GENE

THERAPY
 Basics
 mankaran singh
 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.
 When genes are
altered so that the
encoded proteins
are unable to carry
out their normal
functions, genetic
disorders can
result.
 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.
 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. 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
 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.
 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.
 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
 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.
 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
 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.
 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.
 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.
 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.
 Gene therapy is a
technique for
correcting defective
genes responsible for
disease development.
Researchers may use
one of several
approaches for
correcting faulty
genes:
 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.
 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".
 Antisense therapy
is not strictly a
form of gene
therapy, but is a
genetically-
mediated therapy
and is often
considered
together with
other methods
 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),
 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.
 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.
 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.
 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.
 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
 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.
 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.
 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.
 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.
 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.
 Lentivirus (lenti-, Latin for "slow") is a
genus of slow viruses of the Retroviridae
family, characterized by a long incubation
period. Lentiviruses 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.
 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
 The vector, now
called a provirus,
remains in the
genome and is
passed on to the
progeny of the cell
when it divides.
 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.
 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.
 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
 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.
 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
 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.
 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)
 Transfection is the
process of introducing
nucleic acids into cells by
non-viral methods. The
term "transformation" is
preferred to describe non-
viral DNA transfer in
bacteria and non-animal
eukaryotic cells;
"transduction" is often used
to describe virus-mediated
DNA transfer.
 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.
 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.
 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
 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.
 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) 
 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
 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
 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
 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?
 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.
 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.
 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.
 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.
 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.
 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
phosphoribosylpyrophosph
ate.
 Neurologix a biotech company announced
that they have successfully completed its
landmark Phase I trial of gene therapy for
Parkinson's Disease.

 This was a 12 patient study with four patients in each

of three dose escalating cohorts. All procedures were
performed under local anesthesia and all 12 patients
were discharged from the hospital within 48 hours of
the procedure, and followed for 12 months. Primary
outcomes of the study design, safety and tolerability,
were successfully met. There were no adverse events
reported relating to the treatment.
 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.
 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.
 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.
 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
Email

doctortvrao@gmail.com