13th Cytology lecture

September 14,2013
DNA technology and Gene therapy
 Recombinant DNA (Rdna) molecules
• are DNA molecules formed by
laboratory methods of
genetic recombination (such
as molecular cloning)
• - to bring together genetic
material from multiple
sources, creating sequences
that would not otherwise be
found in biological organisms.
• Recombinant DNA is possible because DNA
molecules from all organisms share the same
chemical structure; they differ only in the
sequence of nucleotides within that identical
overall structure.
• Consequently, when DNA from a foreign
source is linked to host sequences that can
drive DNA replication and then introduced
into a host organism, the foreign DNA is
replicated along with the host DNA
 Introduction
• Recombinant DNA molecules are
sometimes called chimeric DNA,
because they are usually made of
material from two different
species, like the mythical chimera.
R-DNA technology uses
palindromic sequences and leads
to the production of
sticky and blunt ends.
• The DNA sequences used in the
construction of recombinant DNA
molecules can originate from any
species.
For example, plant DNA may be joined to
bacterial DNA, or human DNA may be joined
with fungal DNA.
In addition, DNA sequences that do not occur
anywhere in nature may be created by the
chemical synthesis of DNA, and incorporated
into recombinant molecules. Using
recombinant DNA technology and synthetic
DNA, literally any DNA sequence may be
created and introduced into any of a very wide
range of living organisms.
• Proteins that result from the expression of
recombinant DNA within living cells are
termed recombinant proteins.
• When recombinant DNA encoding a protein is
introduced into a host organism, the
recombinant protein will not necessarily be
produced.
• Expression of foreign proteins requires the use
of specialized expression vectors and often
necessitates significant restructuring of the
foreign coding sequence.
• Recombinant DNA differs from
genetic recombination in that the
former results from artificial
methods in the test tube, while the
latter is a normal biological process
that results in the remixing of
existing DNA sequences in essentially
all organisms
 Creating recombinant DNA
• Molecular cloning is the laboratory process used to
create recombinant DNA .
• It is one of two widely used methods (along with
polymerase chain reaction, abbr. PCR) used to direct
the replication of any specific DNA sequence chosen
by the experimentalist.
• The fundamental difference between the two
methods is that molecular cloning involves replication
of the DNA within a living cell, while PCR replicates
DNA in the test tube, free of living cells
• Formation of recombinant DNA requires a cloning vector, a DNA
molecule that will replicate within a living cell.
• Vectors are generally derived from plasmids or viruses, and represent
relatively small segments of DNA that contain necessary genetic
signals for replication, as well as additional elements for convenience
in inserting foreign DNA, identifying cells that contain recombinant
DNA, and, where appropriate, expressing the foreign DNA.

The choice of vector for molecular cloning depends on

1. the choice of host organism,
2. the size of the DNA to be cloned, and
3 whether and how the foreign DNA is to be expressed. [5] The

DNA segments can be combined by using a variety of methods, such as

restriction enzyme/ligase cloning or Gibson assembly
• In standard cloning protocols, the cloning of any DNA
fragment essentially involves seven steps: (1) Choice
of host organism and cloning vector, (2) Preparation of
vector DNA,
(3) Preparation of DNA to be cloned,
(4) Creation of recombinant DNA,
(5) Introduction of recombinant DNA into the host
organism,
(6) Selection of organisms containing recombinant
DNA, and
(7) Screening for clones with desired DNA inserts and
biological properties.
 Expression of recombinant DNA
• Following transplantation into the host
organism, the foreign DNA contained within
the recombinant DNA construct may or may
not be expressed.
• That is, the DNA may simply be replicated
without expression, or it may be transcribed
and translated so that a recombinant protein
is produced.
• Generally speaking, expression of a
foreign gene requires restructuring
the gene to include sequences that
are required for producing a mRNA
molecule that can be used by the
host's translational apparatus (e.g.
promoter,
translational initiation signal, and
transcriptional terminator).
Specific changes to the host organism
may be made to improve expression of
the ectopic gene.
In addition, changes may be needed to
the coding sequences as well, to
optimize translation, make the protein
soluble, direct the recombinant protein
to the proper cellular or extracellular
location, and stabilize the protein from
degradation
 Properties of organisms containing
recombinant DNA
• In most cases, organisms containing recombinant
DNA have apparently normal phenotypes.
• That is, their appearance, behavior and
metabolism are usually unchanged, and the only
way to demonstrate the presence of recombinant
sequences is to examine the DNA itself, typically
using a polymerase chain reaction (PCR) test.
