gene therapy understanding basics
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Gene Therapy Understanding BasicsTRANSCRIPT
GENE THERAPY Basics
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
A Tribute to Gregory Mendel
Dr.T.V.Rao MD 2
Gregory Mendel - GENETICS – He used pollen from some
plants to carefully fertilize other
plants
– He was able to observe that
characteristics are inherited in a
predictable manner.
– He determined that alleles of a
single trait segregate (separate)
independently.
– He observed that the alleles for
different traits assort
independently.
– This gave rise to a new
discipline that we now call
Genetics Dr.T.V.Rao MD 3
What Genes can do
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. it‟s the
proteins that perform most life
functions and even make up the
majority of cellular structures. Dr.T.V.Rao MD 4
Why Genetic Disorders
When genes are
altered so that the
encoded proteins
are unable to carry
out their normal
functions, genetic
disorders can
result.
Dr.T.V.Rao MD 5
All of us carry some defective Genes,
some are apparent and many in apparent
Each of us carries about half a dozen
defective genes. We remain blissfully unaware
of this fact unless we, or one of our close
relatives, are amongst the many millions who
suffer from a genetic disease. About one in ten
people has, or will develop at some later stage,
an inherited genetic disorder, and approximately
2,800 specific conditions are known to be
caused by defects (mutations) in just one of
the patient's genes.
Dr.T.V.Rao MD 6
What we Inherit from Parents?
Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all genes, one derived from our mother and the other from our father.
Dr.T.V.Rao MD 7
Why Males differ from Females
• The only exceptions to
this rule are the genes
found on the male sex
chromosomes. Males
have one X and one Y
chromosome, the former
from the mother and the
latter from the father, so
each cell has only one
copy of the genes on
these chromosomes
XX
XY
Dr.T.V.Rao MD 8
Law of Inheritance
In the majority of cases, one normal gene
is sufficient to avoid all the symptoms of
disease. If the potentially harmful gene is
recessive, then its normal counterpart will
carry out all the tasks assigned to both.
Only if we inherit from our parents two
copies of the same recessive gene will a
disease develop.
Dr.T.V.Rao MD 9
Gene therapy is ….
Gene therapy is the insertion of
genes into an individual's cells and
tissues to treat a disease, such as a
hereditary disease in which a
deleterious mutant allele is replaced
with a functional one. Although the
technology is still in its infancy, it has
been used with some success. Dr.T.V.Rao MD 10
What is gene therapy?
• Gene therapy is an experimental technique that uses
genes to treat or prevent disease. In the future, this
technique may allow doctors to treat a disorder by
inserting a gene into a patient‟s cells instead of using
drugs or surgery. Researchers are testing several
approaches to gene therapy, including:
• Replacing a mutated gene that causes disease with a
healthy copy of the gene.
• Inactivating, or “knocking out,” a mutated gene that is
functioning improperly.
• Introducing a new gene into the body to help fight a
disease.
Dr.T.V.Rao MD 11
How It Works
A vector delivers the therapeutic gene into
a patient‟s target cell
The target cells become infected with the
viral vector
The vector‟s genetic material is inserted
into the target cell
Functional proteins are created from the
therapeutic gene causing the cell to return
to a normal state Dr.T.V.Rao MD 12
Gene Therapy is Experimental
Advances in understanding and
manipulating genes have set the
stage for scientists to alter a person's
genetic material to fight or prevent
disease. Gene therapy is an
experimental treatment that involves
introducing genetic material (DNA or
RNA) into a person's cells to fight
disease. Dr.T.V.Rao MD 13
Majority are Trails
Gene therapy is
being studied in
clinical trials
(research studies
with people) for many
different types of
cancer and for other
diseases. It is not
currently available
outside a clinical
trials
Dr.T.V.Rao MD 14
Vivo to Vitro
Dr.T.V.Rao MD 15
What Gene therapy can Achieve
Replacing a mutated gene that causes disease with a healthy copy of the gene.
Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
Introducing a new gene into the body to help fight a disease.
