CYSTIC

FIBROSIS

GENE THERAPY

NAME: CHAN SIE KEI

CLASS: BIO 2-1
 INTRODUCTION
Cystic Fibrosis
• Cystic Fibrosis (CF) is a lethal genetic disease. It is an
autosomal recessive disease. The gene for CF is not
found in the sex chromosomes and two abnormal CF
genes have to appear together in a chromosome for an
individual to develop symptoms of CF (Cystic Fibrosis
Foundation 1995).

• CF mainly affect the patients’ glands which results in the

production of more mucus which favours the growth of
bacteria. The mucus produced is stickier and thicker
than normal individual without CF. The respiratory and
digestive system are the most affected systems, making
them more prone to bacterial infection which may lead
to organ damage (National Institutes of Health 1995).
• Symptoms of CF ( Cystic Fibrosis Foundation 1995).
– Breathing difficulties and wheezing
– Lung infections including bronchitis and pneumonia
– Congested lungs or digestive tract
– Poor growth and weight gain

• The cystic fibrosis transmembrane conductance regulator (CFTR) gene is responsible for
the production of a protein that regulates transmembrane conductance. The mutated
CFTR gene on human chromosome 7 is the main cause of Cystic Fibrosis. It was
discovered in the Pseudomonas Genome Project, 1998 (Knudson 2004).

• Normal CFTR gene ensures a higher chloride concentration gradient outside the cell to
attract water to the mucus layer that traps dirt outside the cell. In CF, mutated CFTR
prevent Chloride ions from being transported out of the cell and hence water will leave
the mucus layer and instead enter the cell which has higher ion concentration gradient.
The mucus layer loses water, becomes thick and sticky and later prevent the cilia from
clearing debris, and this causes infection ( UTAH 2014).
Gene Therapy
• Gene therapy is a technique that uses genes to cure diseases. A few approaches to
gene therapy have been researched and this include:
 Introducing healthy gene to replace mutated and disease causing gene.
 Halt the activities of mutated gene that is functioning abnormally.

• Types of genetic therapies involved in the treatment of cystic fibrosis:

– Somatic gene therapy
• Example: Virus mediated gene transfer systems and non-viral gene transfer systems
• Non reproductive, will not be passed down to subsequent generations
• Gene transfer tales place in the targeted cells
– Germline gene therapy
• Results in permanent changes that are inherited to the next generations.
• Gene transfer takes place in cells of developing embryo
• Multiple social issues arise as some people are anxious that the change of gene
might lead to unforeseen deleterious effects on the future generations and view
gene therapy as ‘playing God’. Other than that, some research has studied that the
effects of somatic cell therapy might not last as the tissues die and are replaced by
new cells. Successful germline therapies can eliminate some heredity diseases in a
particular family but this has also raised controversy. These social issues will be
further discussed later.
 BACKGROUND
• Summarised Timeline of the discovery of Gene therapy for Cystic Fibrosis

• The first evidence of the existence of cystic fibrosis

1595 (CF).

• Genetic engineering was first used in animal and plant

1932 breeding

• It was discovered that the sweat of patient with CF has

1948 a high concentration of salt

• After the discovery of sweat electrolyte defect, CF was

1959 dismissed from being a disorder of mucus

• First gene was transferred into animals and humans via viral
1960s vectors or genetically modified cultured cells
1970
• Herbert Boyer and Stanley Cohen first accomplish the first
1973 direct transfer of DNA from one organism to another Herbert Boyer and Stanley Cohen

• Genetically modified bacteria successfully produce somatostatin

1978 and insulin after commercialisation of the technology
• Gene therapy started receiving approval and became widely
1980s accepted

• CF genes that result in the basic defect, CFTR are found

1989
• Treatment for cystic fibrosis through the gene replacement
1990s therapies were carried out
History of the discovery of Genes and Gene Therapy ( Kenyon
College n.d.)