Significant exceptions exist, and are discussed
below
• If the rDNA sequences encode a gene that is expressed,
then the presence of RNA and/or protein products of
the recombinant gene can be detected, typically using
RT-PCR or western hybridization methods.
• Gross phenotypic changes are not the norm, unless the
recombinant gene has been chosen and modified so as
to generate biological activity in the host organism.
• Additional phenotypes that are encountered include
toxicity to the host organism induced by the
recombinant gene product, especially if it is
over-expressed or expressed within inappropriate cells
or tissues
• In some cases, recombinant DNA can have deleterious effects
even if it is not expressed.
• One mechanism by which this happens is insertional
inactivation, in which the rDNA becomes inserted into a host
cell’s gene. In some cases, researchers use this phenomenon to
“knock out” genes in order to determine their biological
function and importance.
• Another mechanism by which rDNA insertion into
chromosomal DNA can affect gene expression is by
inappropriate activation of previously unexpressed host cell
genes. This can happen, for example, when a recombinant DNA
fragment containing an active promoter becomes located next
to a previously silent host cell gene, or when a host cell gene
that functions to restrain gene expression undergoes
insertional inactivation by recombinant DNA
 Applications of recombinant DNA
technology
• Recombinant DNA is widely used in biotechnology, medicine
and research.
• Today, recombinant proteins and other products that result
from the use of rDNA technology are found in essentially every
western pharmacy, doctor's or veterinarian's office, medical
testing laboratory, and biological research laboratory. In
addition, organisms that have been manipulated using
recombinant DNA technology, as well as products derived from
those organisms, have found their way into many farms,
supermarkets, home medicine cabinets, and even pet shops,
such as those which sell GloFish and other genetically modified
animals
• The most common application of recombinant DNA is in basic
research, in which the technology is important to most current
work in the biological and biomedical sciences.
• Recombinant DNA is used to identify, map and sequence
genes, and to determine their function.
• rDNA probes are employed in analyzing gene expression
within individual cells, and throughout the tissues of whole
organisms.
• Recombinant proteins are widely used as reagents in
laboratory experiments and to generate antibody probes for
examining protein synthesis within cells and organisms.[2]
• Many additional practical applications of recombinant DNA are
found in industry, food production, human and veterinary
medicine, agriculture, and bioengineering.[2] Some specific
examples are identified below
 Recombinant chymosin
• found in rennet, is an enzyme required to manufacture cheese.
It was the first genetically engineered food additive to be used
commercially. Traditionally, processors obtained chymosin from
rennet, a preparation derived from the fourth stomach of milk-
fed calves. Scientists engineered a non-pathogenic strain (K-12)
of E. coli bacteria for large-scale laboratory production of the
enzyme. This microbiologically produced recombinant enzyme,
identical structurally to the calf derived enzyme, costs less and
is produced in abundant quantities. Today about 60% of U.S.
hard cheese is made with genetically engineered chymosin. In
1990, FDA granted chymosin "generally-recognized-as-safe"
(GRAS) status based on data showing that the enzyme was safe
 Recombinant human insulin
• almost completely replaced insulin obtained
from animal sources (e.g. pigs and cattle) for
the treatment of insulin-dependent diabetes.
A variety of different recombinant insulin
preparations are in widespread use.[12]
Recombinant insulin is synthesized by
inserting the human insulin gene into E. coli,
which then produces insulin for human use
Recombinant human growth hormone (HGH,
somatotropin)
• administered to patients whose pituitary glands
generate insufficient quantities to support normal
growth and development. Before recombinant HGH
became available, HGH for therapeutic use was
obtained from pituitary glands of cadavers. This
unsafe practice led to some patients developing
Creutzfeldt-Jacob disease. Recombinant HGH
eliminated this problem, and is now used
therapeutically.[14] It has also been misused as a
performance enhancing drug by athletes and others
 Recombinant blood clotting factor VIII
• a blood-clotting protein that is administered to patients
with forms of the bleeding disorder hemophilia, who
are unable to produce factor VIII in quantities sufficient
to support normal blood coagulation.
• Before the development of recombinant factor VIII, the
protein was obtained by processing large quantities of
human blood from multiple donors, which carried a very
high risk of transmission of
blood borne infectious diseases, for example HIV and
hepatitis B
 Recombinant hepatitis B vaccine 
• hepatitis B infection is controlled through the
use of a recombinant hepatitis B vaccine,
which contains a form of the hepatitis B virus
surface antigen that is produced in yeast cells.
