gene therapy and its potential in treating cystic fibrosis


Dec 12, 2012 (4 years and 8 months ago)


Meyers 2:00


University of Pittsburgh

March 1, 2012

Swanson School of Engineering



Sean O’Connor (
), Kevin Hulbert (


Genetic Engineering has been changing the world
of research and medicine since the late 1900’s
. This paper is
about the ever
changing field of gene therapy and its
to cure
cystic fibrosis. It will describe the complex
processes and transformations involved in the gene therapy
treatments of cystic fibrosis. This research

done on cystic
fibrosis is
the best model for gene therapy treatment of other
illnesses (Wagner 203). This therapy raises ethical issues
with social and

political ramifications. These ethical
concerns will be discussed in the paper.

Current methods of somatic gene therapy transfer
involve the processes of viral and non
viral injections into
the cells of the human body. The viral method, using viruses
as v
ectors, has become the more successful of the two
(Dhillon 1). Their viral transformation and how they work in
treatment of CF will be d
iscussed in more detail in this
paper. Understanding the cystic fibrosis trans
conductance regulator gene (CFTR
) is a major aspect in the
treatment of CF. This gene is crucial in regulating proteins
that secrete chloride ions. When mutated, defective protein
processing and production may ensue, as well as defective
chloride secretion. This may cause a buildup of pr
otein in
the lungs and subsequent fatal lung disease. As a target for
gene therapy development, CF is one of the most extensively
researched genetic diseases (Wagner 203). Unfortunately,
the progress of this gene therapy research has been slowed
because of

the complexity of multi
genetic diseases (Bosone
1). Research is still in progress

Key Words

Gene Therapy, Cystic Fibrosis, Genetic
Engineering, Ethics, Cystic Fibrosis Trans
Conductance Regulator, Bioengineering, Animal Research,
Viral Transf



Before gene therapy, permanent cures for some of the
deadliest diseases were hopeless and unlikely to ever occur.
Now with research in gene therapy, these diseases have the
opportunity to be permanently cure
Cystic Fibrosis is a
genetic disease that is well known and has caused a lot of
suffering and death in the United States and around the
world. It is fairly common disease, affecting
1 in 2500
Caucasian newborns [1].

Cystic fibrosis is the result in a v
ariety of genetic
mutations of the Cystic Fibrosis Trans
Conductance Regulator gene (CFTR) in the human genome.

The CFTR gene is found in region q31.2 on the long (q) arm
of human chromosome 7.






Two scientists, Collins and Tsui, discovered the location of
the gene at Yale University in 1989.
When functioning
CFTR codes for the production of a protein made
up of 1480 amino acids [2]. This protein

product is a channel
for chloride ion
s and is located in the epithelial cells lining
the lungs, liver, pancreas, reproductive tracts, intestines, and
For this gene to fire and secrete Cl

phosphorylation must occur (PO43

The exact structure
of the CFTR protein product has n
ot been determined
through research but after being compared to other
transporter proteins, it is the only one with a fifth regulatory

The vast majority of Cystic Fibrosis cases are due to a
deletion mutation of three base pairs in the gene.
deletion causes loss of the amino acid phenylalanine located
at position 508 in the protein; therefore, this mutation is
referred to as delta F508
. The protein will not fold properly
and will eventually degrade. Also found is that

a mutation in
the S3
41A position can alter
the affinity and voltage
dependence of the protein, vital to blocking and opening the
channels of CFTR [3]. This change in affinity and voltage
may cause less phosphorylation, rendering the CFTR protein
almost useless.

With this bein
g said, mucus and bacteria may
build up

causing lung disease. The lack of chlorine in the
cells of the pulmonary tract
, when one has CF, inhibits
airway surface liquid (mediated by Cl
) to adjust, in turn
preventing periciliar liquid layer (PCL
) to from at

a correct
height [4
]. This PCL is what “lubricates” airways allowing
mucus and bacteria to pass through without clotting/blocking
the airway. Without this CF becomes one of the deadliest
diseases on the planet.

