiency of
gene transfer and expression.

15. MAMMALIAN VIRAL VECTORS
BECAUSE OF THEIR HIGH EFFICIENCY
OF GENE TRANSFER

16. ONCORETROVIRAL
VECTORS
•Retroviruses are RNA viruses which possess a reverse
transcriptase function, enabling them to synthesize a
complementary DNA form.
•Following infection (transduction), retroviruses deliver a
nucleoprotein complex (pre integration complex) into the
cytoplasm of infected cells.
•This complex reverse transcribes the viral RNA genome
and then integrates the resulting DNA copy into a single
site in the host cell chromosomes.
• Retroviruses are very efficient at transferring DNA into
cells, and the integrated DNA can be stably propagated,
offering the possibility of a permanent cure for a disease.

17. ONCORETROVIRAL
VECTORS
•Because of these properties, retroviruses were considered
the most promising vehicles for gene delivery and
currently about 60% of all approved clinical protocols
utilize retroviral vectors.
•Since all the viral genes are removed from the vector, the
viruses cannot replicate by themselves.
•They can accept inserts of up to 8 kb of exogenous DNA
and require a variety of packaging systems to enclose the
viral genome within viral particles
•Oncoretroviruses can only transduce cells that divide
shortly after infection: the pre integration complex is
excluded from the nucleus and can only reach the host cell
chromosomes when the nuclear membrane is fragmented
during cell division.

18. gene therapy

19. ADENOVIRUS VECTORS
•Adenoviruses are DNA viruses that produce infections of the
upper respiratory tract and have a natural tropism for
respiratory epithelium, the cornea and the gastrointestinal tract.
•Adenovirus vectors have been the second most popular delivery
system in gene therapy (with extensive applications in gene
therapy for cystic fibrosis and certain types of cancer).
•They are human viruses which can be produced at very high
titers in culture, and they are able to infect a large number of
different human cell types including nondividing cells.
•Entry into cells occurs by receptor-mediated endocytosis and
transduction efficiency is very high (often approaching 100%
in vitro).
•They are large viruses and so have the potential for accepting
large inserts (upto 35 kb).

20. ADENOVIRUSES ENTER CELLS BY
RECEPTOR-MEDIATED
ENDOCYTOSIS
Binding of viral coat protein to a specific receptor on the plasma membrane of
cells is followed by endocytosis. Subsequent vesicle disruption by adenovirus
proteins allows virions to escape and migrate towards the nucleus where viral
DNA enters through pores in the nuclear envelope.

21. DISADVANTAGES OF
ADENOVIRUSES
•The inserted DNA does not integrate, and so expression of
inserted genes can be sustained over short periods only.
•For example, the recombinant adenoviruses used in cystic
fibrosis gene therapy trials showed that transgene expression
declined after about 2 weeks and was negligible after only 4
weeks.
•Because they can infect all human cells, adenovirus vectors
may be risky in some therapies that are designed to kill
cancer cells without causing toxicity to normal surrounding
cells.
•Most importantly, first generation adenovirus vectors can
generate unwanted immune responses, causing chronic
inflammation.

22. HERPES SIMPLEX VIRUS
VECTORS
•HSV vectors are tropic for the central nervous system
(CNS) and can establish lifelong latent infections in
neurons.
•They have a comparatively large insert size capacity
(>20 kb) but are non integrating and so long-term
expression of transferred genes is not possible.
•Their major applications are expected to be in delivering
genes into neurons for the treatment of neurological
diseases, such as Parkinson's disease, and for treating
CNS tumors.

23. LENTIVIRUSES
•The lentivirus family, which includes HIV (human
immunodeficiency virus), are complex retroviruses that
infect macrophages and lymphocytes.
•Unlike oncoretroviruses, lentiviruses are able to
transduce non dividing cells.
•In the case of HIV, for example, the pre integration
complex contains nuclear localization signals that permit
its active transport through nuclear pores into the
nucleus during interphase.
•Because of their ability to infect non dividing cells and
to integrate into host cell chromosomes, considerable
efforts are now being devoted to making lentivirus
vectors for gene therapy.

24. MOST COMMON VIRAL VECTORS
Retroviruses
Adenoviruses
Adeno-associated viruses
Herpes simplex viruses
can create double-stranded DNA copies of their RNA genomes. Can integrate into genome. HIV,
MoMuLV, v-src, Rous sarcoma virus
dsDNA viruses that cause respiratory, intestinal, and eye infections
in humans. Virus for common cold
ssDNA viruses that can insert their genetic material
at a specific site on chromosome 19
dsDNA viruses that infect a neurons. Cold sores virus

25. METHODS OF GENE DELIVERY
(THERAPEUTIC CONSTRUCTS)
-- Injection of DNA via Mechanical and electrical strategies
include microinjection
-- DNA transfer by liposomes
(delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)
-- DNA coated on the surface of gold pellets
which are air-propelled into the epidermis
(gene-gun), mainly non applicable to cancer
-- Biological vehicles (vectors) such as viruses and bacteria.
Viruses are genetically engineered
so as not to replicate once inside the host.
They are currently the most efficient means of gene transfer.

26. MECHANICAL AND ELECTRICAL
TECHNIQUES
•Mechanical and electrical strategies of introducing DNA
into cells include microinjection, particle bombardment,
the use of pressure, and electroporation.
•The direct injection (microinjection) of naked DNA (i.e.,
uncomplexed DNA) into a cell nucleus is perhaps the most
simple, and therefore appealing, approach to gene
delivery.