The development of the recombinant subunit
vaccine was an important and necessary
development because hepatitis B virus, unlike
other common viruses such as polio virus,
cannot be grown in vitro.
 Diagnosis of infection with HIV 
• each of the three widely used methods for
diagnosing HIV infection has been developed using
recombinant DNA. The antibody test (ELISA or
western blot) uses a recombinant HIV protein to test for
the presence of antibodies that the body has produced in
response to an HIV infection.
• The DNA test looks for the presence of HIV genetic
material using reverse transcriptase polymerase chain
reaction (RT-PCR). Development of the RT-PCR test was
made possible by the molecular cloning and sequence
analysis of HIV genomes
 Golden rice 
• a recombinant variety of rice that has been
engineered to express the enzymes
responsible for β-carotene biosynthesis.
• This variety of rice holds substantial promise
for reducing the incidence of
vitamin A deficiency in the world's population.
• Golden rice is not currently in use, pending
the resolution of regulatory issues
 Herbicide-resistant crops
• commercial varieties of important agricultural
crops (including soy, maize/corn, sorghum,
canola, alfalfa and cotton) have been developed
which incorporate a recombinant gene that
results in resistance to the herbicide glyphosate
(trade name Roundup), and simplifies weed
control by glyphosate application.
• These crops are in common commercial use in
several countries
 Insect-resistant crops
• Bacillus thuringeiensis is a bacterium that naturally
produces a protein (Bt toxin) with insecticidal properties.
[

• The bacterium has been applied to crops as an insect-

control strategy for many years, and this practice has
been widely adopted in agriculture and gardening.
Recently, plants have been developed which express a
recombinant form of the bacterial protein, which may
effectively control some insect predators. Environmental
issues associated with the use of these transgenic crops
have not been fully resolved
 History of recombinant DNA
• The idea of recombinant DNA was first proposed by Peter
Lobban, a graduate student of Prof. Dale Kaiser in the
Biochemistry Department at Stanford University Medical School.
The first publications describing the successful production and
intracellular replication of recombinant DNA appeared in 1972
and 1973.[
• Stanford University applied for a US patent on recombinant DNA
in 1974, listing the inventors as Stanley N. Cohen and
Herbert W. Boyer; this patent was awarded in 1980.
• The first licensed drug generated using recombinant DNA
technology was human insulin, developed by Genentech and
Licensed by Eli Lilly and Company
 Controversy
• Scientists associated with the initial development of recombinant DNA
methods recognized that the potential existed for organisms containing
recombinant DNA to have undesirable or dangerous properties. At the
1975 Asilomar Conference on Recombinant DNA, these concerns were
discussed and a voluntary moratorium on recombinant DNA research was
initiated for experiments that were thought to be particularly risky. This
moratorium was widely observed until the National Institutes of Health
(USA) developed and issued formal guidelines for rDNA work.
• Today, recombinant DNA molecules and recombinant proteins are usually
not regarded as dangerous. However, concerns remain about some
organisms that express recombinant DNA, particularly when they leave the
laboratory and are introduced into the environment or food chain. These
concerns are discussed in the articles on genetically modified organisms
and genetically modified food controversies
 Gene therapy
• 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
.
• The most common form of gene therapy involves using DNA that
encodes a functional, therapeutic gene 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. In gene therapy, DNA that encodes a
therapeutic protein is packaged within a "vector", which is used to
get the DNA inside cells within the body. Once inside, the DNA
becomes expressed by the cell machinery, resulting in the production
of therapeutic protein, which in turn treats the patient's disease
• 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
Creutzfeldt-Jacob disease.
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
• Although early clinical failures led many to dismiss gene therapy
as over-hyped, clinical successes since 2006 have bolstered new
optimism in the promise of gene therapy. These include
successful treatment of patients with the retinal disease
Leber's congenital amaurosis,[X-linked SCID,[8] ADA-SCID,[9]
adrenoleukodystrophy,[10] chronic lymphocytic leukemia (CLL),
[11] acute lymphocytic leukemia (ALL),[12] multiple myeloma[13]

and Parkinson's disease.[14] These recent clinical successes have

led to a renewed interest in gene therapy, with several articles
in scientific and popular publications calling for continued
investment in the field.
• 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
Gene therapy using an adenovirus vector. A new gene is
inserted into a cell using an adenovirus. If the treatment
is successful, the new gene will make functional protein
to treat a disease
 Approach
• Scientists have taken the logical step of trying to introduce
genes directly into human cells, focusing on diseases caused by
single-gene defects, such as cystic fibrosis, haemophilia,
muscular dystrophy, thalassemia, and sickle cell anemia.