Sean O’Connor

Kevin Hulbert


Treatment of the Disease before Gene Therap

Before gene therapy research, there seemed to be no hope of
curing cystic fibrosis and those who are unfortunately
diagnosed with it were cursed to live a shortened life filled
with pain and infection. The build of mucous in respiratory
canals often cau
sed infection and made it very difficult to
breathe. Many patients end up with continuous
hospitalizations, endless antibiotics, and enzyme
supplements. In the 1990’s, it was predicted that babies
diagnosed with cystic fibrosis could
live up to about 40
ars old [5

Many antibiotic drugs have been developed to
help such as ticarcillin, piperacillin, and ceftazidime. The
use of inhaled antibiotics such as coliston has proved to be
successful in improving pulmonary function in the past.
However there are us
ually cons involved with most medical
treatments. The development of multidrug resistant
microorganisms rises everyday and continues to cause
problems in treating cystic fibrosis.

This occurs because this
treatment method involves large amounts of intakes
antibiotics making it easier to become resistant.

Different methods of physical therapy and exercise have
been used to try and clear obstructed airways in patients.

Patients are often put in positions that allow for drainage of
the mucus formed in t
he lungs while a percussion machine
pats the back or chest to dislodge mucus from the walls of
the airways. Medications such as b
mucolytics, and decongestants

are available on the mark
but can prove to be costly [5

The patient is encour
aged to
exercise consistently because it also helps clear the lungs.
Children often need to be treated by their family members
but with age, may be able to use these techniques on their

Another method used is lung transplantation. This is a

more sever method that is very expensive and is hard
to come by because of the large need for it. It is suggested
mainly for patients that experience severe respiratory failure.
This treatment, if successful, allows for normal CFTR
proteins to be formed b
ut normal transplant complications
are always possibility. All of the older methods can help
prolong the inevitable among patients but over the years,
they become very costly and do not provi
de a permanent

An increase in research in gene therapy
and the
development of higher technologies continues to allow for a
promising future.

If gene therapy is perfected in treating
cystic fibrosis, then a truly permanent treatment may finally
be possible.


Genetic engineering research has and continues to put a lot
of work into developing gene therapy and the technology
involved with it.
Gene therapy is a process involved in
genetic engineering that allows for modification of genes
internally and externally

to treat diseases. The attention of
cystic fibrosis is mainly
on the respiratory tracts because it
proves to be the most problematic. The defect gene for
CFTR is cloned and modified. Chloride transport was made
possible and this new product is called wild
type CFTR.
process of cloning the DNA is made easier with real
quantitative PCR (Q

PCR is a polymerase chain
reaction and its products can now be measured in real time.
A fluorescent signal is generated from the reaction and is
captured by
instruments such as TaqMan Probes, Molecular
beacons, and SYBR intercalating green dyes. The probes are
capable of determining the
expression level of specific allele
in comparison to the allele one nucleotide away.
It measures
mRNA expression, DNA copy nu
mbers, transgene copy
number and expression analysis, allele discrimination, and
viral titers [6]. This technology has only recently become a
useful factor in gene therapy and provides for the
opportunity of using less expensive instruments. Q
s exponentially a graph of DNA copies until it
plateaus off.

The measurements that are collected allow for
knowledge of exact placement and expression of the CFTR



External strategies prove harder for the cells of the lungs
so an in
vivo process is needed.
Vectors must be applied
directly to the target cells for in
vivo methods. Vectors are
modes of transport for the newly modified healthy genes.
Many different v
ectors show promise in being successfully
taken in by h
umans, but viral vectors
show the most
There are many different types of viral vectors,
different from the other in regards to DNA copying,
administration into the
subject, and how it bon
ds to the
CFTR. There are retroviruses, adenoviruses, lentiviruses,
associated viruses, and
engineered substances.
Adenoviral and retroviral vectors have been more successful
when tested than other forms of viral vectors.
When the viral
vector r
oute is taken, t
he virus DNA originally starts in the
form of a plasmid, a ring of DNA. The goal is to get this

Sean O’Connor

Kevin Hulbert


virus to deliver the healthy CFTR to a target cell but not to
replicate or form more viruses. The replication gene and the
capsid gene are remov
ed from the virus.