• 1865, Gregor Mendel first discovered genetic inheritance

• 1898, The name “gene” was created by Hugo de Vries and in 1905,William
Bateson coined the term “genetics”.
• Walter Sutton and Theodore Boveri discovered chromosomes using dyes.
• 1932, Genetic engineering was first used in animal and plant breeding.
• 1944, Avery, MacLeod and McCarty identify the “transforming principle” as DNA.
• 1953, Rosalind Franklin and Maurice Wilkins discovered the double helix structure
of DNA. Thomas Watson and Frances Crick pointed out the structure of the
base pairs which allow DNA replication.
• 1960s, The gene was first transferred into animals and humans using viral vectors or
genetically modified cultured cells. Viral genomes were used to develop efficient
methods for gene transfer into mammalian cells.
• Late 1970s, Selection system for cultured cells and recombinant DNA technology
were combined with early transfection techniques.
• 1973, Herbert Boyer and Stanley Cohen first accomplish the direct transfer of DNA
from one organism to another.
• 1976, the advent of genetically modified bacteria that produce somatostatin and
insulin in 1978 took place after the technology were commercialised.
• 1980s, gene therapy started receiving approval and became widely accepted.
History of the discovery of Cystic Fibrosis (CF) and its treatment
(Pamela, 2006)

• 1595, the first clear evidence of the existence of this disease where patients who suffered
CF are described to have salty skin and damage of pancreas. European folklore warned
“woe is the child who tastes salty from a kiss on the brow, for he is cursed, and soon
must die” (Nick 2012)
• 1938, This disease is then named ‘Cystic Fibrosis of the pancreas’ by Dr. Dorothy
Anderson who first described the disorder in medical literate (Nick 2012). CF was
differentiated from celiac disease and is categorised as pathologic diagnosis with life
expectancy of 6 months.
• 1948, after observing infants that were admitted to the emergency room due to
dehydration during a heat wave in that summer that year, Dr. Paul di Sant’Agnese
discovered that the sweat of children with CF has high salt concentration.
• 1953, Sweat electrolyte defect is discovered
• 1959, Identification of milder case which happened after the standardization of the sweat
test. CF was then denied from being a disorder of mucus.
• 1980, Discovery of the fact that inflammation contributes to lung disease and
constitutes a therapeutic target.
• 1983, Paul Quinton first used sweat duct to identify chloride transport as the basic
defect in CF. The basic physiologic CF defect which is the chloride transport was
identified.
• 1989, CF genes that result in the basic defect are found in a cAMP-regulated chloride
channel. The gene responsible, CF Transmembrane conductance Regulator (CFTR)
were identified and its genetic code was sequenced.
• 1990’s, treatment for cystic fibrosis through the gene replacement therapies were
carried out
GENETIC TECHNIQUE: VIRAL
MEDIATED GENE TRANSFER
• In this genetic technique, gene therapy is done by manipulating viruses that carry viral genes to
contain “good genes” which will infect the cells to code for needed proteins, hormones, or enzymes
(Luke 2009).
• The Adenoviral vector is used in this technique to transfer the gene of healthy cystic fibrosis
transmembrane conductance regulator (CFTR) to the target cells involved in Cystic Fibrosis
(CF), the respiratory epithelial cells lining the lung (Greisenbach & Alton 2009) in order to
produce more CFTR to recover the normal chloride ions transportation.
• Among the vectors available, the adeno-associated virus (AAV) vector, specifically AAV2H22 is
the most suitable vector that can efficiently transfer healthy CF transmembrane conductance
regulatory gene (CFTR) directly to the CF patients’ lungs (Tebbutt 2000).
• Advantages of AAV vector include low immunogenicity, no risk of pathogenicity, tropism, cell
infectivity, easy manipulation, capable of infecting a broad range of cell types and infection is not
dependent on active host cell division (Steines n.d.).
1. IDENTIFICATION OF CYSTIC FIBROSIS GENE (DRUMM 1990)

1. The technique of chromosome jumping was constructed and applied to a marker closely
linked to the cystic fibrosis (CF) gene as a model system. This results in a clone mapping
closer to the CF locus than the marker.
2. Using Southern blotting and pulsed-field gel electrophoresis, a long-range restriction map
is developed to determine the distance to be crossed in order to detect the CF gene.
3. Chromosome jumping was focused on the region estimated, ultimately landing next to a
CpG, or HTF, island which was at the terminal of the CF gene, and was used to detect
cDNA copies of the gene.
4. This gene encode a membrane bound protein called the cystic fibrosis transmembrane
regulatory (CFTR).
5. A full length copy of the gene is constructed in cDNA form to carry out studies.
6. The normal sequence of the gene is poisonous to the E.coli cloning hosts, hence, an
excisable 10bp fragment which contains a stop codon is needed to treat the DNA.
2. Cloning of Cystic fibrosis gene
Vector gene cloning
1. The DNA sequence is inserted into a plasmid and is replicated
along with the rest of the plasmid DNA in the process known
as DNA cloning.
2. The specific DNA sequence of 50-100 base pairs must be
present in a plasmid for it to replicate as it is the replication
origin (ORI). Host-cell enzymes bind to ORI, initiating
replication of circular plasmid.
3. Once DNA replication is initiated at ORI, it continues around
the circular plasmid regardless of its nucleotide sequence.