• However, this has proven more difficult than
genetically modifying bacteria, primarily because of the
problems involved in carrying large sections of DNA and
delivering them to the correct site on the gene. Today, most
gene therapy studies are aimed at cancer and hereditary
diseases linked to a genetic defect. Antisense therapy is not
strictly a form of gene therapy, but is a related, genetically
mediated therapy
• The most common form of genetic engineering involves the
insertion of a functional gene at an unspecified location in
the host genome. This is accomplished by isolating and
copying the gene of interest, generating a construct
containing all the genetic elements for correct expression,
and then inserting this construct into a random location in
the host organism. Other forms of genetic engineering
include gene targeting and knocking out specific genes via
engineered nucleases such as zinc finger nucleases,
engineered I-CreI homing endonucleases, or nucleases
generated from TAL effectors. An example of gene-knockout
mediated gene therapy is the knockout of the human CCR5
gene in T-cells to control HIV infection.This approach is
currently being used in several human clinical trials
Types of gene therapy
 1. Somatic gene therapy
• In somatic gene therapy, the therapeutic genes are
transferred into the somatic cells (non sex-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 the therapeutic DNA transgene (either
integrated in the genome or as an external episome
or plasmid) is used to treat a disease in an individual
 2. Germ line gene therapy
• In germ line gene therapy, germ cells (sperm
or eggs), are modified by the introduction of
functional genes, which are integrated into
their genomes.
• Germ cells will combine to form a zygote
which will divide to produce all the other cells
in an organism and therefore if a germ cell is
genetically modified then all the cells in the
organism will contain the modified gene.
• This would allow the therapy to be heritable and passed
on to later generations. Although this should, in theory, be
highly effective in counteracting genetic disorders and
hereditary diseases, some jurisdictions, including
Australia, Canada, Germany, Israel, Switzerland, and the
Netherlands prohibit this for application in human beings,
at least for the present, for technical and ethical reasons,
including insufficient knowledge about possible risks to
future generations[21]and higher risk than somatic gene
therapy (e.g. using non-integrative vectors).
• The USA has no federal legislation specifically addressing
human germ-line or somatic genetic modification (beyond
the usual FDA testing regulations for therapies in general)
 Viruses
• All viruses bind to their hosts and introduce their genetic
material into the host cell as part of their replication
cycle.
• Therefore this has been recognized as a plausible strategy
for gene therapy, by removing the viral DNA and using
the virus as a vehicle to deliver the therapeutic DNA.
• A number of viruses have been used for human gene
therapy, including retrovirus, adenovirus, lentivirus,
herpes simplex virus, vaccinia, pox virus, and adeno
-associated virus
 Non-viral methods
• Non-viral methods can present certain advantages over viral
methods, such as large scale production and low host
immunogenicity.
• Previously, low levels of transfection and expression of the gene
held non-viral methods at a disadvantage; however, recent
advances in vector technology have yielded molecules and
techniques that approach the transfection efficiencies of viruses.
• There are several methods for non-viral gene therapy, including
the injection of naked DNA, electroporation, the gene gun,
sonoporation, magnetofection, and the use of oligonucleotides,
lipoplexes, dendrimers, and inorganic nanoparticles
 Major
developments
in gene therapy
 1970s and earlier
• In 1972 Friedmann and Roblin authored a
paper in Science titled "Gene therapy for
human genetic disease?" Rogers (1970) was
cited for proposing that exogenous good DNA
be used to replace the defective DNA in those
who suffer from genetic defects
 1990s
• The first approved gene therapy case in the
United States took place on 14 September 1990,
at the National Institute of Health, under the
direction of Professor William French Anderson
It was performed on a four year old girl named
Ashanti DeSilva. It was a treatment for a genetic
defect that left her with ADA-SCID, a severe
immune system deficiency. The effects were
only temporary, but successful
• New gene therapy approach repairs errors in
messenger RNA derived from defective genes.
