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Cystic Fibrosis and Gene Therapy
Lecture Notes
Biol 100 – K.Marr
• Topics for this Lecture
–
•
Gene Therapy as a treatment for Cystic Fibrosis
Reading assignments in Essential Biology
–
Chapter 12: DNA Technology:
• Restriction Enzymes (p. 225)
• Gel Electrophoresis (p. 229)
• PCR (Polymerase Chain Reaction) p. 228
• DNA Fingerprinting (pp. 227-230
• Human Gene Therapy (pp. 235-236
Optional Reading
• Cystic Fibrosis Foundation: Gene Therapy and CF
– http://www.cff.org/about_cf/gene_therapy_and_cf.cfm
• Center for Gene Therapy and other Genetic Diseases
– http://genetherapy.genetics.uiowa.edu/
• Cystic Fibrosis Research Directions
– http://www.niddk.nih.gov/health/endo/pubs/cystic/cystic.htm
• See "Lecture Related Resources and Enrichment" at
the class website
What is the hope for people with cystic fibrosis?
Do you want to have a healthy child?
1. Screen potential carriers of CF
• E.g. Use DNA Probe
2. Screen for CF gene in embryo
•
Only implant embryos without CF allele
Do you want a cure for yourself?
3. Gene Therapy
• Use a viral vector to insert the normal CFTR gene into
the lungs cells of people with CF
• Somatic vs. Germ line gene therapy
Normal Genotype
Abnormal Genotype
DNA probe complementary to mutant gene
•
How to use a DNA
Probe to Screen
for the CF Gene
1.
Isolate DNA from
patient
Heat to separate DNA
strands
Add labeled probe
that has
complementary base
sequence to mutant
gene
Add restriction
enzymes (cuts DNA
into fragments) and
separate by gel
electrophoresis
2.
3.
Single stranded DNA from patient
4.
Probe does not
bind to DNA
Probe binds to DNA
Separation of DNA fragments by Gel electrophoresis
a.)
DNA samples from PCR of
Snowball’s DNA
Well
DNA and dye
are loaded in a
well on a gel, and
an electric field
is placed across
the gel.
Direction of
electric field
b.)
DNA fragments move
through the gel, shorter
fragments faster than
longer fragments.
c.)
Place photographic film over
gel to detect DNA labeled
with the probe
Gel
(+) Electrode attracts
negatively charged
DNA fragments
Separation of DNA fragments by Gel electrophoresis
•
•
Smaller fragments move faster than larger fragments through
the porous gel.
Use photographic film to locate DNA fragments bound with
radioactive probe
DNA fingerprints from a murder case
Two Kinds of Gene Therapy
Replace defective gene in.....
1. Body cells: Somatic Cell Gene Therapy
•
2.
Permanent cure for individual
Egg cell: Germ line Gene Therapy
•
•
Permanent cure for future generations
Banned by most countries!! Why?

Can’t control where gene inserts. Possible Consequences:
 Abortive or defective embryos! Why?
 Could cause cancer! Why?
What’s Necessary for Gene Therapy to Work?
1. Identify the defective gene
•
e.g. CFTR gene discovered in 1989
2. Use PCR to make copies of good gene
•
PCR = polymerase chain reaction
3. Get good gene into the right cells (need vector)
•
Use a viral vector
4. Get the cells to transcribe and translate the good
gene
•
Must make the right amount of protein at the right time
and get it to the right place
Thermocycler
heats sample
to near boiling
(~94oC)
Heat breaks
hydrogen bonds
and strands
separate
Thermocycler
lowers
temperature
to 55 °C
Thermocycler
raises
temperature
to 72 °C
Doublestranded
DNA
Singlestranded
DNA
taq DNA
polymerase
DNA Primers bind
to their
complementary
sequence
DNA
Polymerase
copies DNA
PCR—what’s needed
1.
DNA—only a tiny
amount is needed!
2.