A capsid is a shell
that surrounds the virus. The removal of these genes can be
done recombinant DNA (rDNA) technology.

enzymes are used to cleave the DNA at a specific site. This
leaves two “sticky” ends on the plasmid.

CFTR gene is
then inserted into the plasmid

and is joined by a DNA ligase
enzyme [7]
. Once the vector has been built. The vector is
placed into a packaging cell line that contains a helper virus
and the replication and capsid genes that were removed
. This

packaging cell line combines the elements with the vector
and aids in replicating the virus with the new plasmid in it.
Cells become full of the virus and they burst. This is when
they are collected into a pure liquid solution of viruses. The
new virus ve
ctor is then put into the infected unhealthy cells
of the lungs.

Methods of injection into cells for gene therapy
include heat shock, electroporation, viruses (produced by
above description),
a gene gun. Heat shock involves
heating the host cells sudde
nly causing its membrane to
become permeable enough for the new DNA to pass
through. Electroporation involves a similar process where
electric shock allows for increased permeability. A gene gun
fires gold particles coated with foreign DNA segments.

or injected DNA

infects the cells and
the new DNA
becomes integrated into a specific location on chromosome
19. This new DNA successfully codes for the correct mRNA
to produce healthy CFTR cells.

This is the general way a
viral vector works, but adenov
iral and retroviral vectors
work slightly differently.

Adenoviruses are medium sized icosahedral viruses
composed of a nucleocapsid and a double
stranded DNA
genome [8]. A nucleocapsid is a viral sac that is made up of
a nucleic acid and protein coat.

The nucleic acid is what
holds the DNA.
Adenoviruses represent the largest

virus, as it is just small enough to be transmitted
through the endosome of the epithelial. These viruses are
transmitted into the subject through respiratory droplet
s or
through fecal routes. Once inside the cell the adenovirus
attaches onto the coxsackie
adenovirus receptor
the mutated cell’s surface through one of its sharp


is a protein that acts like a hand, grabbing the
adenovirus and
pull it into the epithelial cells of the mutated
After it passes through the surface of the host cell, the
virus lets the DNA free into the nucleus of the host. This
process has two parts to it. The first interaction with the host
involves the fibre
of the virus binding to the cox
receptor of the host. This first process is fairly simple, but
the second process is far more

complex. Once the
adenovirus is bonded to the CAR, a penton base protein in
the adenovirus binds to an integrin molecule, w
hich acts as a
receptor to the CAR. This binding causes internalization
of the virus into the host cell. This binding stimulates the
cell to signal and causes actin polymerization to occur, in
turn allowing the virus to enter an endosome of the host cel
Actin polymerization is the process of comb
ining DNA
monomers into a polymer

so the actin protein in the host can
move the virus into the cell endosome.

After this the
endosome can acidify and break apart releasing the
adenovirus into the cytoplasm of the host. The virus is then
transported to the nuclear pore complex (openings in the
nucleus), where the adenovirus can break open allowing the
DNA of the v
irus to finally enter the nucleus of the mutated
cell [8].

There are some disadvantages to gene therapy. Cost is one
of the major problems. This kind of treatment will become
expensive if the patient needs to keep receiving the
treatment. The issue of
response to the treatment is another
The more times the treatment is administered to try
and achieve long
term affects, the more likely the body is to
become immune to the treatment.
Current treatments of
cystic fibrosis have not proven to be perma
nent. The affects
will work for a few days but then the cells will turn back to
their incorrect coding of CFTR.
The target cells have been
debated a lot and determination of the exact “parent” cells
has not occurred.