This technique has the potential to treat the
blood disorder thalassaemia, cystic fibrosis,
and some cancers.[26] Researchers at
Case Western Reserve University and
Copernicus Therapeutics are able to create
tiny liposomes 25 nanometers across that can
carry therapeutic DNA through pores in the
nuclear membrane
• Sickle-cell disease is successfully treated in mice.[28]
The mice – which have essentially the same defect
that causes sickle cell disease in humans – through
the use a viral vector, were made to express the
production of fetal hemoglobin (HbF), which
normally ceases to be produced by an individual
shortly after birth. In humans, the use of h
ydroxyurea to stimulate the production of HbF has
long been shown to temporarily alleviate the
symptoms of sickle cell disease. The researchers
demonstrated this method of gene therapy to be a
more permanent means to increase the production
of the therapeutic HbF
• In 1992 Doctor Claudio Bordignon working at the Vita-Salute
San Raffaele University, Milan, Italy performed the first
procedure of gene therapy using hematopoietic stem cells
as vectors to deliver genes intended to correct hereditary
diseases. In 2002 this work led to the publication of the first
successful gene therapy treatment for adenosine
deaminase-deficiency (SCID). The success of a multi-center
trial for treating children with SCID (
severe combined immune deficiency or "bubble boy"
disease) held from 2000 and 2002 was questioned when two
of the ten children treated at the trial's Paris center
developed a leukemia-like condition. Clinical trials were
halted temporarily in 2002, but resumed after regulatory
review of the protocol in the United States, the United
Kingdom, France, Italy, and German
• In 1993 Andrew Gobea was born with
severe combined immunodeficiency (SCID). Genetic screening before
birth showed that he had SCID. Blood was removed from Andrew's
placenta and umbilical cord immediately after birth, containing stem
cells. The allele that codes for ADA was obtained and was inserted
into a retrovirus. Retroviruses and stem cells were mixed, after which
the viruses entered and inserted the gene into the stem cells'
chromosomes. Stem cells containing the working ADA gene were
injected into Andrew's blood system via a vein. Injections of the ADA
enzyme were also given weekly. For four years T cells (white blood
cells), produced by stem cells, made ADA enzymes using the ADA
gene. After four years more treatment was needed
• The 1999 death of Jesse Gelsinger in a gene-therapy experiment
resulted in a significant setback to gene therapy research in the
United States.As a result, the U.S. FDA suspended several clinical
trials pending the re-evaluation of ethical and procedural practices in
the field
 2003
• 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.
• RNA interference or gene silencing may be a new way to treat
Huntington's disease. Short pieces of double-stranded RNA
(short, interfering RNAs or siRNAs) are used by cells to degrade
RNA of a particular sequence. If a siRNA is designed to match
the RNA copied from a faulty gene, then the abnormal protein
product of that gene will not be produced.
 2006
• Scientists at the National Institutes of Health (
Bethesda, Maryland) have successfully treated metastatic
melanoma in two patients using killer T cells genetically
retargeted to attack the cancer cells. This study constitutes
one of the first demonstrations that gene therapy can be
effective in treating cancer.[37]
• In March 2006 an international group of scientists announced
the successful use of gene therapy to treat two adult patients
for a disease affecting myeloid cells. The study, published in
Nature Medicine, is believed to be the first to show that gene
therapy can cure diseases of the myeloid system
• In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown
from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in
Milan, Italy reported a breakthrough for gene therapy in which they
developed a way to prevent the immune system from rejecting a newly
delivered gene.
• Similar to organ transplantation, gene therapy has been plagued by the
problem of immune rejection. So far, delivery of the 'normal' gene has been
difficult because the immune system recognizes the new gene as foreign and
rejects the cells carrying it.
• To overcome this problem, the HSR-TIGET group utilized a newly uncovered
network of genes regulated by molecules known as microRNAs. Dr. Naldini's
group reasoned that they could use this natural function of microRNA to
selectively turn off the identity of their therapeutic gene in cells of the
immune system and prevent the gene from being found and destroyed. The
researchers injected mice with the gene containing an immune-cell microRNA
target sequence, and the mice did not reject the gene, as previously occurred
when vectors without the microRNA target sequence were used. This work
will have important implications for the treatment of hemophilia and other
genetic diseases by gene therapy.
• In November 2006 Preston Nix from the
University of Pennsylvania School of Medicine reported on VRX496,
a gene-based immunotherapy for the treatment of
human immunodeficiency virus (HIV) that uses a lentiviral vector
for delivery of an antisense gene against the HIV envelope. In the
Phase I trial enrolling five subjects with chronic HIV infection who
had failed to respond to at least two antiretroviral regimens, a
single intravenous infusion of autologous CD4 T cells genetically
modified with VRX496 was safe and well tolerated. All patients had
stable or decreased viral load; four of the five patients had stable
or increased CD4 T cell counts. In addition, all five patients had
stable or increased immune response to HIV antigens and other
pathogens. This was the first evaluation of a lentiviral vector
administered in U.S. Food and Drug Administration-approved
human clinical trials for any disease.[40] Data from an ongoing Phase
I/II clinical trial were presented at CROI 2009
 2007
• On 1 May 2007 Moorfields Eye Hospital and
University College London's Institute of Ophthalmology announced
the world's first gene therapy trial for inherited retinal disease. The
first operation was carried out on a 23 year-old British male,
Robert Johnson, in early 2007.[42] Leber's congenital amaurosis is an
inherited blinding disease caused by mutations in the RPE65 gene.
