Gene therapy. Adenovirus Retrovirus In vivo and Ex vivo methods Molecular techniques Alternative vectors Biolistic missile Homologous recombination

1. Gene therapy:
Strategies and clinical applications
Dr. Ishan Y. Pandya
PhD. Biotechnology, MSc, MBA
Head Biology, Dedicated academy, Jamnagar,
Gujarat, India

2. Introduction to gene therapy:
•Genes, which are carried on chromosomes, are the
basic physical and functional units of heredity.
•Genes are specific sequences of bases that encode
instructions on how to make proteins.
•Although genes get a lot of attention, it’s the
proteins that perform most life functions and even
make up the majority of cellular structures.
•When genes are altered so that the encoded
proteins are unable to carry out their normal
functions, genetic disorders can result.

3. What is gene therapy ?
•More than 200 genes responsible for a variety of
the genetic disorders in the humans are identified.
•The causes of the disorders in the form of specific
mutations in these genes have also been
elucidated.
•Therefore, if the know defects in the genes are
rectified, or if the defective genes in the each case
replaced by the normal healthy gene, then the
corresponding genetic disorder can be corrected .
•Such a treatment in the human is described as
gene therapy .

4.  In the 1980s, Scientists began to look
into gene therapy.
◦ They would insert human genes into a
bacteria cell.
◦ Then the bacteria cell would transcribe and
translate the information into a protein.
◦ Then they would introduce the protein into
human cells.
The Beginning…

5. Gene therapy has the potential to treat all of
the listed classes of disorder:
•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).

6. Subdivision of gene therapy
approaches is as follows:
•Classical gene therapy. The rationale of this type of
approach is to deliver genes to appropriate target cells with
the aim of obtaining optimal expression of the introduced
genes. Once inside the desired cells in the patient, the
expressed genes are intended to do one of the following:
1.Produce a product that the patient lacks;
2.Kill diseased cells directly, e.g. by producing a toxin
which kills the cells;
3.Activate cells of the immune system so as to aid killing
of diseased cells.
•Nonclassical gene therapy. The idea here is to inhibit the
expression of genes associated with the pathogenesis, or to
correct a genetic defect and so restore normal gene
expression.

8. Somatic cell gene therapy:- Repair or replace the
defective gene in some or all body cell of an
individual.
Germ line gene therapy :-Repair or replace the
defective gene in the germ line cells. Repaired gene
would be inherited.
Present condition: Current gene therapy is
exclusively somatic gene therapy, the
introduction of genes into somatic cells of an
affected individual. The prospect of human
germ line gene therapy raises a number of
ethical concerns, and is currently not
sanctioned.

9. Requirements for the gene
therapy:
•Identification of specific allele associated
with the disease.
•Mapping, sequencing and study of the
genes.
•Ability to get dna into appropriate cells.
•Specific cells involved in the disease, for the
somatic cell therapy germ line cells for the
germ line therapy.
•Ability to directly replace a defective allele
with the functional one.
•Stable maintenance of the normal allele.

11. Ex vivo gene transfer:
•This initially involves 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
In vivo gene transfer:
•Here 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.

12. Methods used for gene delivery:
There are two methods used:
• Using viral vectors.
• Using non viral vectors.

13. Vectors:
• RNA virus (retrovirus)
1.Murine leukamia virus (MuLV)
2.Human immunodeficiency virus (HIV)
3.Human T cell lymphotropic virus (HTLV)
• DNA virus vectors
1.Adenovirus
2.Adeno associated virus vector (AAV)
3.Herpes simplex vector (HSV)
4.Pox virus

14. Retro virus:

15. How it works:

16. •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 (preintegration 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.
•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.
•The retrovirus vectors that have traditionally been used in
gene therapy are derived from simple retroviruses
(oncoretroviruses), notably murine leukemia virus.
Retro viruses:

17. 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

18. •Oncoretroviruses can only transduce cells that divide shortly
after infection: the preintegration complex is excluded from
the nucleus and can only reach the host cell chromosomes
when the nuclear membrane is fragmented during cell
division.
•This therefore limits potential target cells. Only certain blood
cells (but not stem cells) and the cells lining the
gastrointestinal tract are continually in division; other cell
types undergo division but not continually and some important
cell targets never divide (e.g. mature neurons).
•The property of transducing only dividing cells can, however,
be beneficial to gene therapy for cancers of tissues that
normally have nondividing cells: the actively dividing cancer
cells can be selectively infected and killed without major risk
to the nondividing cells of the normal tissue.