Studies have shown that some patients
defections in 100% of their CFTR genes,
and other
patients have
defections in
as little

as six percent [9

natural response of the body when foreign material enters it
is to fight it and either kill or remove it. Infecting cells with
the new forei
gn DNA in the vectors
may cause the patients
body to produce an immune response against it causing the
patient to become ill. Using viruses as vectors also creates a
risky situation because there is always the chance that the
virus will regain its old abil
ity to spread disease in the
patient. The new DNA may not make it to the right cells that
need to be altered and if it does make it to the right cell, the
vector DNA might not be placed correctly into the incorrect
DNA. There are barriers in the lung that
make it more
difficult to get the new DNA to the correct lung cells in the
patient. The mucocillary clearance defense system can be
slowed down with new drugs such as rhDNase [1].
Finding a
safe method to apply this treatment to the patient
s mucus
filled l
ungs has been the main

in gene therapy
being affective for good

A lot of research has been going
on in
current and recent

years to help find the best way to
administer the healthy gene into the cell

and once that
occurs, making the cure permane

Both are detrimental to
making gene therapy a trusted cure for diseases, but
advances are being made.


Researchers have been working very hard to find out a
way to make gene therapy successful. If it were to be found
uccessful, a plethora of deadly diseases all across the world
would have a great opportunity to be cured. Diseases such
as: AIDS, cancer, Alzheimer’s might be things of the past.
That is why a lot of research goes into gene therapy. In
1989, after it was f
ound that the CFTR gene was the cause of
cystic fibrosis

, gene therapy research began on a large

Sean O’Connor

Kevin Hulbert


Was it just a coincidence? Or was the fact that a
mutated CFTR gene was found to

be the culprit of the
disease, which

resulted in the first trials o
f gene therapy on
humans to occur that same year. In 1989, Dr. Steven A.
Rosenberg administered the first trial of gene therapy to be
done on a human [9].
The Rosenberg trial involved
introducing a retrovirus into five patients with melanoma.
This trial di
dn’t involve cystic fibrosis, yet it was a huge step
in curing the disease, as the number of trials in administering
gene therapy rose exponentially.
“This study demonstrated
the feasibility of using retroviral gene transduction in
humans and set the stage

for further studies” [9]. Since the
first study in 1989, almost 1,000 clinical gene therapy trials
have been completed or have been approved by doctors and
researchers all over the world. In 1990, another major gene
therapy trial ensued. The first therape
utic gene therapy trial
was allowed to be performed. Up until 2004, there had been
gene therapy trials in 24 different countries, with the United
States administering the majority of them.

These trials used
many different vectors to send the healthy genes
into the
cells of subjects. The majority of these being viral vectors

Of all trials, 28% of trials involved retroviral vectors
and 26% involved adenoviruses [9]. Non
viral vectors are
used because they are less dangerous to the subject than viral
ctors, yet aren’t as effective. Many diseases had been
tested upon as well; cancer being the most common. Ten

of the trials were involved in monogenetic diseases,
with one
third of those being cystic fibrosis [9].

Adenoviral Vectors

In 1995 a
clinical trial involving adenoviral vectors in
treating cystic fibrosis was directed on 1
2 patients with
cystic fibrosis [10].

The adenovirus was
inserted into the
right nostril first, then the left nostril of the subjects. The
adenovirus was administered
in the epithelial of the nostrils
with increasing dosage. After about an hour it was found that
only 58% of the adenovirus solution remained in the nose.
After the trials statistics showed that there was no real
potential difference in the epithelial of th
e nostril between
the four cohorts of subjects. The fourth cohort (most
dosage), didn’t differ much at all from the first cohort (least
dosage). The researchers at the end of the study concluded
that the gene transfer through adenoviruses in the nostrils
as not efficient [10]. Chloride ion transport was not
increased in the CFTR, only inflammation was. All in all it
was not a

successful test, but in 1999, a

test using
a similar
transport method was successful

At Stanford University researchers began
a clinical trial
in which adeno
associated viruses were administered to the
patient via aerosols [11]. These aerosols were inserted
through the nostrils of the patients. Including inflammation
of the lungs in patients with CF, the sinuses are affected
tively as well. When the adeno
associated virus was
inserted into the nostrils, it was found that there was a
“significant reduction in inflammation” [11]. This study
didn’t show gene transfer would be successful in the
epithelial of the lungs and cells, b
ut it was a big confidence
booster, that would help pave the way for more CF gene
therapy research in the coming years.

