2. (1) Introduction
(2) Classes of gene therapy
(3) Strategies for gene therapy
(4) Methods used for gene therapy
(5) Factors that have kept gene therapy ineffective for
treatment of genetic diseases
(6) Developments in gene therapy research
(7) RNA interference
(8) Ethical concerns
3. Introduction
Gene therapy is a technique that involves insertion of
normal genes to correct defective genes responsible
for disease development.
It is the use of DNA as a pharmaceutical agent to treat
disease.
It derives its name from the idea that DNA can be
used to supplement or alter genes within an
individual's cells as a therapy to treat disease
4. The most common form of gene therapy
involves using DNA that encodes a functional,
therapeutic gene in order to replace a mutated
gene.
Other forms involve directly correcting a
mutation or using DNA that encodes a
therapeutic protein drug (rather than a natural
human gene) to provide treatment.
5. Gene therapy was first conceptualized in
1972, with the authors urging caution before
commencing gene therapy studies in
humans.
The first FDA-approved gene therapy
experiment in the United States occurred in
1990, when Ashanti DeSilva was treated for
ADA-SCID. Since then, over 1,700 clinical
trials have been conducted using a number
of techniques for gene therapy.
6. Researchers may use one of several 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
7. An abnormal gene could be swapped for a
normal gene through homologous
recombination.
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
8. Somatic Gene Therapy
In somatic gene therapy, the therapeutic genes are
transferred into the somatic 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 mRNA is
used to treat a disease in an individual.
9. In germ line gene therapy, Germ cells, i.e., sperm or eggs,
are modified by the introduction of functional genes, which
are integrated into their genomes.
This would allow the therapy to be heritable and passed on
to later generations (offsprings).
Although this should, in theory, be highly effective in
counteracting genetic disorders and hereditary diseases,
many jurisdictions prohibit this for application in human
beings for a variety of technical and ethical reasons
12. (3) Strategies for Gene Therapy
1. Ex vivo, which means “outside the body”
Cells from the patient’s blood or bone marrow are
removed and grown in the laboratory.
They are then exposed to a virus carrying the
desired gene. The virus enters the cells, and the
desired gene becomes part of the DNA of the cells.
The cells are allowed to grow in the laboratory
before being returned to the patient by injection into
a vein or bone marrow.
13. 2. In vivo, which means “inside the body”
No cells are removed from the patient’s body.
Instead, vectors are used to deliver the desired
gene to cells in the patient’s body.
3. In Situ
Vector is placed directly into the affected tissues.
14. List of Common Genetic Disorders
Disorder Mutation Chromosome
Angelman syndrome DCP 15
Color blindness P X
Cystic fibrosis P 7
Down syndrome C 21
Haemophilia P X
Klinefelter's syndrome C X
15.
16. Two methods are involved
(ii) Non-viral mediated gene delivery method
(iii) Viral mediated gene delivery method
17. (I)Non-viral mediated gene delivery method
(i)Direct injection of therapeutic DNA into target cells
This approach is limited in its application because it can
be used only with certain tissues and requires large
amounts of DNA
(ii) Liposomes
Creation of an artificial lipid sphere with an aqueous core
In this method, the liposome, which carries the
therapeutic DNA, is capable of passing the DNA
through the target cell's membrane
18. (iii) Inorganic nanoparticles.
Chemically linking the DNA to a molecule that will bind to special
cell receptors. Once bound to these receptors, the therapeutic
DNA constructs are engulfed by the cell membrane and passed
into the interior of the target cell
(iv) Researchers also are experimenting with introducing a
47th (artificial human) chromosome into target cells.
This chromosome would exist autonomously alongside the
standard 46, not affecting their workings or causing any
mutations.
A problem with this potential method is the difficulty in delivering
such a large molecule to the nucleus of a target cell.
19. There are other
several methods for non-viral gene therapy, including,
electroporation, the gene gun, sonoporation,
magnetofection etc.
E.g In 2003 a University of California, Los Angeles research team
inserted genes into the brain using liposomes coated in a polymer called
polyethylene glycol. The transfer of genes into the brain is a significant
achievement because viral vectors are too big to get across the
blood-brain barrier. This method has potential for treating
Parkinson's disease.
20. Gene gun
OR
microprojectile
bombardment
“biolistics”
Tiny DNA-
coated particles
are shot into
the cells
21. The Gene Gun Helium chamber
Rupture disk
Macrocarrier
DNA coated
gold particle
Stopping screen
Focusing device
PDS1000 Microparticle Delivery System Target tissue
22. This method employs viruses as vehicles to transport
the desired genes to the target cells (These are
recombinant viruses (sometimes called
biological nanoparticles
Different types of viruses used as vectors
for gene therapy
Retroviruses
A class of viruses that can create double-stranded
DNA copies of their RNA genomes
23. Adenoviruses - A class of viruses with double-
stranded DNA genomes that cause respiratory,
intestinal, and eye infections in humans
Adeno-associated viruses - A class of small, single-
stranded DNA viruses that can insert their genetic
material at a specific site on chromosome 19
Herpes simplex viruses
A class of double-stranded DNA viruses that infect a
particular cell type, eg. Neurons
24. In most gene therapy studies, a "normal" gene is
inserted into the genome to replace an "abnormal,"
disease-causing gene
A carrier molecule called a vector must be 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
25. Scientists have manipulated the virus genome to
remove disease-causing genes and insert therapeutic
genes.
When target cells are infected with the viral vector.
The vector then unloads its genetic material
containing the therapeutic human gene
Then, generation of a functional protein product from
the therapeutic gene restores the target cell to a
normal state.
26.
