Genetic Engineering, Genetic

Counseling, & Gene Therapy

 
1,2
Marhaen Hardjo
1
Head of Biochemistry Department, Medical Faculty of Hasanuddin University
2
Director of Stem Cell Center Hasanuddin University Hospital
 

DEPARTMENT OF BIOCHEMISTRYY
MEDICAL FACULTI OF
HASANUDDIN UNIVERSITY
 Part One:
Genetic Engineering
 Genetic Engineering
• Genetic Engineers can alter • Selective Breeding
the DNA code of living
organisms. • Recombinant DNA

• PCR

• Gel Electrophoresis

• Transgenic Organisms
 Selective Breeding

• Breed only those plants

or animals with
desirable traits

• People have been using

selective breeding for
1000’s of years with
farm crops and
domesticated animals.
 Recombinant DNA

• The ability to combine

the DNA of one
organism with the DNA
of another organism.

• Recombinant DNA
technology was first
used in the 1970’s with
bacteria.
 Genetically modified organisms are called
transgenic organisms.

TRANSGENIC ANIMALS

1. Mice – used to study human immune

system

2. Chickens – more resistant to

infections

3. Cows – increase milk supply and

leaner meat

4. Goats, sheep and pigs – produce

human proteins in their milk
 Transgenic Goat

.
This goat contains a human
gene that codes for a blood
clotting agent. The blood
Human DNA in
clotting agent can be harvested
a Goat Cell
in the goat’s milk.
 How to Create a
Transgenic Animal

Desired DNA
is
added to an
egg cell.
Ha Ha Ha!
Genetic Engineering and
Crime Scenes……
 Polymerase Chain Reaction
PCR
• PCR allows scientists to
make many copies of a piece
of DNA.

1. Heat the DNA so it “unzips”.

2. Add the complementary

nitrogenous bases.

3. Allow DNA to cool so the

complementary strands can
“zip” together.
 Gel Electrophoresis

• This technology
allows scientists to
identify someone’s
DNA!
 Steps Involved in Gel Electrophoresis
1. “Cut” DNA sample with restriction
enzymes.

2. Run the DNA fragments through a

gel.

3. Bands will form in the gel.

4. Everyone’s DNA bands are unique

and can be used to identify a person.

5. DNA bands are like “genetic

fingerprints”.
• Dalam rekayasa genetika  DNA dan RNA
• DNA (deoxyribonucleic Acid) : penyimpan informasi
genetika
• Informasi  melambangkan suatu keteraturan
kebalikan dari entropi yaitu ketidakteraturan atau
acak
• Komputer penyimpan, pengolah dan penarikan
informasi, bahasa komputer: unit informasi bit
(binary digit)
• Jumlah dan jenis informasi yg terdapat di dalam sel
manusia masih melampaui sel pembuatan program
oleh komputer
• 20 asam amino  bukan hanya 20 unit penyandi
karena setiap asam amino dpt memberikan arti
yang berbeda-beda pada protein
• Contoh: serin  memberikan gugus esensial pada
sisi aktif suatu protein (tripsin) atau gugus
esensial pd sisi pengaturan (glikogen fosforilase)
atau molekul pembawa gugus fosfat
• DNA : molekul yang sangat panjang terdiri dari ribuan
deoksinukleotida (4 jenis) yang bergabung dalam suatu
urutan yg bersifat khas bagi setiap organisme
• Sel eukariotik memiliki DNA yang lebih besar
dibandingkan dgn prokariotik, DNA ini membentuk
kromosom dalam nukleus yang dikelilingi membran
• Virus terdapat RNA atau DNA
• RNA dalam sel : RNA ribosom (r RNA), RNA pemindah
(t RNA) dan RNA data (m RNA)
• DNA RNA
• Purin : adenin (A) Adenin (A)
Guanin (G) Guanin (G)
Pirimidin: Sitosin (C) Sitosin (C)
Timin (T) Urasil (U)
Ikatan fosfodiester menggabungkan nukleotida
berikutnya
DNA double helix
• Teknologi DNA rekombinan atau rekayasa genetika
memungkinkan ilmuwan utk mengindentifikasi, mengisolasi
dan memperbanyak fragmen bahan genetik DNA
• Cara pengerjaan ini dilakukan in vitro dgn bantuan bahan
biologi enzim, plasmid dan virus
• Teknologi rekombinan dpt dikatakan merupakan penjabaran
proses evolusi dan penyesuaian diri secara alamiah terhadap
lingkungan
• Populasi organisme dpt menyesuaikan diri karena populasi
tersebut bersifat heterogen akibat mutasi gen
• Proses alamiah restrukturisasi genetika membentuk pokok2
teknologi DNA rekombinan dengan perbedaan yg penting
yaitu hasil di laboratorium tergantung pada ilmuwan, tidak
lagi pada seleksi alam
Plasmid
• DNA (sirkular) di luar kromosom
• Biasanya mengandung sifat penciri, seperti ketahanan
terhadap antibiotika
• Plasmid hasil rekayasa dipakai sebagai vektor/kendaraan
pembawa gen asing di dalam kloning. Pada plasmid rekayasa
ditambahkan beberapa lokasi penting, seperti tempat yang DNA bakteri
dan virus