Cationic Liposomes

Current research falls heavily on new ways of delivering the
genes efficiently.
Whit regards to efficiency, a lot of
research has turned to developing viral psuedotyping, a new
generation of cationic liposomes, and the use of non
vectors with a powerful transfection levels such as the
Sendai virus [1].
Cationic liposome research

mixing cationic lipids with cholesterol and

These are bonded to
DNA electrostatically and are allowed to interact and pass
through cell membranes. Research has proven that this
method has shown transfection in t
he lungs epithelial cells of
mice. This was tried in a human trial by nasal perfusion and
resulted in partial correction of nasal differences but the
patient did succumb to a fever. Other t
rials had similar
results and

modifications of the cationic liposom
es are
needed. Some ideas for modification include reducing non
essential CpG to reduce inflammatory response by patients
Research on extending the length of time of the effects
is also crucial because the main goal is for this treatment to
cure cyst
ic fibrosis permanently. Adenovirus vectors have
proven to be the most efficient with this part of the process.
The latest of developments in adenovirus vectors has shown
high transgenic effects from treatment lasting up to 15 weeks
in mice [12]. This is g
ood progress but is still a long step
from the intended goal.

epithelial Cells

Research on treating alternative non
epithelial cells is also
currently in progress. The CFTR defect in immune cells can
prevent them from clearing pathogens. Because of t
defect, patients become considerably more vulnerable to
pathogens in the air that may cause illness. Correcting the
defect in these cells would prove to be effective and
beneficial to the patient.

Research in mutated, CFTR
lacking, mice has risen quest
ions about whether or not
inflammatory signals are due to mutated epithelial cells or
deficient lymphocytes [12]. Correcting CFTR can cause the
lymphocyte channel to work properly again. It is important
to protect the effectiveness of the immune system in
patient with cystic fibrosis because they are consistently
faced with mucus build up and respiratory infection. Studies
on cystic fibrosis lung
transplant recipients have shown that
they exhibit a larger decline in health than those without the
t. Correcting the CFTR in non
epithelial cells has
been relatively unexplored but offers potential in aiding
against treating patients with cystic fibrosis.

Sean O’Connor

Kevin Hulbert




Genetic Engineering is a big part of the medical field. The
medical field

has always been limited due to ethics and laws
passed by the government. Many of the considerations that
go into aspects of the medical field apply to genetic
engineering as well. It is important to realize that not
everyone agrees with genetic engineerin
g, specifically when
it comes to altering the DNA of a fetus.

The government has
passed laws on any time of germ line therapy in humans
because the alterations would affect future outcomes

many generations [13

The government has not chose to
prevent s
omatic gene therapy to treat disease so this gives
research the potential to improve. Genetic engineering is a
fearful advancement because of its risk and its potential.
Many fear that the excitement of the potential of this type of
treatment has led to re
search trials that may have been too
Researchers are not sure of what the best way to
transfer the correct genes is and it will take many years of
trials to determine it.
Researchers have to make sure of all
there research and answer many questions
before being
allowed access to human trials.
Many people hold the belief
that we should be fated to what we were born with and that it
is unnatural to try and alter nature’s course. Some find that
going against the natural way of things is unethical and th
people should not try and play “god”.
People are afraid that
instead of gene therapy being used to cure diseases that the
rich will take it to the extreme and try to purchase “super”

Many are in favor of gene therapy as long as it is
d to curing diseases that causes pain and suffering to

the families and the public [13
]. It is normal for people to
want the medical field to continue to advance so that all
disease will be cured. It is also understandable that there are
real fears among p
eople about growing technologies in the
medical field. If the wrong people in government decided to
change the laws, then all around the world, super
will begin to rise.

Many people also worry about insurance
companies using genetic testing to deter
mine who gets
and who does not get covered [13
]. This is a real
concern because insurance companies could potentially
condemn those with “bad” genes to poor medical care and
the inability to afford medical treatment.