27. (i) Short-lived nature of gene therapy
-Problems with integrating therapeutic DNA into the
genome and the mortal nature of many cells prevent gene
therapy from achieving any long-term benefits. Patients
will have to undergo multiple rounds of gene therapy
(ii) Immune response
- Anytime a foreign object is introduced into human
tissues, the immune system is designed to attack the
invader
28. Furthermore, the immune system's enhanced response
to invaders it has seen before makes it difficult for
gene therapy to be repeated in patients
(iii) Problems with viral vectors
-Though viruses are the carriers of choice in most gene
therapy studies, there some possible potential
problems to the patient; toxicity, immune and
inflammatory responses and the fear that the viral
vector, once inside the patient, may recover its ability
to cause disease.
29. (iv) Multigene disorders
-Conditions or disorders that arise from
mutations in a single gene are the best
candidates for gene therapy
-Unfortunately, some of the most commonly
occurring disorders, such as heart disease,
HBP, arthritis, and diabetes, are caused by the
combined effects of variations in many genes.
- Multigene or multifactorial disorders such as
these would be especially difficult to treat
effectively using gene therapy.
30. If the DNA is integrated in the wrong place in the
genome, for example in a tumor suppressor gene, it
could induce a tumor
This has occurred in clinical trials for X-linked severe
combined immunodeficiency (X-SCID) patients, in
which hematopoietic stem cells were transduced with a
corrective transgene using a retrovirus and this led to
the development of T cell leukemia in 3 of 20 patients.
31. Since gene therapy is relatively new and at an
experimental stage, it is an expensive treatment to
undertake.;
This explains why current studies are focused on
illnesses commonly found in developed countries,
where more people can afford to pay for treatment.
It may take decades before developing countries
can take advantage of this technology.
32.
33. Scientists currently know the functions of
only a few genes. Hence, gene therapy can
address only some genes that cause a
particular disease.
Worse, it is not known exactly whether genes
have more than one function, which creates
uncertainty as to whether replacing such
genes is indeed desirable.
34. Generally, Gene therapy is very promising
Sickle cell was successfully treated in mice.
(2002)
Thalassaemia, cystic fibrosis, and some
cancers. (October 11, 2002).
35. Nanotechnology + gene therapy yields treatment
to cancer. March, 2009. The School of Pharmacy
in London is tested a treatment in mice, which
delivers genes wrapped in nanoparticles to target
and destroy hard-to-reach cancer cells
Results of world's first gene therapy for inherited
blindness show sight improvement. 28 April
2008. UK researchers from the UCL Institute of
Ophthalmology and Biomedical Research Centre
announced results from the world’s first clinical
trial
36. Leber's congenital amaurosis, a type of inherited
childhood blindness caused by a single abnormal gene.
The procedure has already been successful at restoring
vision for dogs. This is the first trial to use gene therapy
in an operation to treat blindness in humans.
Gene Therapy cured deafness in guinea pigs(Feb
2005)-A gene, called Atoh1, which stimulates the hair
cells' growth, was delivered to the cochlea by an
adenovirus
37. Aug. 24, 2011 -- Nine years after getting gene therapy
for "bubble boy disease," 14 out of 16 children are doing
well, UK researchers report.
"Bubble boy disease" — more accurately called severe
combined immunodeficiency, or SCID — results from a
malfunctioning gene in the bone marrow, which
produces immune cells.
Because the immune system is so severely weakened,
any infection or disease could result in death. The
children are therefore forced to live very sheltered,
isolated lives.
38. 11 children with “bubble boy disease.”
1 child did not respond to treatment
8 children cured
2 children came down with leukemia
39. THERAPY IN NEWS
‘Slimming Gene’
Discovered That
Regulates Body
Fat
40. (7) RNA INTERFERENCE OR GENE SILENCING
May be a new way to treat Huntington's d’se.
Basic mechanism
Short pieces of double-stranded RNA (siRNAs) are
used by cells to degrade mRNA of a particular
sequence.
If a siRNA is designed to match the mRNA copied
from a faulty gene, then the abnormal protein product
of that gene will not be produced. (March 13, 2003).
41. RNAi is an RNA-dependent gene silencing process that is
controlled by the RNA-induced silencing complex (RISC)
and is initiated by short double-stranded RNA molecules in
a cell's cytoplasm, where they interact with the catalytic
RISC component argonaute.
RNAi is initiated by the enzyme Dicer, which cleaves long
double-stranded RNA (dsRNA) molecules into short
fragments of ~20 nucleotides that are called siRNAs.
Each siRNA is unwound into two single-stranded (ss)
ssRNAs, namely the passenger strand and the guide
strand.
42. The passenger strand will be degraded, and the
guide strand is incorporated into the RNA-induced
silencing complex (RISC)
The most well-studied outcome is post-
transcriptional gene silencing, which occurs when
the guide strand base pairs with a complementary
sequence of a messenger RNA molecule and
induces cleavage by Argonaute, the catalytic
component of the RISC complex.
In 2006, Andrew Fire and Craig C. Mello shared the
Nobel Prize in Physiology for their work on RNAi
interference in the nematode worm C. elegans,
which they published in 1998.
43. Some Questions to Consider
What is normal and what is a disability or disorder,
and who decides?
Are disabilities diseases? Do they need to be cured
or prevented?
Does searching for a cure demean the lives of
individuals presently affected by disabilities?
44. Ethical Considerations cont…
Who should pay?
Preliminary attempts at gene therapy are exorbitantly
expensive. Who will have access to these therapies?
Who will pay for their use?
Gene therapy currently cost up to $200,000 per
treatment.
Unforeseen consequences
Is it possible that eliminating defective genes from
our genome may have unforeseen consequences
45. Religious Objections
Majority of the world believes in some type of
higher power.
The majority of religions states that everyone
should be happy with what they have.
Do we have the right to play GOD?