bisa dikenali oleh beberapa enzim pemotong DNA (Endonuklease

restriksi).
Replikasi DNA
Gambar Keseluruhan Replikasi
Sense strand
TRANSLASI, DARI RNA KE PROTEIN

protein
Dari RNA ke
KODE GENETIK
• Perkembangan dan aplikasi nyata metode DNA
rekombinan pd organisme prokariotik dan eukariotik
didorong oleh berbagai penemuan biokimiawi,
terutama penemuan enzim endonuklease restriksi yg
mula2 berhasil diisolasi dari golongan prokariotik
• Enzim ini memecah untai ganda DNA secara spesifik
pada daerah DNA dengan deret tertentu
• Penemuan lain adalah penemuan enzim transkriptase
kebalikan (reverse transkriptase) yaitu enzim yg
mengkatalisis reaksi biosintesis DNA dari RNA
sehingga dapat dibuat molekul DNA komplemneter
(cDNA) dari molekul mRNA yg telah dimurnikan
terlebih dahulu
 Teknik umum DNA rekombinan
1. Kloning
• Teknik inti dlm DNA rekombinan adalah kloning molekler yaitu
isolasi dan pengembangbiakan fragmen DNA sejenis
• Kloning terdiri 2 tahap yaitu
1. DNA yg diinginkan atau DNA yg dimasukan, digabungkan dgn
molekul DNA yg disebut vektor kloning utk membentuk DNA
rekombinan atau klon
2. Molekul DNA rekombinan dimasukan ke dlm sel melalui proses
transformasi. Sel inang yg menangkap molekul DNA disebut
transforman atau sel yg ditransformasi.
• Suatu transforman mengalami banyak siklus
pembelahan sel utk mendapatkan koloni sel yg
berasal dari sel inag pemula (asli)
• Pd setiap siklus pembelahan, DNA
rekombinan di dalam sel juga ikut membelah.
• Pada saat koloni sudah terbentuk sempurna,
molekul DNA rekombian tunggal telah
membelah secara sempurna bahkan sampai
beberapa kali.
RESTRICTION ENDONUKLEASE DAN
DASAR REKAYASA GENETIKA
• Endonuklease restriksi
memotong DNA pada sisi
spesifik.
• Dasar rekayasa genetika
adalah memotong DNA
target dan vektor
pembawa/plasmid dengan
endonuklease restriksi pada
sisi spesifik, menyambung
dengan DNA ligase dan
transformasi ke dalam
bakteri inang yang cocok
untuk diperbanyak dan
KLONING 1

diekspresikan.
KLONING 2
 Figure:  Cloning a Restriction Fragment into a Plasmid

                                                                                                                                                                                                                
2.  Mutagenesis
Memodifikasi gen pada organisme tersebut dengan
mengganti sekuen basa nitrogen pada DNA yang ada
untuk diganti dengan basa nitrogen lain sehingga terjadi
perubahan sifat pada organisme tersebut, contoh: semula
sifatnya tidak tahan hama menjadi tahan hama.