Many religious people say

that it is unholy to alter the
genes that god gave a person.
It is a sin and is wrongful to
alter nature with new genetic engineering technology.
that have been taken show that 58% of people disagree with
the religious view on gene therapy

. It
is understandable
to take into account people

s religious beliefs but when it
es to passing laws against it, their beliefs

influence the decision because of separation of church and
Who gets to decide what diseases are worthy of being
ted by gene therapy? This is a common question brought
up because it concerns ethics. It is tough to decide
specifically what is a suitable disease for gene therapy
because restricting another disease may cause a person pain
and suffering when there is the

potential to cure that person.
It is important for the citizens of the world to stay active in
the debates on gene therapy and to voice their opinions
because it is not right for any one man to make the decisions
concerning gene therapy.

Gene therap
y has not been perfected so it can be
unpredictable at times. Using treatment on humans where
there is the potential of viral vectors turning to their natural
viral tendencies is unethical because it could cause the
patient to become even sicker with aller
gic reaction or
inflammation. It has become one of the main goals of gene
therapy researchers to perfect making the transfer of healthy
genes safe and consistent.

People believe that treating
patients with gene therapy may be unethical becau
se of all
the r
isks involved [13
]. In the medical field, there are always
risks. Progress has been made over the years because risks
have been taken. It is important to take the right risks. After
a considerable amount of research has been conducted and
the animal trials

have been successful, human trials are
necessary for the advancement of the medical field. People
have always dreamed about a world without cancer, aids,
and other painful diseases such as cystic fibrosis. The same
risks are often involved in other medica
l procedures. A lung
transplant to try and treat cystic fibrosis has many of the
same risks as gene therapy if not more risks. They both share
the possibility of rejection by the immune system and
The lung transplant will be very expensive
d will require many treatments to make sure that
everything is working correctly and that the body is
accepting it.

There is always the risk that the new DNA will
make its way to the egg and sperm cells. This causes a lot of
concern because that would affe
ct future generations. There
has been know indication of egg or sperm cells being
affected in conducted research trials


Gene therapy,
when perfected, will involve one treatment that may be
painless and will cure cystic fibrosis for the rest of the

s life.


Genetic engineering has a very promising future. Research
continues to be well funded and new breakthroughs and
positive results are imminent. Using gene therapy to treat
diseases and cancers will transform the medical field and the
world. If scientists an
d engineers can perfect the vector
methods used to administer healthy
genes into a
patients cell, cystic fibrosis can be cured.

The gene gun and
aerosol injection are prominent technologies for delivering
the vector but better methods need to be found
New ideas to
aid in the treatment of cystic fibrosis are being developed
such as treating non
epithelial affected cells and cationic
Developing newer and more efficient technology
for delivery is essential in the prog
ress of the therapy.

rch needs to be geared towards finding away for cells
to sustain the effects of gene therapy treatment permanently

Sean O’Connor

Kevin Hulbert


or for long periods of time.

Fifteen weeks of positive results
is nice but is nowhere near as good as giving a patient
suffering with cystic
fibrosis a lifetime without the disease.

Overcoming the barriers provided by the immune response
is also one of the tougher tasks.

The body is always going to
try and fight foreign substances that enter it. This
significantly increases the risks of gene
therapy and slows
down trials and research.

Many suffer from the disease and
many would like to see it as well as other diseases
eliminated from the world. Gene therapy faces many
struggles such as religious beliefs, government laws and
ethics, and the pub
lic fear of the its potential to play the role
of god.

New technology, new treatments, and new ideas
scare a lot of the general public. Majorities of people do not
like change even if it is for the greater good.
More human
and animal trials need to be cond
ucted and the government
needs to continue to supply sufficient funding. It is clear that
since the discovery of the human genome the future of
medicine would be involved with it. Gene therapy is the
solution that mankind has been waiting for many years an
its technology and processes need to be fully supported by
the public in order for it to be successful.


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Kevin and I

would like to thank our grader, Carol, for all of
her insight and advice on our paper. We would like to t
Janelle for meeting with Kevin and I

and discussing ways to
improve ou
r paper throughout the process. Finally, w
would like to thank
our co
chair for her
encouragement and

Sean O’Connor

Kevin Hulbert