Dr.T.V.Rao MD 16
Uses of gene therapy
Replace missing or defective genes;
Deliver genes that speed the destruction of cancer cells;
Supply genes that cause cancer cells to revert back to normal cells;
Deliver bacterial or viral genes as a form of vaccination;
Provide genes that promote or impede the growth of new tissue; and;
Deliver genes that stimulate the healing of damaged tissue.
Dr.T.V.Rao MD 17
Genes are Medicine ?
Gene therapy is „the use of genes as
medicine‟. It involves the transfer of a
therapeutic or working gene copy into
specific cells of an individual in order to
repair a faulty gene copy. Thus it maybe
used to replace a faulty gene, or to
introduce a new gene whose function is to
cure or to favourably modify the clinical
course of a condition.
Dr.T.V.Rao MD 18
Delivering desired Genes
Dr.T.V.Rao MD 19
Gene Therapy Corrects
Gene therapy is a
technique for
correcting defective
genes responsible for
disease
development.
Researchers may
use one of several
approaches for
correcting faulty
genes: Dr.T.V.Rao MD 20
Goal of Gene therapy A normal gene may be inserted into a non-specific
location within the genome to replace a non-functional gene. This approach is most common.
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.
Dr.T.V.Rao MD 21
Steps in Gene Therapy
Dr.T.V.Rao MD 22
Dr.T.V.Rao MD 23
Manipulation corrects the
Defective Genes
Dr.T.V.Rao MD 24
Gene Therapy delivers
Proteins Today, gene therapy
is the ultimate
method of protein
delivery, in which the
delivered gene
enters the body's
cells and turns them
into small "factories"
that produce a
therapeutic protein
for a specific disease
over a prolonged
period.
Dr.T.V.Rao MD 25
Antisense therapy
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off".
Dr.T.V.Rao MD 26
Antisense Therapy
Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods
Dr.T.V.Rao MD 27
Dr.T.V.Rao MD 28
First Approved Gene Therapy
On September 14, 1990 at the U.S.
National Institutes of Health, W. French
Anderson M.D. and his colleagues R.
Michael Blaese, M.D., C. Bouzaid, M.D.,
and Kenneth Culver, M.D., performed the
first approved gene therapy procedure on
four-year old Ashanthi DeSilva. Born with
a rare genetic disease called severe
combined immunodeficiency (SCID),
Dr.T.V.Rao MD 29
What did they do In Ashanthi's gene
therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the laboratory, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream.
Dr.T.V.Rao MD 30
A success story
As of early 2007, she was still in
good health, and she was attending
college. Some would state that the
study is of great importance despite
its indefinite results, if only because it
demonstrated that gene therapy
could be practically attempted
without adverse consequences. Dr.T.V.Rao MD 31
Gene Therapy Depends on
Delivery of Corrective Genes
Viral vectors are a tool commonly used
by molecular biologists to deliver genetic
material into cells. This process can be
performed inside a living organism (in
vivo) or in cell culture (in vitro). Viruses
have evolved specialized molecular
mechanisms to efficiently transport their
genomes inside the cells they infect.
Dr.T.V.Rao MD 32
Viruses are used as Delivery
Tolls
Viruses are used as vectors to introduce
the genetic material inside the bodies.
These viruses are inactivated, they are
not able to reproduce
Adenoviruses
Herpes viruses DNA tumor viruses
Retroviruses RNA tumor viruses
Dr.T.V.Rao MD 33
Making the new Genetic Material
Functional
Gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is used to introduce the therapeutic gene into the patient's target cells. The most common vector that is used is a virus that has been genetically altered to carry normal human DNA. Viruses cause diseases in humans by encapsulating and delivering the genes into cells.
Dr.T.V.Rao MD 34
Somatic and Germ Line Gene
Therapy
Gene therapy can target somatic (body)
or germ (egg and sperm) cells. In somatic
gene therapy the recipient's genome is
changed, but the change is not passed on
to the next generation; whereas with germ
line gene therapy the newly introduced
gene is passed on to the offspring.
Dr.T.V.Rao MD 35
Safety
Safety: Although
viral vectors are
occasionally
created from
pathogenic
viruses, they are
modified in such
a way as to
minimize the risk
of handling them. Dr.T.V.Rao MD 36
Making safe Protocols
Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects.
Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. Dr.T.V.Rao MD 37
Cell type specificity
Cell type specificity:
Most viral vectors are
engineered to infect
as wide a range of
cell types as
possible.
However, sometimes
the opposite is
preferred. The viral
receptor can be
modified to target the
virus to a specific kind
of cell.
Dr.T.V.Rao MD 38
Lentivirus
Lentivirus (lenti-, Latin for "slow") is a
genus of slow viruses of the Retroviridae
family, characterized by a long incubation
period. Lent viruses can deliver a
significant amount of genetic information
into the DNA of the host cell, so they are
one of the most efficient methods of a
gene delivery vector. HIV, SIV, and FIV
are all examples of lentiviruses.
Dr.T.V.Rao MD 39
Retroviruses
Retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme
Dr.T.V.Rao MD 40
Vectors deliver the Genetic
Materials
The vector, now
called a
provirus,
remains in the
genome and is
passed on to the
progeny of the
cell when it
divides. Dr.T.V.Rao MD 41
Adenoviruses
As opposed to lenti
viruses, adenoviral
DNA does not
integrate into the
genome and is not
replicated during cell
division. Adenoviral
vectors are
occasionally used in
in vitro experiments.
Dr.T.V.Rao MD 42
Choosing non infective
Adenovirus Their primary applications are in gene
therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.
Dr.T.V.Rao MD 43
Adeno-associated viruses
Adeno-associated virus (AAV) is a small virus
which infects humans and some other primate
species. AAV is not currently known to cause
disease and consequently the virus causes a
very mild immune response. AAV can infect
both dividing and non-dividing cells and may
incorporate its genome into that of the host cell.
These features make AAV a very attractive
candidate for creating viral vectors for gene
therapy
Dr.T.V.Rao MD 44
Limitation of Direct Gene
Induction The simplest method
is the direct
introduction 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.
Dr.T.V.Rao MD 45
Nonviral approach
Nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane
Dr.T.V.Rao MD 46
Nonviral Vectors:
Liposomes less Immunogenic
DNA/lipid complexes are easy to prepare and
there is no limit to the size of genes that can be
delivered. Because carrier systems lack
proteins, they may evoke much less
immunogenic responses. More importantly, the
cationic lipid systems have much less risk of
generating the infectious form or inducing
tumorigenic mutations because genes delivered
have low integration frequency and cannot
replicate or recombine.
Dr.T.V.Rao MD 47
Nanotechnology in Gene therapy
• Nanotechnology", is the study of the control of matter on an atomic and molecular scale. Generally nanotechnology deals with structures of the size 100 nanometres or smaller, and involves developing materials or devices within that size
• The nanotechnology-based approach used by the researchers has minimal toxic side effects to normal cells
Dr.T.V.Rao MD 48
Nano engineered substances
Nonviral substances
such as Ormosil have
been used as DNA
vectors and can
deliver DNA loads to
specifically targeted
cells in living animals.
(Ormosil stands for
organically modified
silica or silicate)
Dr.T.V.Rao MD 49
Gene Therapy should not
Interfere Germ Line
The germ line of a mature or developing individual is the line (sequence) of germ cells that have genetic material that may be passed to a child
Germ line cells are immortal, in the sense that they can reproduce indefinitely. This is largely due to the activity of the enzyme known as telomerase. This enzyme extends the telomeres of the chromosome, preventing chromosome fusions and other negative effects of shortened telomeres. Dr.T.V.Rao MD 50
Creating New Chromosome
Researchers are also experimenting with
introducing a 47th artificial chromosome
to the body.
It would exist autonomously along side of
the other 46, not affecting their workings
or causing any mutations.
It would be a large vector capable of
carrying substantial amounts of genetic
information and the body‟s immune
system would not attack it.
Dr.T.V.Rao MD 51
ADA deficiency was selected for the first approved
human gene therapy trial for several reasons
The disease is caused by a defect in a single gene, which increases the likelihood that gene therapy will succeed.
The gene is regulated in a simple, “always-on” fashion, unlike many genes whose regulation is complex.
