Cystic Fibrosis and Gene TherapyLecture 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

• How to use a DNA Probe to Screen for the CF Gene

1. Isolate DNA from patient

2. Heat to separate DNA strands

3. Add labeled probe that has complementary base sequence to mutant gene

4. Add restriction enzymes (cuts DNA into fragments) and separate by gel electrophoresis

Single stranded DNA from patient

DNA probe complementary to mutant gene

Normal Genotype Abnormal Genotype

Probe does not bind to DNA

Probe binds to DNA

a.) DNA and dye are loaded in a well on a gel, and an electric field is placed across the gel.

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

(+) Electrode attracts negatively charged DNA fragments

GelWell

DNA samples from PCR ofSnowball’s DNA

Direction ofelectric field

Separation of DNA fragments by Gel electrophoresis

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

• Permanent cure for individual

2. 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

Double-strandedDNA

Single-strandedDNA

taq DNApolymerase

DNA Primers bindto theircomplementarysequence

DNAPolymerase

copies DNA

Thermocyclerheats sampleto near boiling

(~94oC)

Thermocyclerlowerstemperatureto 55 °C

Thermocyclerraisestemperatureto 72 °C

Heat breakshydrogen bonds

and strandsseparate

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

Thermocyclerautomaticallyrepeats steps2, 3, and 4over 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. Retroviruses (RNA viruses)2. Adenoviruses (DNA viruses)3. Liposomes4. 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)

Disadvantages1. 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

Micropipette containing DNA

Pipette holding cell

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

– 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

• Viruses—genes in packages!

– Very small—about the size of a ribosome

• Viruses sit on the fence between life and nonlife

What is a virus?

Importance of Viruses1. 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 of 3)

Flu Viruses Currently infecting... • Humans: H1N1, H1N2, and H3N2• Avian Flu Virus: H5N1

1. Flu viruses are named by the type of surface proteinsa. Hemagglutinin

• Helps virus enter cell

• Type A infects humans, birds and pigs

• Type A has ~ 20 different sub types

Background: Influenza Virus Structure (2 of 3)

2. Named for the type of surface proteinsb. Neuraminidase

• Helps virus exit cell

• 9 subtypes

• Currently infecting Humans:

H1N1, H1N2, and H3N2

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?

Background: Influenza Virus Structure (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 SubtypesAntigenic 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

Antigenic Drift: mutations result in changes to the Hemagglutinin (HA) molecules

Where do the “new flu” viruses come from?

- RNA replication is error prone- New HA types are created frequently- Requires new vaccine every “season”- What is a vaccine?

Vaccines: Protection against viruses1. 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!

• The first viruses studied were bacteriophages

Bacteriophages: Viruses that attack bacteria

Head

Tail

Tail fiber

DNA of virusBacterialcell

Bacteriophages (phages) have two reproductive cycles

Phage DNA

1

2

3

4

Phage DNA circularizes

New phage DNA andproteins are synthesized

5 Phage DNA inserts into the bacterialchromosome by recombination

Cell lyses,releasing phages

6 Lysogenic bacteriumreproduces normally,replicating the prophageat each cell division

7Occasionally a prophagemay leave the bacterialchromosome

Bacterial chromosome (DNA)

Lytic cycle Lysogenic

cycle

Many cell divisions

• Lytic Cycle and Lysogenic Cycle

• The Herpes viruses and HIV both carry out these to reproductive cycles

Prophage

(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 1 of 2)

(d) Virus particles assemble. (e) Cell bursts, releasing new virus particle.

The Lytic Cycle of a Bacteriophage (slide 2 of 2)

AIDS: Acquired Immunodeficiency Syndrome

HIV infecting a Helper T-Cell

• AIDS—caused by HIV infection

• HIV = Human Immunodeficiency Virus

AIDS around the world (Source: UNAIDS)

Part of the World People with HIV

New HIV cases in

2002

Deaths from Aids in 2002

Children (under 15) with Aids by end of

2002

North America 980,000 45,000 15,000 10,000

Sub-Saharan Africa

29.4m 3.5m 2.4m 2.8m

South & Southeast Asia

6m

India: 3.9 m

700,000 440,000 240,000

Latin America 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

RNA (2 copies of its genome)

Capsid made of protein

Protein

Lipid envelope

Carbohydrate

Envelope protein

Reverse Transcriptase

The Structure of HIV: A Retrovirus (RNA virus)

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 1.) Binding 2.) Fusion 3.) Infection

Envelopeprotein

Capsid

Plasma membrane of T-helper cell

Helper protein

CD4Receptorprotein

Cytoplasm of white blood cell(T-Helper Cell)

RNA

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?

1

23

4

5

6

DNA strand

Viral RNAReverse transcriptase

Cytoplasm

Double-stranded DNA

Viral RNA and proteins

Nucleus

Overview of HIV’s Reproductive Cycle

DNA of host cell

Provirus DNA

What’s happening?

1.

2.

3.

4.

5.

6.

EntryHIV

Reverse transcriptase

Viral RNAcopiedto viral DNA

Viral DNAintegrates intocell chromosomesand makes moreviral RNA

Protease cleaves largeproteins into smaller ones

Synthesisof HIV proteins

HIV envelope proteinscome to cell surface

HIV assemblesand buds from

cell

Viral RNA

Viral RNA

Integrase

Reproductive Cycle of HIV—the details!

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

Treatments for HIV

Vaccines: Protection against viruses1. 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

Plasmid DNA

Bacterial Chromosome

Bacteria have two types of DNA• Bacterial Chromosome—contains the genes necessary for life

• Plasmid DNA—contains genes that give resistance to antibiotics

Fig. 7.17-1

Extract mRNA

mRNAHuman cell

cDNA

Use reverse transcriptase to

make cDNA

Insert cDNAinto plasmid

Cutplasmids

Transform recombinantplasmids into bacterial cells

PlasmidDNA

Recombinant DNA Technology

(1 of 2)• Bacterial production of

human protein

e.g. insulin, growth hormone

1. Extract the desired mRNA

2. Use reverse transcriptase make complementary DNA (cDNA)

3. Insert cDNA into bacterial plasmid

4. Transform bacteria with recombinant plasmid

• Some, but not all cells contain the recombinant plasmid

Culture therecombinantbacteria

Make radiolabeledprobe for human gene Probe binds

to human genein the colonythat has it

Hybridized probeto colonies

Grow the bacteriacontaining the human gene, then isolate & purify the human protein

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!

Using a restriction enzyme and DNA ligase to make recombinant DNA

1. Cut bacterial plasmid DNA with restriction enzyme

2. 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.

1.) Parental DNA Molecules

Bacterial plasmid DNA Human gene to clone

Discarded

“Restriction enzyme”

How bacterial plasmids are used to clone genes (slide 1 of 2)

2.) Cut Parental DNA Molecules with a Restriction Enzyme

5.) DNA Ligase joins fragments

3.) Mix plasmid and parental DNA molecules

Plasmid DNA Human Gene

4.) Recombinant DNA Molecule

How bacterial plasmids are used to clone genes (slide 2 of 2)

Inject recombinantDNA into goatZygote (fertilized egg)

Human gene of interest

Promoter

Using “Pharm” Animals to Produce Pharmaceuticals (1 of 2)

“Pharm” animal

Transfer the injectedembryo into theuterus of a surrogatemother goat

Test the offspringfor the presenceof the human DNA

Isolate human proteinfrom the milk

Mate the animalswith the human geneand establish ahomozygousbreeding stock

Using “Pharm” Animals to Produce

Pharmaceuticals (2 of 2)

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