Gambar 2. Proses Mutagenesis

Recombinant
1.
Bacteria
Remove bacterial DNA (plasmid).

2. Cut the Bacterial DNA with

“restriction enzymes”.

3. Cut the DNA from another organism

with “restriction enzymes”.

4. Combine the cut pieces of DNA

together with another enzyme and
insert them into bacteria.

5. Reproduce the recombinant bacteria.

6. The foreign genes will be expressed

in the bacteria.
 Benefits of Recombinant Bacteria

1. Bacteria can make human insulin or human

growth hormone.

1. Bacteria can be engineered to “eat” oil spills.

 The DNA of plants and animals
can also be altered.
PLANTS

1. disease-resistant and
insect-resistant crops

2. Hardier fruit

3. 70-75% of food in
supermarket is genetically
modified.
How to Create a Genetically
Modified Plant
1.Create recombinant
bacteria with desired
gene.

2. Allow the bacteria to

“infect" the plant cells.

3. Desired gene is
inserted into plant
chromosomes.
What do you think about eating genetically
modified foods?
 Part Two:
Genetic Counceling
 What is Genetic Counseling?
• communication process
• address individual concerns relating to development /
transmission of hereditary disorder
• consultand = individual who seeks genetic counseling
• strong communicative and supportive element so that
those who seek information are able to reach their own
fully informed decisions without undue pressure or stress
What Information should be provided?
• medical diagnosis and its implications in terms of
prognosis and possible treatment

• mode of inheritance of disorder and the risk of

developing and/or transmitting it

• choices or options available for dealing with the risks

 Steps in Genetic Counseling
• Diagnosis - based on history, examination
and investigations
• Risk assessment
• Communication
• Discussion of Options
• Long-term contact and support
 Establishing the Diagnosis
• most crucial step in any genetic counseling
• if incorrect - totally misleading information could be
given with tragic consequences
• reaching diagnosis involves three fundamental steps
– taking a history
– examination
– undertaking appropriate investigations
 Establishing the Diagnosis
• Information about consultand’s family is obtained by skilled
genetics nurse or counselor
• pre-clinic telephone or home visit is helpful
• clinic visit - full examination
• appropriate tests - chromosomes, molecular studies, referral to
specialists (neurology, ophthalmology)
• PROBLEM - Genetic Heterogeneity, and etiologic
heterogeneity
 Genetic Heterogeneity
• def - disorder that can be caused by more than one
genetic mechanism

• Ehlers Danlos AD, AR, XR

• Charcot-Marie-Tooth AD, AR, XR
• Retinitis Pigmentosa AD, AR, XR
 Genetic Heterogeneity
• Charcot-Marie-Tooth - also known as
hereditary motor and sensory neuropathy type I
(HMSN I) has been shown to result from a
small duplication on short arm of chromosome
17