The amount of ADA present does not need to be precisely regulated. Even small amounts of the enzyme are known to be beneficial, while larger amounts are also tolerated well
Dr.T.V.Rao MD 52
Problems of Large Gene
It would be a large vector capable of
carrying substantial amounts of genetic
code, and scientists anticipate that,
because of its construction and autonomy,
the body's immune systems would not
attack it. A problem with this potential
method is the difficulty in delivering such a
large molecule to the nucleus of a target
cells.
Dr.T.V.Rao MD 53
Gene Therapy Uses AIDS Virus to Fight
AIDS
In the study, immune
cells were removed from
the patients' bodies,
modified with a disabled
AIDS virus known as a
lentivirus, and then
intravenously returned.
The genetically altered
cells disseminated anti-
HIV material and
prevented HIV from
reproducing( 07
November, 2006)
Dr.T.V.Rao MD 54
Cystic Fibrosis needs Correction-
Gene therapy can be best option
Dr.T.V.Rao MD 55
Technical Difficulties in Gene
Therapy
Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells. Delivery of genes for cancer therapy may also be complicated by the disease being present at several sites. Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have been more successful
Dr.T.V.Rao MD 56
Problems with Gene Therapy
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
Dr.T.V.Rao MD 57
Law interferes in Gene Therapy
Dr.T.V.Rao MD 58
Aldo Leopold The First
Bioethicist
A thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. It is wrong when it tends otherwise." - Aldo Leopold, 1949, A Sand County Almanac
Dr.T.V.Rao MD 59
What are the ethical issues in
Gene therapy?
How can “good” and “bad” uses of gene therapy be distinguished?
Who decides which traits are normal and which constitute a disability or disorder?
Will the high costs of gene therapy make it available only to the wealthy?
Could the widespread use of gene therapy make society less accepting of people who are different?
Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?
Dr.T.V.Rao MD 60
The Future of Gene Therapy
Current uses of gene therapy focus on
treating or curing existing conditions. In
the future, the focus could shift to
prevention. As more of the human
genome is understood, medicine will know
more about which genes contribute to or
cause disease. With that knowledge in
hand, gene therapy could be used to head
off problems before they occur.
Dr.T.V.Rao MD 61
Creating 47th Chromosome
Researchers are also experimenting with
introducing a 47th artificial chromosome
to the body.
It would exist autonomously along side of
the other 46, not affecting their workings
or causing any mutations.
It would be a large vector capable of
carrying substantial amounts of genetic
information and the body‟s immune
system would not attack it.
Dr.T.V.Rao MD 62
Several Diseases have Genetic
basis
Gene mutations probably play a role in many of
today's most common diseases, such as heart
disease, diabetes, immune system disorders,
and birth defects. These diseases are believed
to result from complex interactions between
genes and environmental factors. When genes
for diseases have been identified, scientists can
study how specific environmental factors, such
as food, drugs, or pollutants interact with those
genes.
Dr.T.V.Rao MD 63
Last two decades made rapid
progress Over the last 20
years, the initial thoughts of gene therapy have been transformed into reality with more than 175 clinical trials and 2,000 patients already treated . Yet with all the trials, there is still no conclusive evidence for efficacy. Dr.T.V.Rao MD 64
Attempting on Most
Disabling Diseases
Dr.T.V.Rao MD 65
Lesch-Nyhan syndrome,
The most likely candidates for future gene therapy trials will be rare diseases such as Lesch-Nyhan syndrome, a distressing disease in which the patients are unable to manufacture a particular enzyme. This leads to a bizarre impulse for self-mutilation, including very severe biting of the lips and fingers. The normal version of the defective gene in this disease has now been cloned.
Dr.T.V.Rao MD 66
X-linked recessive Disease
LNS is transmitted as and X-linked recessive
trait. Female carriers do
not show the symptoms.
LNS is characterized by
self-mutilating
behaviours such as lip
and finger biting and/or
head banging. The
deficiency of HPRT
activity leads to
accumulation of
phosphoribosylpyrophos
phate. Dr.T.V.Rao MD 67
Lesch-Nyhan syndrome
(an X-linked recessive disease)
Dr.T.V.Rao MD 68
Successful One Year Gene Therapy Trial For
Parkinson's Disease
A successful Documentation
Neurologix a biotech
company announced
that they have
successfully
completed its
landmark Phase I
trial of gene therapy
for Parkinson's
Disease.