The results of the Moorfields/UCL trial were published in New
England Journal of Medicine in April 2008. [43] They researched the
safety of the subretinal delivery of recombinant adeno-associated
virus (AAV) carrying RPE65 gene, and found it yielded positive
results, with patients having modest increase in vision, and,
perhaps more importantly, no apparent side-effects
 2008

• In May 2008, two more groups, one at the University

of Florida and another at the University of
Pennsylvania, reported positive results in
independent clinical trials using gene therapy to
treat Leber's congenital amaurosis.
• In all three clinical trials, patients recovered
functional vision without apparent side-effects.
These studies, which used adeno-associated virus,
have spawned a number of new studies investigating
gene therapy for human retinal disease
 2009
• In September 2009, the journal Nature reported
that researchers at the University of Washington and
University of Florida were able to give trichromatic
vision to squirrel monkeys using gene therapy, a
hopeful precursor to a treatment for color blindness
in humans.[44] In November 2009, the journal Science
reported that researchers succeeded at halting a
fatal brain disease, adrenoleukodystrophy, using a
vector derived from HIV to deliver the gene for the
missing enzyme
 2010
• A paper by Komáromy et al. published in April 2010, deals with gene therapy for a
form of achromatopsia in dogs. Achromatopsia, or complete color blindness, is
presented as an ideal model to develop gene therapy directed to cone
photoreceptors. Cone function and day vision have been restored for at least 33
months in two young dogs with achromatopsia. However, the therapy was less
efficient for older dogs.
• In September 2010, it was announced that a patient in France had been cured of
beta-thalassemia. Headed by Dr. Phillipe Leboulche, the study used a lentiviral
vector to transduce the human β-globin gene into purified blood and marrow
cells obtained from the patient.[47] Between 21 and 33 months after the
transplant, the percentage of vector-bearing cells in his blood gradually increased,
and today, the 18-year-old patient is transfusion independent (ever since June
2008).[47] His hemoglobin levels are presently stable at 9 to 10 g/dL and about a
third of the hemoglobin contains the form introduced by the viral vector. [47] The
clinical trial is, therefore, being heralded as a success around the world
 2011

• In 2007 and 2008, a man being treated by Gero Hütter was cured of HIV by
repeated Hematopoietic stem cell transplantation (see also Allogeneic
stem cell transplantation, Allogeneic bone marrow transplantation,
Allotransplantation) with double-delta-32 mutation which disables the CCR5
receptor; this cure was not completely accepted by the medical community
until 2011. This cure required complete ablation of existing bone marrow
which is very debilitating.
• In August 2011, two of three subjects of a pilot study were confirmed to
have been cured from chronic lymphocytic leukemia (CLL). The study carried
out by the researchers at the University of Pennsylvania used genetically
modified T cells to fight the disease.[11]
• Human HGF plasmid DNA therapy of cardiomyocytes is being examined as a
potential treatment for coronary artery disease as well as treatment for the
damage that occurs to the heart after myocardial infarction
 2012
• In July 2012, the European Medicines Agency recommended
approval of a gene therapy treatment for the first time in either
Europe or the United States. The treatment, called Glybera,
compensates for lipoprotein lipase deficiency, which can cause
severe pancreatitis.[53] The recommendation was endorsed by the
European Commission in November 2012 and commercial rollout is
expected in late 2013.[54]
• In December 2012, it was reported that 10 of 13 patients with
multiple myeloma were in remission "or very close to it" three
months after being injected with a treatment involving genetically
engineered T cells to target proteins NY-ESO-1 and LAGE-1 which
exist only on cancerous myeloma cells. This procedure had been
developed by a company called Adaptimmune
 2013
• In March 2013, Researchers at the Memorial Sloan-Kettering Cancer Center in New
York, reported that three of five subjects who had acute lymphocytic leukemia
(ALL) had been in remission for five months to two years after being treated with
genetically modified T cells which attacked cells with CD19 genes on their surface,
i.e. all B-cells, cancerous or not. The researchers believed that the patients immune
systems would make normal T-cells and B-cells after a couple of months however
they were given bone marrow to make sure. One patient had relapsed and died
and one had died of a blood clot unrelated to the disease. [12]
• In July 2013 the Italian San Raffaele Telethon Institute for Gene Therapy (HSR-
TIGET) reported that six children with two severe hereditary diseases had been
treated with a partially deactivated lentivirus to replace a faulty gene and after 7-32
months the results were promising. Three of the children had metachromatic
leukodystrophy which causes children to lose cognitive and motor skills. [55] The
other children had Wiskott-Aldrich syndrome which leaves them to open to
infection, autoimmune diseases and cancer due to a faulty immune system
 Problems
• Some of the problems of gene therapy include:
• Short-lived nature of gene therapy – Before gene therapy can become a
permanent cure for any condition, the therapeutic DNA introduced into
target cells must remain functional and the cells containing the
therapeutic DNA must be long-lived and stable. Problems with
integrating therapeutic DNA into the genome and the rapidly dividing
nature of many cells prevent gene therapy from achieving any long-term
benefits. Patients will have to undergo multiple rounds of gene therapy.