• If found - this would aid in counseling

 Etiologic heterogeneity
• even though firm diagnosis - several causes may
be possible
• eg. Deafness and non-specific mental
retardation
– environmental or genetic factors
– empirical risks can be used although these are less
satisfactory than risks based on specific diagnosis
 Calculating and Presenting the Risk
• straightforward counseling situations - little more
than knowledge about Mendelian inheritance is needed
• Problems:
– delayed age of onset
– reduced penetrance
– use of linked markers can make calculations more
complex
 Presenting the Risk
• does not simply involve conveying stark risk
figures in isolation
• parents must be given as much background as
possible
• as rule of thumb: recurrence risks should be
quantified, qualified and placed in context
 Quantification
• Most prospective parents will have some concept
of risks
• Experience demonstrates that some common
misinterpretations occur
– a risk of 1 in 4 may be remembered as 4 to 1, 1 in 40,
or even 14% !!!
– the risk only applies to every fourth child !!
 Quantification
• vital to emphasize that the risk applies to each child,
and that chance does not have a memory
• genetic counselors should not be seen
exclusively as prophets of doom
– for example a family with a risk of 1 in 25 for
NTD, should be reminded that in 24 of 25 cases the
child will be normal
 Qualification - Nature of a Risk
• factor which influences parents when deciding
whether to have another child is nature of the long-
term burden associated with a risk rather than precise
numerical value
• “high-risk” of 1 in 2 for a trivial problem
(polydactaly) will not deter many families while a
“low risk” of 1 in 25 for a disabling condition (NTD)
can have a significant deterrent effect
 Discussing the Options
• provide consultands with all information needed to
arrive at their own informed decision
• details of all the choices open to them - include a
complete discussion of reproductive options
• alternative approaches to conception - AID, donor ova
• review of techniques, limitations and risks associated
with methods available for prenatal diagnosis
 Communication and Support
• Communication - two way process
• Counselor provides information
• Receptive to fears and aspirations: expressed
or unexpressed by consultant
• Information - present in clear, sympathetic
and appropriate manner
 Communication and Support
• Individual or couple will be extremely upset
when first made aware of a genetic disorder
• complex psychological and emotional factors can
influence counseling dialogue
• setting - agreeable, private and quiet, with
ample time for discussion and questions
 Counseling
• Session can be so intense and intimidating that
amount and accuracy of information retained is
very disappointing
• Letter summarizing the topics discussed at
counseling session is often sent to family
• Follow-up home visit or clinic appointment to
clarify any confusing issues
 Directive or Non-Directive
• Universal agreement - non-coercive with no attempt to direct
consultand along a course of action
• Non-judgmental - even if decision reached seems ill-advised
• Unwise to answer “What would you do if placed in my
position?” rather consideration should be given to
consequences of each possible course of action
• remember - counsultand has to live with
consequences!!!
 Special Problems in Genetic
Counseling
• Consanguinity and Incest
• Adoption and genetic disorders
• Disputed Paternity
 Consanguinity and Incest
• Consanguineous Marriage is between blood
relatives who have at least one common
ancestor no more remote than great-great
grand parent
• Incest - union between first degree relatives
(brother-sister, parent-child)
 Proportion
Genetics relationship
of Genes
Proportion shared
Shared
Risk of abnormality
of partners genes in offspring

First Degree 1/2 50%

parent-child
brother-sister

Second Degree 1/4 5-10%

uncle-niece
aunt-nephew

double first cousins 1/8 3-5%

Frequencies of three main types of abnormalities
in the children of incestuous relationships

• Mental Retardation 25%

• Autosomal recessive disorder 10-15%
• Congenital malformations 10%
 Marriage Between Blood Relatives
• Increased risk of AR disorders in future offspring
• Probability that first cousins will have a child
with AR disorder is 3%
 Paternity Testing
• genetic fingerprinting using minisatellite
repeat sequence probes
• pattern of DNA fragments generated by those
probes is so highly polymorphic that the
restriction map is unique to each individual
• specific as fingerprints
 Chromosome Disorders
Introduction
• 1956 - technique for chromosome analysis
became reliable
• to date, more than 100 chromosome syndromes
have been reported
• 47, XX/XY, +21
• Klinfelters (47XXY)
• Turners (45,X)
Incidence: Chromosome Abnormalities
• 15 - 20% of all recognized pregnancies
end in spontaneous miscarriages
• 50% of all SAB have a chromosome
abnormality
• incidence of chromosome abnormaility
at conception is 20%
• by birth - 0.5 - 1%
 Chromosome Abnormalities in SAB
Abnormality Incidence (% of total)
Trisomy 13 2
16 15
18 3
21 5
other 25
Monsomy X 20
Tripoloidy 15
Tetraploidy 5
Other 10
 Incidence: Chromosome Abnormality at term
Abnormality Incidence per
10,000 births

Autosomal trisomy 13 2
18 3
21 15
Sex Chromosomes
Female births 45, X 1
47,XXX 10
Male births 47, XXY 10
42, XYY 10
 Chromosome Deletion Syndromes
• Microscopically visible deletions of terminal
portions of:

Chromosome 4p - Wolf Hirshorn

Chromosome 5p - Cri-du-Chat
severe mental retardation
failure to thrive
Both very rare - 1/50,00 births
 Microdeletion Syndromes
• high resolution prometaphase banding and
FISH
• Some microdeletions involve loss of only a few
genes at closely adjacent loci “Contiguous
gene syndromes”
• In others - several loci are involved
 Microdeletion Syndromes
Syndrome Chromosome
Williams 7
Langer-Giedion 8
WAGR 11
Angelman 15
Prader-Willi 15
Rubenstien Taybi 15
Miller-Dieker 17
Smith-Magennis 17
DiGeorge 22
Shprintzen 22
 Lessons form Microdeletion
Syndromes
• Retinoblastoma
• Wilms’ tumor
• Angelman and Prader-Willi S.
• DiGeorge and Shprintzen S.
 Retinoblastoma
• 5% of children with RB had other
abnormalities - ie Mental Retardation
• in several children a constitutional interstitial
deletion of 13 q 14
• this deletion at 13 q 14 is the locus for the AD
form of RB
 Wilm’s tumor
• Wilm’s tumor (hydronephroma)
• Aniridia (absent Iris)
• Genital abnormalities
• Retardation of growth and development
• WAGR syndrome
 WAGR Syndrome
• interstitial deletion of particular region on
short arm of chromosome 11

• gene location - WT1

 Wilms Tumor
• Family cases of AD Wilms’ tumor have
been shown not to be linked to this locus
(WT1)
• rare overgrowth syndrome - Beckwith-
Wiedemann S. is associated with a deletion
and imprinting of a separate locus on 11p.
 Angelman and Prader-Willi S.
Angelman - inappropriate laughter,
convulsions, poor coordination (ataxia) and
mental retardation

Prader-Willi - extremely floppy (hypotonic)

in early infancy, marked obesity, and mild to
moderate mental retardation later in life.
 Imprinting - Angelman + PWS
• If deletion occurs de novo on paternally
inherited number 15 chromosome
– PWS - 15q (15q 11-12)
• If deletion occurs de novo on maternally
inherited number 15 chromosome
– AS - 15q (15q 11-12)
 AS and PWS
• non deletion cases also exist and are often due
to uniparental disomy (UPD)
– AS - both #15 chromosomes being paternal
in origin
– PWS - both #15 chromosomes being
maternal in origin
 AS and PWS
• loss at a critical region from paternal #15
chromosome causes PWS
• loss of identical critical region from
maternally inherited #15 chromosome
causes AS
 Triploidy
69, XXX; 69, XXY; 69, XYY
• relatively common in SAB
• rare in live-born infants
• IUGR: in utero-relative preservation of head
size with small trunk
• syndactaly of 3rd and 4th fingers and/or 2nd and
3rd toes
• dispermy or fertilization by diploid sperm
 Hypomelanosis of Ito
• Mosaicism for diploidy/triploidy identified
• skin: alternating patterns of normally pigmented
and depigmented streaks which correspond to
embryological developemental lines of skin
known as Blashko’s lines
• most are moderately retarded and have
convulsions which are difficult to treat.
 Part Three:
Gene Theraphy
Use of Gene Therapy in The
Treatment of Disease
 DEFINISI
• Terapi gen adalah suatu tehnik untuk
memperbaiki gen yang cacat/ rusak sehingga
dapat menimbulkan penyakit
• Clinical trial terapi gen sudah dimulai sejak tahun
1990
• Gen itu terdapat dalam kromosom merupakan
dasar fungsional dan fisik dari keturunan
• Perintahnya berupa sandi untuk membuat protein
pada struktur seluler.
• Bila terjadi perubahan dari gen, maka protein
yang terbentuk tidak lagi dapat melakukan fungsi
yang normal, sehingga akan menimbulkan
kelainan genetik
• Selama ini obat dibuat dari tumbuh2an, hewan dan dari
bahan kimia
• Para ilmuwan sekarang sudah dapat menentukan gen
yang dapat menimbulkan penyakit/kelainan . Dan sedang
dipelajari pula bagaimana cara memperbaiki atau
mengganti gen tersebut, dengan menggunakan bakteri,
virus yang sudah dimodifikasi secara genetik , dan
dipakai sebagai obat bahkan dapat menggantikan organ
tubuh
 Usaha para peneliti untuk memperbaiki
gen yang cacat
• 1. Memasukkan gen yang normal kedalam lokasi
non spesifik diantara genom untuk pengganti gen
yang tidak berfungsi
• 2. Gen yang cacat diganti dengan gen yang
normal melalui rekombinasi homolugus
• 3. Gen yang cacat diperbaiki dengan mutasi balik
yang selektif sehingga gen kembali ke fungsi
yang normal
Terapi gen adalah tehnik untuk mengoreksi gen
yang cacat yang bertanggung jawab terhadap
suatu penyakit
Misalnya untuk mencegah kematian dengan
penyakit kanker
Pengobatan dengan gen terapi meliputi
• 1. Imunoterapi
• 2. Viro onkolitik
• 3. Transfer gen
• 1. IMUNOTERAPI
Menggunakan sel yang telah dimodifikasi secara
genetik dari partikel virus untuk menstimulir
sistem imun tubuh sehingga mampu mengalahkan
keganasan sel kanker
• 2. VIRO ONKOLITIK
Menggunakan partikel sel virus yang bereplikasi
didalam sel kanker dan menyebabkan sel kanker
menjadi mati.
• 3. TRANSFER GEN
Tehnik ini relatif baru, dengan cara
memperkenalkan gen 2 baru yang dimasukan
kedalam sel kanker atau mengelilingi jaringan
kanker sehingga dapat menghentikan
pertumbuhan dan menghancurkan sel kanker
 What’s gene therapy?
• Imagine that you Many medical conditions result from flaws, or mutations, in one