• Polymerase Chain Reaction (PCR)

1. DNA is replicated without vectors through in vitro method.
2. The template DNA which is the DNA to be copied is mixed with forward and reverse primers
complementary to its terminal, Taq polymerase and nucleotides.
3. Denaturation which separates the strands of DNA molecule, primer annealing which binds
primers to the single-stranded DNA and extension in which nucleotides are added to primers to
form double stranded DNA are repeated.
3. GENETIC MODIFICATION OF ADENOVIRAL VECTOR (USEFUL)

1. The E1 region of the Ad vectors are deleted in the first generation Ad vectors.
2. The tight junction of Ad vectors is transient disrupted to increase the efficiency of
transduction and to decrease the vector dose required dramatically to achieve therapeutic
levels of transduction.
3. The viral genes present in first generation Ad vectors result in pneumonia and therefore in
second generation Ad vectors, other early viral genes such as E2 or E4 are deleted or
mutated, and E1 is further diminished.
4. All viral coding sequences on Ad vectors are deleted, forming helper-dependent adenoviral
vectors (HDAd) to mediate high level and long-term transgene expression without chronic
toxicity as there is no viral protein expression.
5. A larger cloning capacity is enabled for
delivery of whole-genomic loci, large
cis-acting elements and transgenes.
 4. RECOMBINANT TECHNOLOGY
I. Cloning of Gene of Interest (GOI) into the shuttle vector.
1. Identify a suitable restriction site on the CFTR gene and primers that contain restriction sites at the
terminal are designed so that the sticky ends can be created to facilitate directional cloning .
2. A western blot expression analysis of the transgene is performed at this stage to prevent any
unanticipated problems at later stages.
3. A SV40 polyadenylation signal and a promoter is incorporated in the transgene cassette to drive
polyadenylation.
4. A consensus Kozak signal sequence is included for efficient transgene expression.
II. In vivo homologous recombination in bacteria.
1. Electrocompetent cells is used in before recombination process to produce BJ5183 cells in the vector that
show low transformation efficiency and high frequency of homologous recombination than most
conventional strains used for cloning.
2. PCR using bi-partite primers is used to create cassettes encoding a drug-resistance gene to determine the
exact junction sequence of the final construct.
3. The linear drug-resistant cassette made by PCR is transformed into recombination-competent cells and
Red-mediated recombination occurs and the final recombinant will be a gene replacement.
4. After transformation, the bacteria is grown on plate and well-isolated and smallest colonies are
chosen and grown in starter culture containing kanamycin.
5. DNA is isolated and restriction digest with PaCl is performed to confirm the presence of desired
recombinants. After desired recombinant clones are validated, DH10B cells are retransformed
using DNA. Mini-prep is cultured and recombinant adenoviral DNA is extracted.
6. Restriction digest and PCR amplification is carried out to confirm the stability of transgene.
5. PURIFICATION BY ULTRACENTRIFUGATION (E LOFFE 2005)

• Adenoviral vectors have to be purified to remove contamination.

• Purification of Adenoviral vectors by CsCl density gradient can be performed easily
and produce highly pure viral.

1. Adenoviral vectors purification were first based on CsCl density gradient centrifugation.
CsCl salt produce a density gradient when in contact to a strong centrifugal field.
2. Viruses are separated from contaminants when they are centrifuged to equilibrium in a CsCl
salt, and are collected in bands according to their buoyant densities.
3. The viral lysate of from infected cell lysis and DNA digestion is applied to a continuous CsCl
step gradient from a higher density to a lower density.
4. The Adenoviral vectors are collected and mixed with 1.35g/ml CsCl solution and second
round of isopycnic gradient ultracentrifugation is carried out.
6. DELIVERY OF VECTOR
1. The virus is introduced into the host, where they will bind to surface receptor proteins. The
virus then enters into the cell, and inserts its DNA into the nucleus. This DNA is then
transcript using the host machinery, before being translated. These translated genes can then
provide the host with what it needs (Wagner et al.).