• Immune response – Any time a foreign object is introduced into human
tissues, the immune system is stimulated to attack the invader. The risk
of stimulating the immune system in a way that reduces gene therapy
effectiveness is always a possibility. Furthermore, the immune system's
enhanced response to invaders that it has seen before makes it difficult
for gene therapy to be repeated in patients.
• Problems with viral vectors – Viruses, the carrier of choice in
most gene therapy studies, present a variety of potential
problems to the patient: toxicity, immune and inflammatory
responses, and gene control and targeting issues.
• In addition, there is always the fear that the viral vector, once
inside the patient, may recover its ability to cause disease.
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, high blood pressure,
Alzheimer's disease, 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
(although a 2013 trial using genes Ins and Gck reported promise
in treating diabetes in dogs[57]).
• For countries in which germ-line gene therapy is illegal,
indications[58] that the Weismann barrier (between soma and
germ-line) can be breached are relevant; spread to the testes,
therefore could impact the germline against the intentions of
the therapy. Chance of inducing a tumor (insertional
mutagenesis) – 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.[59][60] One possible solution
for this is to add a functional tumor suppressor gene onto the
DNA to be integrated; however, this poses its own problems,
since the longer the DNA is, the harder it is to integrate it
efficiently into cell genomes
• Three patients' deaths have been reported in
gene therapy trials, putting the field under close
scrutiny.
• The first was that of Jesse Gelsinger in 1999,
which represented a major setback in the field.
One X-SCID patient died of leukemia following
gene therapy treatment in 2003.[2] In 2007, a
rheumatoid arthritis patient died from an
infection in a gene therapy trial; a subsequent
investigation concluded that the death was not
related to her gene therapy treatment
 Preventive gene therapy
• Preventive gene therapy is the repair of a gene with a mutation associated
with a progressive disease, prior to the expression of a medical condition, to
prevent that expression.
• One case study of preventive gene therapy: Retinitis Pigmentosa
• Blindness can be caused by multiple genetic diseases. Many gene therapy
efforts have been focused on treating blindness as a result of moderate
success in preventing the loss of vision in multiple animal models. To do this,
blindness must be diagnosed early on before the symptoms begin. The
retina, which is located in the back of the eyeball, is the first step in
processing visual information, accordingly it is a common target in
exploration of the genetic issue that leads to blindness. One autosomal
genetic disease that has been extensively researched is retinitis pigmentosa
(RP) because it has excellent animal models for genetic therapy techniques
to treat blindness
• Within a single disease, there can be multiple preventative gene
therapy strategies used to combat the progression of the
symptoms. Most people who suffer from RP are born with
rod cells that are either dead or dysfunctional, so they are
effectively blind at nighttime, since these are the cells
responsible for vision in low levels of light. What follows often is
the death of cone cells, responsible for color vision and acuity, at
light levels present during the day. Loss of cones leads to full
blindness as early as five years old, but may not onset until many
years later. There have been multiple hypotheses about how the
lack of rod cells can lead to the death of cone cells. Pinpointing a
mechanism for RP is difficult because there are more than 39
genetic loci and genes correlated with this disease. In an effort
to find the cause of RP, there have been different gene therapy
techniques applied to address each of the hypothese
• One hypothesis is that when the rod cells die, there is no longer a
critical growth factor secreted for proper cone function and survival.