So, if a flawed gene

accidentally broke one or more of a person's genes.

caused our "broken window," can you

of your neighbor's "fix" it? What are your options?
windows.
I. Stay silent: ignore the genetic
I. Stay silent: no one will disorder and nothing gets fixed.
ever find out that you are II. Try to treat the disorder with
guilty, but the window drugs or other approaches:
depending on the disorder,
doesn't get fixed. treatment may or may not be a
II. Repair it with some tape: good long-term solution.
not the best long-term III. Put in a normal, functioning copy
of the gene: if you can do this, it
solution. may solve the problem!
III. Put in a new window: not
only do you solve the
 How to fix it

B C A a beneficial gene
A
virus modified virus

• A virus is found which replicates by inserting its genes into the host cell's genome. This virus has three
genes - A, B and C.
• Gene A encodes a protein which allows this virus to insert itself into the host's genome.
• Genes B and C actually cause the disease this virus is associated with.
• Replace B and C with a beneficial gene. Thus, the modified virus could introduce your 'good gene' into the
host cell's genome without causing any disease.
• So we use the modified virus to fix the “broken window”
• KERJA TERAPI GEN
Dalam terapi gen ini kita memerlukan satu
molekul yang berfungsi sebagai karier disebut
sebagai vektor . Vektor inilah yang membawa gen
/DNA yang normal ke sel target pasien , dan
yang biasa dipakai sebagai vektor adalah virus
yang telah diubah secara genetik.
 BEBERAPA JENIS VIRUS YANG
DIGUNAKAN UNTUK TERAPI GEN
• 1. Retro virus
Golongan virus yang dapat membuat rantai ganda DNA
dari genomnya dan disatukan dengan kromosom sel
inangnya mis: HIV (human defisiensi virus)
• 2. Adeno virus
Golongan virus dengan rantai DNA gandanya dapat
menyebabkan infeksi pada saluran pernapasan, saluran
pencenaan dan menimbulkan kematian
mis: virus influenza
• 3.Adeno-assosiated virus.
Virusnya kecil mempunyai single strandid DNA
dan dapat memasukan material genetik di tempat
spesifik pada kromosom 19.
• 4. Herpes simpleks
Golongan virus dengan rantai ganda DNA yang
menginfeksi sebagian dari sel seperti sel neuron
 Kendala terapi gen
• 1. Terapi gen akan efektif bila gangguan terjadi pada gen
tunggal
• 2. Virus yang berfungsi sebagai vektor , dalam tubuh
manusia bisa berubah sifatnya sehingga menimbulkan
penyakit
• 3. Masih banyak terapi gen yang keberhasilannya pendek
sehingga perlu diulang
• 4. Sistem imun tubuh akan mengurangi efek terapi gen .
Begitu pula terapi gen yang berulang akan menyebabkan
sistem imun tubuh akan meningkat daya tolaknya
Gene therapy using an Adenovirus vector.
A new gene is inserted into an adenovirus vector, which is used to introduce the
modified DNA into a human cell. If the treatment is successful, the new gene will
make a functional protein.
 Background
• In the 1980s, advances in molecular biology had already enabled human genes to
be sequenced and cloned. Scientists looking for a method of easily producing
proteins, such as the protein deficient in diabetics — insulin, investigated
introducing human genes to bacterial DNA. The modified bacteria then produce the
corresponding protein, which can be harvested and injected in people who cannot
produce it naturally.
• Scientists took the logical step of trying to introduce genes straight into human
cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis,
hemophilia, muscular dystrophy and sickle cell anemia, optic nerve disease 1,
wound repair and regeneration2, and cardiovascular disease3.
• However, this has been much harder than modifying simple bacteria, primarily
because of the problems involved in carrying large sections of DNA and delivering
it to the right site on the genome.
 Cystic Fibrosis