Heat stable DNA
polymerase (e.g. taq
DNA polymerase)
3.
DNA primers that
bind just outside the
DNA to clone
4.
DNA nucleotides
5.
Thermocycler or
water baths at 94oC,
55oC and 72oC
Thermocycler
automatically
repeats steps
2, 3, and 4
over and over
The polymerase chain reaction (PCR)—another view
1.
heat briefly to 94oC
to break hydrogen
bonds & separate
strands
2 . Cool to 55oC to allow
primers to
hydrogen bond
3.
Taq DNA
polymerase adds
nucleotides to 3’
end of each primer
Cycle 1
produces
2 DNA
molecules
Cycle 2 produces
4 DNA molecules
Cycle 3 produces
8 DNA molecules
Common Vectors used in Gene Therapy
1.
2.
3.
4.
Retroviruses (RNA viruses)
Adenoviruses (DNA viruses)
Liposomes
Naked DNA
1. Modified Retroviruses (RNA viruses)
(1 of 2)
Advantages
• Good at inserting genes into host
chromosome
-
Used with partial success treating Gaucher’s disease
Successfully cured 4 babies of S.C.I.D.S. in early 2000
• Severe Combined Immunodeficiency Syndrome
(Bubble Baby)
Use of Gene
Therapy to modify
blood stem cells
e.g. S.C.I.D.S. and
Gaucher Disease
1. Modified Retroviruses (RNA viruses)
(2 of 2)
Disadvantages
1. Inserts genes randomly. Possible
Consequences?
2. Usually needs an actively dividing host cell
• Therefore, not used for Cystic Fibrosis
3. Modified virus may mutate and cause
serious disease.
2. Liposomes
Liposome
•
•
•
hollow sphere surrounded by a lipid bilayer
Place gene of interest inside
Clinical trials underway with the CFTR gene
Advantages
•
No threat of disease.
Disadvantages
•
Very inefficient at inserting genes into host chromosome
3. Modified Adenoviruses—a DNA viruses
Advantages
• Most adenoviruses don’t cause serious disease.
• Clinical trials are underway with the CFTR gene
Disadvantages
• Inefficient at inserting genes into host
chromosome
4. Naked DNA
Advantages
• No threat of disease
Disadvantages
• Very inefficient at inserting genes into host
chromosome
Injecting DNA into a Cell
Pipette holding cell
Micropipette containing DNA
Problems Doing Gene therapy (1 of 2)
Inefficient gene delivery—not suitable for all genetic
diseases
1. Most effective if Stem cells are involved
•
•
Only to correct a few cells with the gene
E.g. Blood stem cells: SCIDS and Gaucher Disease
2. Less effective or Ineffective if many cells must be
corrected
•
•
Brain cells (Tay-Sacs disease, Huntington’s disease)
Cystic Fibrosis
Problems Doing Gene therapy (2 of 2)
4. Insertion of Gene isn’t always permanent
• e.g. Gaucher Disease: temporary cure until
GCase gene “popped” out of chromosome
5. Insertion of gene into genome could disrupt
other genes.
• Possible consequences?
6. Some viruses elicit immune response or may
cause disease
• E.g. Jesse Gelsinger died in 1999
What is a virus?
• Viruses—genes in packages!
