Transcript Human Gene Therapy

ZEESHAN GAUHAR
PhD SCHOLOR-BIOTECHNOLOGY
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GENE THERAPY
APPROACHES
HISTORY
TYPES
GERMLINE
SOMATIC GENE THERAPY
VECTORS
VIRAL
NON VIRAL
DISADVANTAGES
GENE THERAPY IN SICKLE CELL ANEMIA
ETHICAL AND SOCIAL CONSIDERATION
CONCLUSION
Gene therapy is the use of genes as
medicines, basically to correct defective
genes responsible for genetic disorder.
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IV.
A normal gene could be inserted into a nonspecific
location within the genome to replace the
Nonfunctional gene (most common)
An abnormal gene could be swapped for a normal
gene homologous recombination
An abnormal gene could be repaired through
selective reverse mutation
Regulation (degree to which a gene is turned on or
off) of a particular gene could be altered
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1990, first approved gene therapy case in the United States
took place on a four year old girl for the treatment of ADASCID, a severe immune system deficiency. The effects were
only temporary, but successful.
1992, Claudio Bordignon of Italy performed the first
procedure of gene therapy using hematopoietic stem cells as
vectors to deliver genes intended to correct hereditary
disease.
1993, a new born baby Andrew Gobea, with SCID, was
treated by gene therapy technique using retrovirus vector
carrying ADA gene.
1999, gene therapy suffered a major setback with the death
of 18 year old Jesse Gelsinger who participated in a gene
therapy trial for ornithine transcarboxylase deficiency.
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2003, “FOOD and DRUG ADMINISTRATION” (FDA)
placed a temporary halt on all gene therapy trials using
retrovirus vector in blood stem cells.
Then in April 2003, FDA eased the ban after regulatory
review of the protocol in USA, UK, France, Italy and
Germany.
2003, Los Angeles research team inserted genes into brain
using liposome coated in a polymer called polyethylene
glycol.
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In 2005, scientists were able to repair deafness in guinea pig
by using adenovirus vector.
In 2006 (March), an international group of scientists
announced the successful use of gene therapy to treat two
adult patients for a disease affecting myeloid cells.
In 2007, a team of British doctors from Moorefield’s Eye
Hospital and University college of London, announced the
world’s first gene therapy trial to test a revolutionary gene
therapy treatment for a type of inherited retinal disease.
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In 2009 (March), the School of Pharmacy in London tried
nanotechnology based gene therapy to target and destroy
hard-to-reach cancer cells.
In 2010, a paper by Komaromy et al. report gene therapy
for a form of achromatopsia (complete colour blindness) in
dogs.
In 2012, Glybera became the first gene therapy treatment to
be approved for clinical use in either Europe or the United
States after its endorsement by the European Commission.
1. Germ line gene therapy: germ cells (sperm or egg) are
modified by the introduction of functional genes, which
are integrated into their genome.
2. Somatic gene therapy: therapeutic genes are
transferred into the somatic cells of a patient.
Two types of vectors are used,
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Viral
2.
Non viral
Viruses have evolved a way of encapsulating and delivering
their genes to human cells in a pathogenic manner.
Scientists have tried to harness their ability by
manipulating the viral genome to remove disease causing
genes and insert theurapatic ones.
RNA viruses
 Use integrase to integrate its genome into the host
genome
Problems
 integrase enzyme can cause insertional mutagenesis i.e.
can insert genetic material of the virus into any
arbitrary position in the genome of the host.
successful application till date
 X linked severe combined immune deficiency
 SCID due to ADA deficiency with relative success.
 Cystic fibrosis
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single stranded DNA viruses
 can infects a broad range of cells
 Can insert genetic material at a specific site on
chromosome19 with near 100% certainty
Drawbacks
 A small virus, carrying only 2 genes in its natural state
 can produce unintended genetic damage because the
virus inserts its genes directly into host cell’s DNA
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Double stranded DNA viruses
human neurotropic virus
large genome which enable scientist to insert more than
one therapeutic gene into a single virus
HSV makes an ideal vector as it can infect a wide range
of tissues including muscle, liver, pancreas, and nerve
and lung cells.