• Background
• Cystic fibrosis was first described as a disease in the late 1930s by Dorothy Hansine
Andersen. In 1988, the first mutation for CF, ΔF508, was discovered by Francis
Collins, Lap-Chee Tsui and John R. Riordan on the 7th chromosome of the human
genome. Research has subsequently found over 1000 different mutations that may
cause CF, however ΔF508 accounts for approximately 70% of CF patients in
Europe (this percentage varies regionally).
• CF is an autosomal recessive disease and is the most common lethal genetic disease among
whites. There are 30,000 cases in the United States, 3,000 cases in Canada, and 27,000 cases
in Europe4.

• The mutation for CF on the 7th chromosome

 Cystic Fibrosis

Gene Therapy for Cystic Fibrosis

• Cystic fibrosis should be an ideal candidate for gene
therapy, for four main reasons:
• (1) it is a single gene defect;
• (2) it is a recessive condition, with heterozygotes being
phenotypically normal (suggesting gene dosage effects
are not critical);
• (3) the main pathology is in the lung, which is accessible
for treatment;
• (4) it is a progressive disease with a virtually normal
phenotype at birth, offering a therapeutic window.
 Cystic Fibrosis

Choices of Vectors
• Viral vectors:
• Vetrovirus Non-viral vectors:
• Adenovirus Liposome
• Adeno-associated virus DNA–polymer conjugates
• Herpes Simplex Virus Naked DNA

• The ideal vector system would have the following characteristics:

• (1) an adequate carrying capacity;
• (2) to be undetectable by the immune system;
• (3) to be non-inflammatory;
• (4) to be safe to the patients with pre-existing lung inflammation;
• (5) to have an efficiency sufficient to correct the cystic fibrosis phenotype;
• (6) to have long duration of expression and/or the ability to be safely re-
administered.
 Cystic Fibrosis

1993 vector used: Adenovirus

• The first cystic fibrosis gene therapy clinical trials used
an adenovirus vector to deliver the full-length CFTR
(cystic fibrosis transmembrane regulator) gene to cells.
• Adequate doses of adenovirus vector will probably cause
an immune response. If the adenovirus is to be useful,
researchers need to find ways to both improve the virus’s
ability to enter cells and reduce the chances of immune
response.
 Cystic Fibrosis

1995 liposome
• Trials using liposome-mediated CFTR gene transfer began in
1995.
• Non-viral vectors have the potential to avoid some of the critical
problems observed with viral vectors, such as the immune
response, limited packaging capacity, and random integration5 .
• Liposomes may be mildly effective, but their activity does not last.
For this approach to work, researchers need to figure out how to
improve delivery, make the effects more permanent and reduce the
adverse side effects.
• To date, only cationic liposome-based systems have been tested in
clinical trials in cystic fibrosis subjects6.
 Cystic Fibrosis