19. Adeno virus
Advantages
1. Efficiency of transduction is high
2. High level gene expression
3. Slightly high capacity of the
exogenous gene
Disadvantages
1. Expression may be transient
2. Cell specific targeting difficult to
achieve
3. Safety not ensured

20. Adeno virus as a delivery system:
•Most used is Adeno virus 5, infects dividing and
non dividing cells.
•Carries a ubitiquitous receptor (CAR) so can
enter different call types.

22. •They also have some major disadvantages.
•The inserted DNA does not integrate, and so expression of
inserted genes can be sustained over short periods only.
•All of the adenovirus genes have been deleted from some newer
adenovirus vectors (‘gutless vectors') which then require the
assistance of a helper virus.
•Such a virus provides certain viral functions in trans (e.g.
enzymes involved in viral DNA replication etc.) which are
essential for productive infection (including viral DNA replication,
viral assembly and infection of new cells) by certain natural
viruses, such as AAV, or artificially disabled viruses.
•The risk of immune response to these vectors is negligible.
•This is an important consideration given the need to administer
treatment frequently (because of the inability of adenovirus to
integrate into chromosomal DNA).
•They also have the advantage that they can accept much larger
inserts (up to 35 kb). Unfortunately, however, deletion of
adenoviral genes can also be counterproductive.
•Deletion of the E3 region removes the capacity to encode a
protein that protects the virus from immune surveillance
mechanisms in the host. In addition, fully disabled adenoviral
vectors have much lower transduction efficiencies.

23. Adeno associated virus:
•AAV is a well know defective parvovirus
(dependovirus subgroup) that relies on co-infection
with the herpes virus or helper virus
•AAV has potential as a human theraputic vectior
because its genome intergates at only specific site
in the human genome (chromosome 19)
•AAV vectors can only accommodate inserts up to
4.5 kb, but they have the advantage of providing
the possibility of long-term gene expression
because they integrate into chromosomal DNA.
•They also provide a high degree of safety:
because 96% of the parental AAV genome has
been deleted, the AAV vectors lack any viral genes
and recombinant AAV vectors only contain the
gene of interest.

24. Use of AAV as
a gene
delivery agent.

25. AAV
Advantages:
• Infects both dividing and non
dividing cells
• Not associated with disease
• Can obtain high titred virus stock
• Small genome easy to
manipulate
Disadvantages:
• Can only contain approximately
4.5 kb of DNA

26. Ability to use multiple AAV contructs to
generate proteins that are encoded by
genes larger than 4.5 kb
•Package half of gene of interest in one
particle and the other half in different
particle and co- transduce cells.
•Approach is relatively inefficient.

27. Herpes simplex virus vectors:
 Double stranded DNA viruses
 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.
 Ex. Herpes simplex virus type 1.

28. Pox virus:
 Family members- cow pox, small pox, vaccinia,
monkey pox, canary pox.
 Large, a highly complex and understudied virus.
 Only family of DNA viruses that replicates in the
cytoplasm of cell.
 Vaccinia is used as vaccine against small pox.
 Vaccinia has broad range.
 Through homologous recombination in vaccinia
infected cells can introduce genes into virus allowing:
1. Production of large quantity of recombinant proteins
from the infected cell.
2. Serve as delivery system for recombinant vaccines.

29. Homologous recombination in
vaccinia virus system:
 Vaccinia promoter (several available depending
on when you want your gene of interest
expressed in infected cells- early, intermediate or
late in infection) and a portion of vaccinia genome
that will be target for insertion
 Clone in gene of interest behind the promoter (
upto 25kb )
 Plasmid frequently contains some sort of suitable
marker
1. Eukaryotic antibiotics
2. Beta galactosidase, etc.
 Transfect into vaccinia expressing cell and
homologous recombination will occur about 0.1%

30. Poxvirus life cycle: early, middle &
late gene expression:

31. Pox virus vector:

32. Concerns over the safety of recombinant viruses have
prompted increasing interest in nonviral vector systems
for gene therapy:
•In the case of retrovirus vectors, chromosomal integration
is still possible but, like other replication-incompetent virus
vectors, they should not be able to undergo a productive
infection in which they replicate, assemble new virions and
infect new cells.
• However, there is the remote possibility that the
introduced viruses can recombine with endogenous
retroviruses, resulting in recombinant progeny that can
undergo productive infection.
•Additionally, adenoviruses are generally nonintegrating
and the repeated injections that may be required may
provoke severe inflammatory responses to the recombinant
adenoviruses as has happened recently in a gene therapy
trial for cystic fibrosis.