Dr.T.V.Rao MD 69
Gene therapy for pain:
emerging strategies and future directions
Gene therapy to alleviate pain could appear
surprising and perhaps not appropriate when
opioids and other active molecules are
available. However, the possibility of introducing
a therapeutic protein into some targeted
structures, where it would be continuously
synthesised and exert its biological effect in the
near vicinity of, or inside the cells, might avoid
some drawbacks of "classical" drugs.
Dr.T.V.Rao MD 70
Pain – Cancer a major research area
Numerous other molecules involved in pain processing or associated with chronic pain have been identified and the gene-based techniques might be particularly adapted for the evaluation of the possible therapeutic interest of these new potential targets
Dr.T.V.Rao MD 71
Correction of Maternal Inherited
Mutation of Mitochondrial DNA
• Mutationatioal
disorders
Dr.T.V.Rao MD 72
Risks associated with current
gene therapy
Viruses can infect more than one type of cells. Viral vectors may alter more than the intended cells. Or the new gene might be inserted into the wrong location in the DNA, causing cancer or other damage.
When DNA is injected directly into a tumor there is a chance that some DNA could be introduced into germ cells, producing inheritable changes.
Dr.T.V.Rao MD 73
Risks associated with current
gene therapy
• The gene might be over-
expressed (toxicity); the viral
vector could cause
inflammation or immune
reaction; the virus could be
transmitted to other individuals
or the environment
Dr.T.V.Rao MD 74
Healthy Mitochondria fused with
Oocyst
Dr.T.V.Rao MD 75
A third Parent donates Mitochondria
Dr.T.V.Rao MD 76
Cancer gene therapy
in India
• A cell-based gene
therapy that can be used for to treat renal cell carcinoma, colon, breast, and lung cancer, has been given permission to be used in Indian patients
• The Indian Department of Biotechnology will allow this innovative therapy to be given to patients
Dr.T.V.Rao MD 77
Consent and Law
• No research on or modification of the
human genome, whether the
modification has therapeutic or
diagnostic aims, can be undertaken
without the free and informed consent
of the person concerned. In the case
of minors and others legally
incapacitated, parents or guardians
should give such consent. Dr.T.V.Rao MD 78
What are some of the ethical
considerations for using gene therapy?
• -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?
Dr.T.V.Rao MD 79
Other ethical considerations for using
Gene therapy?
• Is somatic gene therapy (which is done in the
adult cells of persons known to have the
disease) more or less ethical than germ line
gene therapy (which is done in egg and sperm
cells and prevents the trait from being passed on
to further generations)? In cases of somatic
gene therapy, the procedure may have to be
repeated in future generations.
Preliminary attempts at gene therapy are
exorbitantly expensive. Who will have access to
these therapies? Who will pay for their use?
Dr.T.V.Rao MD 80
Nature - UNESCO
• Somatic cell gene
therapy - encouraged
for any disease
- Somatic cell gene
enhancement - not to
be illegal
- Germ-line gene
therapy - not to be
illegal
- Germ-line gene
enhancement -
should not be done Dr.T.V.Rao MD 81
Understanding Genome and Human Genome
Project is a boost to Gene Therapy
Dr.T.V.Rao MD 82
Dr.T.V.Rao MD 83
Dr.T.V.Rao MD 84
Changing from Mending the Bones to Patching the Genes
Dr.T.V.Rao MD 85
Tomorrow’s imagination of life
Dr.T.V.Rao MD 86
Collins et al., Nature 4/24/03 Dr.T.V.Rao MD 87
Do not forget Genes can be
Unpredictable ?
Dr.T.V.Rao MD 88
Are we Distorting the Nature?
Dr.T.V.Rao MD 89
Is Gene therapy totally safe
??? • Although gene therapy is a promising
treatment option for a number of diseases
(including inherited disorders, some types
of cancer, and certain viral infections), the
technique remains risky and is still under
study to make sure that it will be safe and
effective. Gene therapy is currently only
being tested for the treatment of diseases
that have no other cures.
Dr.T.V.Rao MD 90
• Created by Dr.T.V.Rao MD for
Medical and Paramedical
Professionals in the Developing
World
Dr.T.V.Rao MD 91