1998 adeno-associated virus

• Trials using adeno-associated virus to deliver the
CFTR gene began in 1998.
• Because it is safe, the adeno-associated virus – as
we predicted earlier – holds promise for being a
good way to deliver the CFTR gene to patients’
airway cells. But researchers need to learn more
about how the virus infects cells in order to make
it an effective delivery method.
 Cystic Fibrosis

Mode of delivery
• The majority of experience in terms of vector delivery to the lungs
has involved the instillation of large volumes of vector-containing
fluid into the lung via the nose.
• However,
I. this mode of delivery poses safety problems because of the
potential for aspiration.
II. In addition, the instillation of large volumes of fluid leads to
enhanced alveolar exposure, as a result of bulk flow into the lung
parenchyma. This exposure is undesirable because it may induce
adverse reactions.
III. At the same time, it is likely that airway epithelial cells, rather
than alveolar epithelial cells, are the appropriate target for CFTR
gene transfer.
• Another mode of lung delivery for vector-containing fluid is by
oral inhalation of aerosolized vectors.
• However, aerosolization of a fluid is typically achieved by means
of a nebulizer, and most nebulizers have been designed to generate
small particles. This is because most nebulizers have been
developed to deliver drugs to treat patients with asthma, and in
asthma the target region of the lungs is often the peripheral
airways. Small particles enhance delivery to the peripheral airways
and the alveolar region of the lung, but this is again undesirable for
gene vector delivery because of the possibility of inducing adverse
effects.
• One way to avoid alveolar deposition is to generate an aerosol
that is composed primarily of large droplets.
• Delivery of the vector by means of a spray device that is
inserted into a bronchoscope may have another advantage over
nebulization.
• Research suggests that spray delivery of the vector could
provide a means of targeting the larger, central airways ,
avoiding deposition in the smaller airways and alveolar region
, which is more likely with nebulizers that generate small
aerosol particles.
• The findings from studies using spray technology indicate that
efficient and targeted delivery of aerosolized gene vectors to
the lungs may be possible in the future.
 Cystic Fibrosis

Current treatment
• Modern treatment now includes
① the intake of digestion enzymes,
nutritional supplements,
② percussion and postural drainage of
the lungs, improved antibiotics
③ inhalation of aerosols containing
medication.
• The most visible gene therapy drug A typical breathing treatment for
Cystic Fibrosis, using a nebulizer
under development is inhaled and the ThAIRapy Vest
complementary DNA to treat CF.
 Cystic Fibrosis

challenges
• The goal of developing an effective genetic therapy for CF lung disease has led
to the attainment of several milestones in the larger field of gene therapy. These
include:
• the first published in vivo gene transfers with adenovirus (Ad)7, and with
recombinant adeno-associated virus (rAAV), and
• the first phase I clinical trials using each of these vector systems.8
• Choice of vector, mode of delivery to the airways, translocation of genetic
information, and expression of normalized CFTR in sufficient amounts to
correct the CF phenotype in the lungs of CF patients continue to be hurdles in
the development of gene therapy for CF9.
• A few attempts at gene therapy were initially successful, but failed to produce
acceptable long-term results.
References:
• 1. Eye 2004, 18, 1049-1055
• 2. Wound Rep. Reg. 2000, 8, 443-451
• 3. Circulation Journal 2002, 66, 1077-1086
• 4. Respiratory Care 2005, 50, 1161-1174
• 5. Am. J. Med. 2003, 115, 560-569
• 6. Biochem. J. 2005, 837, 1-15
• 7. Science 1991, 252, 431-434
• 8. Hum. Gene Ther. 1996, 7, 1145-1159
• 9. Chest 2001, 120, 124S-131S

Additional resources:
http://gslc.genetics.utah.edu/units/genetherapy/
http://www.asgt.org/
http://www.congrex.se/esgt/
http://web.archive.org/web/20030219034830/http://www.gtherapy.co.uk/
http://www.cheng.cam.ac.uk/research/groups/biosci/index.html
http://www.gene-watch.org/
The End

• THANKS