Double stranded DNA viruses
 DNA molecule is left free in the nucleus of the host
cell, and transcribed just like any other gene
 can infect a broader variety of cells
Applications
Gendicine,first gene therapy product to be licensed to
treat head and neck cancer
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electroporation (creation of electric field induced pores
in plasma membrane)
sonoporation (ultrasonic frequencies to disrupt cell
membrane),
magnetofection (use of magnetic particle complexed
with DNA),
gene guns (shoots DNA coated gold particles into cells
by using high pressure)
receptor mediated gene transfer
use of synthetic oligonucleotides (to inactivate
defective genes by using antisense specific to target
gene)
 lipoplexes (made up of anionic and neutral lipids)
 polyplexes (complex of polymers with DNA)
 Hybrid
methods e.g vibrosomes that combine
liposomes with an inactivated HIV or influenza virus
and viral vectors with cationic lipids
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Short-lived nature of gene therapy
Immune response
Problem with viral vectors
Multigenic disorders
Insertional mutagenesis
sickle cell anemia
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caused by a change in just one amino acid at a specific
site in the β-globin gene.
results in the production of sickle shaped cells which
prevent oxygenated blood from flowing through.
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the first sickle-cell gene therapy to be tested in humans.
At the regenerative medicine and stem cell research
centre of the University of California, Los Angeles,
molecular geneticist and physician Donald Kohn is
developing protocols for a clinical trial of this
technique that is due to start enrolling patients by the
end of 2014.
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first harvest bone marrow from the hip bones of patients
with sickle-cell disease
then extract haematopoietic stem cells from the marrow
Using a viral vector,insert a new, working haemoglobin
gene into the cells’ DNA
the old, faulty haemoglobin gene will still be present, but it
will go silent as the new gene takes over
The modified cells will then be infused back into the
patient’s bloodstream
They will migrate to the bone marrow, where they can
provide a continual source of healthy red blood cells.
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This technique has been tested by injecting modified
human haematopoietic stem cells into mice, and
found that they were free of sickle cells 2 to 3 months
later.(Romero, Z. et al. J. Clin. Invest. 123, 3317–
3330 (2013).
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researchers must harvest the bone marrow
itself, which can be difficult and slow, and
limits the number of cells that can be collected
at one time
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Further away from clinical trials, but potentially a lot
more exciting, is gene editing.
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Zinc finger nucleases(ZFNs)
Transcription
activator-like
nuclease(TALEN)
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CRISPRs (clustered
palindromic repeats)
regularly
effector
interspaced
short
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Zinc-finger nucleases (ZFNs) are artificial restriction
enzymes generated by fusing a zinc finger DNAbinding domain to a DNA-cleavage domain.
engineered zinc finger nucleases (ZFNs) bind to a
specific section of DNA and create a break at both ends
Cells will start to repair the break, at which point a
specific sequence of laboratory-made DNA can be
slotted into the gap.
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Transcription activator-like effector nucleases
(TALENs) are artificial restriction enzymes generated
by fusing a TAL effector DNA binding domain to a
DNA cleavage domain.
Transcription activator-like effectors (TALEs) can be
quickly engineered to bind practically any desired DNA
sequence. By combining such an engineered TALE
with a DNA cleavage domain (which cuts DNA
strands), one can engineer restriction enzymes that are
specific for any desired DNA sequence.
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DNA loci containing short repetitions of base
sequences.
CRISPRs are often associated with cas genes that code
for proteins related to CRISPRs.
used for gene editing since 2013.
Some of the ethical considerations for gene therapy
include:
 Deciding what is normal and what is a disability
 Deciding whether disabilities are diseases and whether
they should be cured
 Deciding whether searching for a cure demeans the live
of people who have disabilities
 Deciding whether somatic gene therapy is more or less
ethical than germ line gene therapy
scientists believe that after 20 years, this will be the last
cure of every genetic disease. Genes may ultimately be
used as medicine and given as simple intravenous
injection of gene transfer vehicle that will seek our
target cells for stable, site-specific chromosomal
integration and subsequent gene expression. And now
that a draft of the human genome map is complete,
research is focusing on the function of each gene and
the role of the faulty gene play in disease. Gene therapy
will ultimately change our lives forever.
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Human Gene Therapy : A Brief Overview of the Genetic Revolution Sanjukta
Misra*
Romero, Z. et al. J. Clin. Invest. 123, 3317–3330 (2013).
Fine, E. J. et al. Nucleic Acids Res. 42, e42 (2014).
Suzuki, K. et al. Cell Stem Cell 15, 31–36 (2014).
THANKS