Some scientists have experimented with treating this issue by injecting
substitute trophic factors into the eye. One group of researchers
injected the rod derived cone viability factor (RdCVF) protein (encoded
for by the Nxnl1 (Txnl6) gene) into the eye of the most commonly
occurring dominant RP mutation rat models. This treatment
demonstrated success in promoting the survival of cone activity, but
the treatment served even more significantly to prevent progression
of the disease by increasing the actual function of the cones. [64] Two of
the major issues encountered when trying to inject these desirable
proteins, is that they are both difficult to purify and difficult to deliver
multiple times as a consistent treatment. Researchers are currently
trying to develop an adeno-associated virus (AAV) vector to use for
such treatments to address these problems. Injection of an AAV vector
encoding this factor might only have to happen once
• Another issue is choosing which protein to inject. Multiple candidates such as brain derived
neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth
factor (bFGF), and pigment epithelium derived factor (PEDF) have demonstrated functional
success within the eye when they are delivered to the subretinal region. One of the most
successful proteins of those aforementioned, is ciliary neurotrophic factor (CNTF). CNTF has
shown success of slowing retinal degradation in 13 different animal models. CNTF is
currently being used as a treatment in human clinical trials.[65] In Phase I of the clinical trial,
there were 10 participants suffering from advanced RP who each had one of their eyes
implanted with CNTF encapsulated cell devices for six months, followed by explantation.
Five of the participants received higher dose implants of CNTF, and the other five received
an approximately five times smaller dose implant. Multiple participants displayed improved
acuity both while the implant was in the eye, and one month later when retested. In some
of the other animal models, high doses of CNTF have demonstrated increased
photoreceptor metabolic activity, which suggests that it is possible for CNTF to increase
photoreceptor metabolic activity in human damaged cones as well. CNTF could improve
metabolic activity in photoreceptor cones to the extent that it improves visual acuity, as
seen in several of the participants who performed significantly better on the letter
recognition task compared to their respective pretreatment baseline. Although not all of
the participants experienced a significant increase in visual acuity, this method of
encapsulated implantations poses an intriguing line of research. It offers the advantage of
being reversible, as one can remove the encapsulated cells should anything go wrong. Gene
therapy using viral vectors to deliver a therapeutic gene is not obviously reversible
 In popular culture
• In the TV series Dark Angel gene therapy is mentioned as one of the practices
performed on transgenics and their surrogate mothers at Manticore, and in the
episode Prodigy, Dr. Tanaka uses a groundbreaking new form of gene therapy to turn
Jude, a premature, vegetative baby of a crack/cocaine addict, into a boy genius. Gene
therapy is a crucial plot element in the video game Metal Gear Solid, where it has
been used to illegally enhance the battle capabilities of soldiers within the US
military, and their Next Generation Special Forces units. Gene therapy plays a major
role in the sci-fi series Stargate Atlantis, as a certain type of alien technology can only
be used if one has a certain gene which can be given to the members of the team
through gene therapy involving a mouse retrovirus. Gene therapy also plays a major
role in the plot of the James Bond movie Die Another Day, where a scientist has
developed a means of altering peoples' entire appearances through the use of DNA
samples acquired from others- generally homeless people that would not be missed-
that are subsequently injected into the bone marrow, the resulting transformation
apparently depriving the subjects of the ability to sleep
• Gene therapy plays a recurring role in the present-time sci-fi television program
ReGenesis, where it is used to cure various diseases, enhance athletic performance
and produce vast profits for bio-tech corporations. (e.g. an undetectable
performance-enhancing gene therapy was used by one of the characters on himself,
but to avoid copyright infringement, this gene therapy was modified from the tested-
to-be-harmless original, which produced a fatal cardiovascular defect)
• Gene therapy is the basis for the plot line of the film I Am Legend.[66]
• Gene therapy is an important plot key in the game Bioshock where the game
contents refer to plasmids and [gene] splicers.
• The book Next by Michael Crichton unravels a story in which fictitious biotechnology
companies experiment with gene therapy. In the television show Alias, a
breakthrough in molecular gene therapy is discovered, whereby a patient's body is
reshaped to identically resemble someone else. Protagonist Sydney Bristow's best
friend was secretly killed and her "double" resumed her place. In the 2011 film
Rise of the Planet of the Apes, a fictional gene therapy called ALZ-112 was a drug that
was a possible cure for Alzheimer's disease, the therapy increased the host's
intelligence and made their irises green, along with the revised therapy called 113
which increased intelligence in apes yet was a deadly, internal virus in humans
The
End