– Very small—about the size of a ribosome
• Viruses sit on the fence between life and nonlife
– Exhibit some but not all
characteristics of living
organisms
– No cellular Structure
– No cell organelles
– Can’t carry out metabolism or
reproduce by itself
– Can only reproduce inside a
host cell
Importance of Viruses
1. Cause many diseases in plants, animals & humans
•
Some viruses are easily controlled with a vaccine

•
Mumps, Measles, Smallpox, Polio
Some viruses are difficult to control with a vaccine

Retroviruses (HIV: ssRNA  dsDNA)

Common cold, Influenza (Flu), HIV
2. Used as vectors in biotechnology
•
Used to insert therapeutic genes into a host cell chromosome
•
Use viruses with provirus in life cycle
Herpes (DNA Virus)
Cold sores
Herpes virus may rest inactive
inside host cells for long
periods
Adenovirus
(DNA Virus)
Adenoviruses cause various
respiratory diseases
Polio Virus
(ssRNA serves as mRNA)
Polio is easily prevented with a vaccine
Measles (ssRNA template for mRNA synthesis)
Measles: a childhood disease that can be prevented with a vaccine
Couple at AIDS quilt (HIV: ssRNA  dsDNA)
HIV is very difficult to control with a vaccine
1918 Influenza epidemic (ssRNA template for mRNA synthesis)
>20 million died of the flu during WW I
A new influenza vaccine must be developed yearly
Influenza Today
Enter H5N1, the avian
flu virus
Why do new strains of influenza
and bird flu arise in Asia?
Background: Influenza Virus Structure
1.
(1 of 3)
Flu viruses are named
by the type of surface
proteins
a.
Hemagglutinin
•
Helps virus
enter cell
•
Type A infects
humans, birds
and pigs
•
Type A has ~ 20
different sub
types
Flu Viruses Currently infecting...
• Humans: H1N1, H1N2, and H3N2
• Avian Flu Virus: H5N1
Background: Influenza Virus Structure
2.
Named for the type of
surface proteins
b.
•
Neuraminidase
•
Helps virus exit
cell
•
9 subtypes
Currently infecting
Humans:
H1N1, H1N2, and H3N2
(2 of 3)
Background: Influenza Virus Structure
3.
Influenza viral genome
•
•
•
ssRNA
8 segments (pieces)
One gene per segment
Avian Flu Virus: H5N1
•
•
•
Transmitted from birds to
humans
No evidence of human to
human transmission
Antiviral drugs: Tamiflu
 a neuraminidase
inhibitor
 Consequences of its
action?
(3 of 3)
Genetic Changes in Influenza Viruses
1. Antigenic drift – due to errors in replication and lack of
repair mechanism to correct errors
–
Results in ___________________ changes
2. Antigenic shift - reassortment of genetic materials when
concurrent infection of different strains occurs
–
Results in ___________________ changes
Emergence of New Influenza Subtypes
Antigenic shift due to genome reassortment within intermediate hosts drives flu
epidemics and pandemics
Key
Solid arrows:
current
transmission
pathways
Dashed arrows:
possible future
transmission
pathways
Numbers:
sequence of
transmission
pathways
Where do the “new flu” viruses come from?
Antigenic Drift: mutations result in changes to the Hemagglutinin (HA) molecules
- RNA replication is error prone
- New HA types are created frequently
- Requires new vaccine every “season”
- What is a vaccine?
Vaccines: Protection against viruses
1. What is a vaccine?
2. Vaccines stimulate the production of memory cells
•
Give long-term protection against a specific antigen
3. Why are vaccines ineffective against the flu virus?
•
Why will this year’s flu vaccine be ineffective next year?
4. Why are vaccines effective against DNA viruses?
-
e.g. small pox and polio virus
Smallpox
(dsDNA  dsDNA)
Smallpox has been irradiated worldwide due
to a very successful vaccine
Why are vaccines for DNA viruses so
successful?
Hepatitis B—an RNA virus
Hepatitis B
Infections may lead to
liver cancer
Emerging viruses
Ebola Virus
Hanta Virus
Both viruses: ssRNA template for mRNA synthesis
Either virus usually results in death within days!
Deer Mouse: Carries Hanta virus in Feces
Mottling of Squash and Tobacco by the Mosaic Virus
Viruses can spread easily from
cell to cell via the
plasmodesmata junctions
between cells
Comparing the size of a virus, a bacterium, and a eukaryotic cell
Viral Size
Millions can fit
on pinhead
Smaller than a
ribosome!