33. Alternative non viral vector system
for the gene transfer:
 Direct introduction of therapeutic DNA
◦ But only with certain tissue
◦ Requires a lot of DNA
 Creation of artificial lipid sphere with aqueous core, liposome
◦ Carries therapeutic DNA through membrane
 Chemically linking DNA to molecule that will bind to special
cell receptors
◦ DNA is engulfed by cell membrane
◦ Less effective 
 Trying to introduce a 47th chromosome
◦ Exist alongside the 46 others
◦ Could carry a lot of information
◦ But how to get the big molecule through membranes?

34. Electroporation:
•Electroporation can be used to transform cells of animals,
yeast, plants and bacteria. Plant cells are first treated to remove
their cell walls. Animal cells don’t have cell walls, so don’t require
this first step.
•The cells are placed in a solution with the new DNA that is to be
added. The solution is then subjected to a high voltage electric
shock for a fraction of a second. This causes small holes to form
in the cell membrane, through which DNA can enter.
•The cells are then placed in a nutrient solution, allowing them to
repair their membranes and cell walls and recover their normal
functions.

35. Microinjections
•The most commonly used method
to transfer DNA directly into animal
cells such as egg cells is to inject the
DNA directly into a newly- fertilised
egg cell using a glass capillary tube.
This is called microinjection.
•The egg cell containing the new
DNA is implanted into a female
animal. Because the gene was
inserted into the DNA at the egg
stage, when the cell divides, every
cell in the growing embryo will
contain the new DNA.

36. Biolistics:
•Tiny tungsten or gold particles are coated with the
DNA to be transferred.
•The particles are usually about 0.004 of a
millimetre in diameter.
•A blast of high-pressure helium gas or gunpowder
shoots the particles carrying the DNA into the cells
that are to be transformed.

37. Liposome:
•Liposome are
synthetic vesicles
which can form
spontaneously in
aqueous solution
following artificial
mixing of lipid
molecules.
•In some cases, a
phospholipids bilayer is
formed, with hydrophilic
phosphate groups
located on the external
surfaces and
hydrophobic lipids
located internally.

38. SMART:

39. Dominantly inherited disorders:
•In addition to the above, targeted inhibition of gene expression may
offer the possibility of treating certain dominantly inherited disorders.
•If a dominantly inherited disorder is the result of a loss-of-function
mutation, treatment may be difficult using conventional gene
augmentation therapy: given that heterozygote with 50% of normal
gene product can be severely affected, very efficient expression of the
introduced genes would be required for the gene therapy to be
successful.
• However, dominantly inherited disorders which arise because of a
gain-of-function mutation may not be amenable to simple addition of
normal genes.
•Instead, it may be possible, in some cases, to inhibit specifically the
expression of the mutant gene, while maintaining expression of the
normal allele.
• Such allele-specific inhibition of gene expression would be facilitated
if the pathogenic mutation results in a significant sequence difference
between the alleles.

40. Recessively inherited disorders:
•Recessively inherited disorders have been of particular interest as candidates
for gene therapy because the mutations are almost always simple loss-of-
function mutations.
•Affected individuals have deficient expression from both alleles and so the
disease phenotype is due to complete or almost complete absence of normal
gene expression.
• Heterozygotes, however, have about 50% of the normal gene product and are
normally asymptomatic.
•Additionally, there is, in at least some cases, wide variation in the normal levels
of gene expression, so that a comparatively small percentage of the average
normal amount of gene product may be sufficient to restore the normal
phenotype.
•It is also often observed that the severity of the phenotype of recessive
disorders is inversely related to the amount of product that is expressed.
•As a result, even if the efficiency of gene transfer is low, modest expression
levels for an introduced gene may make a substantial difference.
•This is quite unlike dominantly inherited disorders where heterozygotes with
loss-of-function mutations have 50% of the normal gene product and may yet be
severely affected.
•Although recessively inherited disorders are, in principle, amenable to gene
augmentation therapy, certain disorders are less amenable than others.

41. Limitations
 Short Lived
◦ Hard to rapidly integrate therapeutic DNA into genome and
rapidly dividing nature of cells prevent gene therapy from long
time
◦ Would have to have multiple rounds of therapy
 Immune Response
◦ new things introduced leads to immune response
◦ increased response when a repeat offender enters
 Viral Vectors
◦ patient could have toxic, immune, inflammatory response
◦ also may cause disease once inside
 Multigene Disorders
◦ Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because you need to introduce more
than one gene
 May induce a tumor if integrated in a tumor suppressor gene
because insertional mutagenesis

42. Take home message
“human gene therapy is a symbol of hope in a
vast sea of the human suffering due to hereditary
”- John c Fletcher and W
.French Anderson