27. BIOLISTIC DNA INJECTION (GENE GUNS)
Invented for DNA transfer to plant cells
Fully applicable to eukaryotic cells
plasmid DNA shown here

28. BIOLISTIC PARTICLE DELIVERY
•Particle bombardment, which is also called biolistic particle
delivery, can introduce DNA into many cells (including cell-
walled plant cells) simultaneously.
•In this procedure, DNA-coated microparticles (composed of
metals such as gold or tungsten) are accelerated to high
velocity to penetrate cell membranes or cell walls.
•Bombardment is widely employed in DNA vaccination,
where limited local expression of delivered DNA (in cells of
the epidermis or muscle) is adequate to achieve immune
responses.
•Because of the difficulty in controlling the DNA entry
pathway, this procedure is applied mainly adherent cell
cultures and has yet to be widely used systemically.

29. ELECTROPORATION
•An alternative approach is to use high-voltage electrical pulses to
transiently permeabilize cell membranes, thus permitting cellular
uptake of macromolecules.
•This process, called electroporation, was first used to deliver DNA
to mammalian cells in 1982 .
•Since that time, electroporation has been used to deliver DNA to
myriad cell types in vitro, including bacteria and yeast.
•It is one of the most efficient gene transfer methods, but it is
limited because of the high mortality of cells after high-voltage
exposure and difficulties in optimization.
•Although electroporation is difficult to apply in vivo, some
progress has been achieved in skin, corneal endothelium, and
muscle.

30. CHEMICAL METHODS
•The use of uptake-enhancing chemicals-which is arguably
the easiest, most versatile,most effective, and most
desirable of the DNA delivery methods—was demonstrated
more than 30 years ago.
•The general principle is based on complex formation
between positively charged chemicals (usually polymers)
and negatively charged DNA molecules.
•These techniques can be broadly classified by the chemical
involved : 2-(diethylamino) ether (DEAE)-dextran, calcium
phosphate, artificial lipids, protein, dendrimers, or others.

31. DEAE DEXTRAN
AND CALCIUM PHOSPHATE
•DEAE dextran and calcium phosphate, which interact with DNA to form
DEAE-dextran–DNA and calcium phosphate–DNA complexes,
respectively.
•After the complexes are deposited onto cells, they are internalized by
endocytosis.
•DEAE-dextran and calcium phosphate methods are simple, effective, and
still widely used in the laboratory for in vitro transfection.
•Even so, both methods are hampered by cytotoxicity and the difficulty of
applying them to in vivo studies. In addition, DEAE dextran can be used
neither with serum in culture medium nor for stable transfection.
•The calcium phosphate method also suffers from variations in calcium
phosphate–DNA sizes, which causes variation among experiments.

32. LIPOSOMES
Lets’ wrap it in something safe
to increase transfection rate
Therapeutic drugs
Lipids – are an obvious idea !

33. DNA DELIVERY OF GENES BY LIPOSOMES
Cheaper than viruses
No immune response
Especially good
for in-lung delivery (cystic fibrosis)
100-1000 times more plasmid DNA needed
for the same transfer efficiency as for viral vector
Complexation of cationic lipids with DNA
was first described in 1987, and ‘lipofection’
was reported to be 5- to >100-fold more efficient
than the earlier calcium phosphate or the DEAE
dextran transfection techniques. Cationic lipids are
highly soluble in aqueous solution, forming positively
charged micellar structures termed liposomes

34. LIPOFECTINS
•Felgner and colleagues developed the cationic lipid
Lipofectin in 1987.
•Lipofectin–DNA complexes can be handled easily
and, therefore, became one of the first chemical
systems that could be used in animals.
•In addition, DNA has been successfully complexed
with cationic, anionic, and neutral liposomes, as
well as various mixtures.

35. LIPOSOMES
•Lipid based systems are probably the most commonly used methods of
DNA delivery and have been used in human clinical trials.
•Still, lipid based systems have important drawbacks, including the lack
of targeting, the poorly understood structure of DNA–lipid complexes,
and variations arising during fabrication.
•The major limitation of the above approaches is toxicity upon systemic
administration.
•Furthermore, a major problem with the application of most nonviral
systems, including lipoplexes, is their poor efficiency at transfecting
nonproliferating cells. This is thought to be mainly a result of the
integrity of the nuclear membrane providing a physical barrier to entry.
•Lipoplexes are seen with diameters of 100–200 nm, and also elongated,
‘spaghetti’-shaped, lipoplexes. Large aggregates or ‘meatball’ lipoplexes
are also observed, and thought to comprise numerous lipid and DNA
molecules.

36. POLYETHYLENIMINE
•PEI is a branched polymer with high cationic potential that is capable of
effective gene transfer in nondifferentiated COS-1 cells; however, it can
also be extremely cytotoxic due to induction of apoptosis.
•The high transfection efficiency of PEI can be attributed to the buffering
effect or the “proton sponge effect” of the polymer caused by the
presence of amino groups in the molecule.
• The strong buffering effect of the polymer helps in rapid endosome
escape.
•The cytotoxicity and transfection efficiency of PEI are directly
proportional to its molecular weight.
•Efforts to reduce the toxicity by synthesis of PEI with graft copolymers
such as linear poly(ethylene glycol), incorporation of low molecular
weight PEI, and PEI glycosylation are under way.

37. Fig; Gene Therapy of
mouse showing
modification in its tail by
removing the defected
gene or replacing the
defected gene with the
functional gene

38. APPLICATION