Bacteriophages: Viruses that attack bacteria
• The first viruses studied were bacteriophages
Head
Tail
Tail fiber
Bacterial
cell
DNA of virus
Bacteriophages (phages) have two reproductive cycles
• Lytic Cycle and Lysogenic Cycle
• The Herpes viruses and HIV both carry out these to
reproductive cycles
Bacterial chromosome
(DNA)
Phage DNA
4 Cell lyses,
releasing phages
1
Many cell divisions
7
Lytic
cycle
Occasionally a prophage
may leave the bacterial
chromosome
Lysogenic
cycle
2 Phage DNA circularizes
Prophage
3 New phage DNA and
proteins are synthesized
6 Lysogenic bacterium
reproduces normally,
replicating the prophage
at each cell division
5 Phage DNA inserts into the bacterial
chromosome by recombination
The Lytic Cycle of a Bacteriophage (slide 1 of 2)
(a) Virus lands on
bacterium.
(b) Virus injects its genes
into the cell.
(c) Virus DNA replicates,
and directs the synthesis
of new virus proteins.
The Lytic Cycle of a Bacteriophage (slide 2 of 2)
(d) Virus particles assemble.
(e) Cell bursts, releasing
new virus particle.
AIDS: Acquired Immunodeficiency Syndrome
• AIDS—caused by HIV
infection
• HIV = Human
Immunodeficiency Virus
HIV infecting a Helper T-Cell
AIDS around the world
(Source: UNAIDS)
Part of the World
People with HIV
New HIV
cases in
2002
North America
980,000
45,000
15,000
10,000
Sub-Saharan
Africa
South &
Southeast Asia
29.4m
3.5m
2.4m
2.8m
6m
700,000
440,000
240,000
India: 3.9 m
1.5m
150,000
60,000
45,000
East Asia &
Pacific
1.2m
270,000
45,000
4,000
Caribbean
440,000
60,000
42,000
20,000
Latin America
Deaths from
Aids in 2002
Children (under 15)
with Aids by end of
2002
The Structure of HIV: A Retrovirus (RNA virus)
Envelope protein
Carbohydrate
Lipid envelope
Reverse
Transcriptase
Protein
Capsid made of
protein
RNA
(2 copies of its genome)
Animation of HIV Life Cycle
Questions to Address:
1. Why does HIV only infect a specific cell type,
T-helper cells (CD-4 cells)?
2. What is HIV’s Genetic material?
3. What are the roles of Reverse Transcriptase and
protease?
4. Reverse Transcriptase does not “proof read” like
DNA polymerase does.
a. What are the consequences?
b. Of what adaptive value is this?
HIV primarily infects T-Helper Cells!
• Why does HIV have a narrow host range?
• Why does the virus that causes rabies have a broad host range?
HIV
1.) Binding
2.) Fusion
3.) Infection
Envelope
protein
Capsid
CD4
Receptor
protein
Plasma membrane of
T-helper cell
RNA
Helper protein
Cytoplasm of white blood cell
(T-Helper Cell)
Overview of HIV’s Reproductive Cycle
Reverse
transcriptase
Viral RNA
DNA
strand
Doublestranded
DNA
1
DNA of host cell
Nucleus
Provirus
DNA
What’s happening?
1.
2.
2
3
4
3.
5
4.
Viral
RNA and
proteins
Cytoplasm
6
5.
6.
Reproductive Cycle of HIV—the details!
HIV
Entry
Reverse transcriptase
Viral RNA
Integrase
Viral RNA
Synthesis
of HIV proteins
HIV envelope proteins
come to cell surface
HIV assembles
and buds from
cell
Viral RNA
copied
to viral DNA
Viral DNA
integrates into
cell chromosomes
and makes more
viral RNA
Protease cleaves large
proteins into smaller ones
Treatments for HIV
1. Reverse Transcriptase Inhibitors  Block viral DNA
formation from viral RNA
2. DNA base analogs (e.g. AZT, 3TC)  Block DNA
elongation
3. Protease Inhibitors  Block enzymes that process envelope
proteins
4. Why use a “Shotgun” approach?
5. Possible future treatments:
•
Plug drugs—drugs that plug receptors for HIV on surface of
host cell
•
Vaccines
Vaccines: Protection against viruses
1. What is a vaccine?
2. Vaccines stimulate the production of memory cells
•
Give long-term protection against a specific antigen
3. Why are vaccines not effective against retroviruses
such as HIV?
4. Why are vaccines effective against DNA viruses?
-
e.g. small pox and polio virus
DNA Technology Lecture Notes
Biol 100 – K.Marr
• Topic for the next lecture
–
•
DNA technology
Reading assignments in Essential Biology
–
–
Viruses: pp. 188-193;
Chapter 12: DNA Technology
An overview of how bacterial plasmids are used to clone genes
Bacteria have two types of DNA
• Bacterial Chromosome—contains the genes necessary for life
• Plasmid DNA—contains genes that give resistance to antibiotics
Plasmid DNA
Bacterial Chromosome
Recombinant DNA
Technology (1 of 2)
•
Bacterial production of
human protein
-
1.
2.
3.
4.
•
Extract mRNA
mRNA
Human cell
e.g. insulin, growth
hormone
Extract the desired
mRNA
Use reverse transcriptase
make complementary
DNA (cDNA)
Insert cDNA into
bacterial plasmid
Transform bacteria with
recombinant plasmid
Some, but not all cells
contain the recombinant
plasmid
cDNA
Use reverse
transcriptase to
make cDNA
Transform recombinant
plasmids into bacterial cells
Fig. 7.17-1
Cut
plasmids
Insert cDNA
into plasmid
Plasmid
DNA
Recombinant DNA
Technology (2 of 2)
Steps 5-7:
•
Isolation of the
bacterial cells that
contain the
recombinant plasmid
•
The use of bacteria to
produce insulin and
other pharmaceuticals
is very expensive!
Culture the
recombinant
bacteria
Make radiolabeled
probe for human gene
Hybridized probe
to colonies
Grow the bacteria
containing the human
gene, then isolate & purify
the human protein
Probe binds
to human gene
in the colony
that has it
Using a restriction enzyme and DNA ligase to make recombinant DNA
1.
2.
Cut bacterial plasmid
DNA with restriction
enzyme
Add human gene that
was cut out by the same
restriction enzyme.
Human gene sticks to
plasmid by
complementary base
pairing of “sticky ends”
3.
Use DNA ligase to join
the strands.
How bacterial plasmids are used to clone genes
(slide 1 of 2)
1.) Parental DNA Molecules
Bacterial plasmid DNA
Human gene to clone
2.) Cut Parental DNA Molecules with a Restriction Enzyme
“Restriction enzyme”
Discarded
How bacterial plasmids are used to clone genes
(slide 2 of 2)
3.) Mix plasmid and parental DNA molecules
Plasmid DNA
Human Gene
4.) Recombinant DNA Molecule
5.) DNA Ligase joins fragments
Using “Pharm” Animals to Produce Pharmaceuticals
(1 of 2)
Human gene of interest
“Pharm” animal
Promoter
Inject recombinant
DNA into goat
Zygote (fertilized egg)
Using “Pharm”
Animals to Produce
Pharmaceuticals (2 of 2)
Transfer the injected
embryo into the
uterus of a surrogate
mother goat
Test the offspring
for the presence
of the human DNA
Mate the animals
with the human gene
and establish a
homozygous
breeding stock
Isolate human protein
from the milk
Cystic Fibrosis, Gene Therapy, Viruses and
DNA Technology Lecture Notes
Biol 100 – K.Marr
• Topics for the next few lectures
–
–
–
•
Gene Therapy as a treatment for Cystic Fibrosis
Biology of Viruses
DNA technology
Reading assignments in Essential Biology
–
–
–
Viruses: pp. 188-193;
Sabotaging HIV : p. 171;
Chapter 12: DNA Technology