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8/3/2019 Maitreyee Mittal- Gene Therapy for Parkinsons Disease
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Gene Therapy
A novel technique to treat genetic disorderst at cannot e treate t roug me cat on
An attempt to cure/treat these diseases by: Replacing the damaged gene with a healthy copy
Repairing a mutated gene
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How Gene Therapy Works
A vector carries therapeutic gene to target cells
rote n pro uct o gene restores ce to norma
state -
Non-viral Vectors
Can be produced in large amounts
Minimal immunological problems
Problem: cannot be transferred efficiently to the
cells
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Viral Vectors
ruses are use to e ver genet c mater a tothe cells
Can target and infect specific cells
dangerous genes and include genes ofinterest
Can efficiently insert viral genome into host
genome
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Transduction of the Target Cell
Infection that introduces genetic informationex ressed from recombinant vectors into tar et cell
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Transduction
Vector containing therapeutic genesequences n s to ce
Genome to enter the nucleus ec or genome ecomes n egra e n o os
genome
protein product follows
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Parkinsons Disease
Neurodegenerativedisease
Neurons are destroyed inparts of the brain stem
Thought to be caused by
genetic mutations
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Dopamine
An essentialneurotransmitter Involved in movement
control and informationprocess ng
a lack of dopamine leadsto slower movements
Destroyed neuronsleads to an insufficient
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Gene Therapy for Parkinsons
To stimulate an increase of dopamine A study was conducted using the adeno-
associated virus (AAV) to deliver GDNF -
blocks the toxin-induced degeneration of thedo amine neurons
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Adeno-Associated Virus (AAV)
Can integrate and stablyex ress trans ene roduct
The packaging capacity ofAAV is about 5.0 kb, a majorlimitation of the AAV
96% of viral genome has tobe removed
Absence of viral enesminimizes expression offoreign proteins Avoids risk of triggering host
mmune responses
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The Problem with AAV
Not all types of neurons are
AAV requires binding to twodifferent receptors
If one is missing, AAV cannotn
High affinity for thesubstantia nigra
striatum Striatum is important because
it prevents degradation of
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Solution: Hemagglutinin Protein
Envelope protein of the influenza virus
Responsible for host cell binding and fusion of viral and
host membranes n s o s a c ac res ues on arge ce sur aces
After virus enters the cell through endocytosis,acidification of vesicle induces chan es in the roteinthat promotes fusion with the vesicle membrane
Releasing the flu virus into the host cytoplasm
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Researchable question
Can the Hema luttinin rotein beadded to the AAV vector so that
neurons and transfer the gene
effectively?
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scFv Phage Library
antibody
Libraries ofantibody genesthat are easier to
Used to select forbindin to aspecific target
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Method
1. Expose scFv 2. Wash away 3. Use buffer to alter pH
AAV vectors
did not bind
did bind are removed
5. Createprotein productsfrom ene
4. Attach scFv gene to
6. Attach HAprotein withscFv to AAV
7. Transduceneurons wtransformedAAV
8. Test the transduction
efficiency of the new AAV
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Materials
AAV vectors with GFP gene
svFv phage library
HA gene PCR machine
Bacteria and plasmid
UV light
Neurons
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1-3:ac ng
scFv to
Vectors
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gene
Use PCR machine to remove scFv gene fromp age
Obtain a copy of HA gene from influenzav rus
Ligate both genes using PCR
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5/6: Protein Products
Insert HA/scFv gene into a
Transform E. colibacteria
with the transformedplasmid
Purify the protein products
Attach HA protein to AAVthrou h the s ecificantibody
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7/8: Transduction
Cells with GFP
Infect neurons on a plate with the new AAV
(as a control)
Use a UV li ht to detect the fluorescence of
GFP
If the virus entered the cell, then it will glow
Can see how many cells were transduced
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Potential Problems
No phage from the library binds to the AAV
All the phages are washed away, including thosethat did bind
,occurs
Not enough HA protein is made
lost
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Works Cited
Bjrklund, A., et al. "Towards a neuroprotective gene therapy for Parkinsonsdisease: use of adenovirus, AAV and lentivirus vectors for gene transfer of
". .886.1-2 (2000): 82-98.
Eckert, Debra M., et al. Mechanisms of Viral Membrane Fusion and ItsInhibition. Annual Review of Biochemistry. 70 (2001): 777-810.
Kay, Mark A., et al. Viral Vectors for Gene Therapy: The Art of Turningn ec ous gen s n o e c es o erapeu cs. a ure e c ne. . :
33-40. Kordower, Jeffrey H. In Vivo Gene Delivery of Glial Cell Line-Derived
Neurotrophic Factor for Parkinsons Disease. Annals of Neurology. Vol. 53.2003: 120-134.
Maynard, Jennifer, et al. Antibody Engineering. Annual Review ofBiochemistry. 2 (2000). 339-376. Panno, Joseph. Gene Therapy: Treating Diseases By Repairing Genes. New
York: Facts on File, 2005. Print.
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Pictures Cited
Slide 4: http://www.armageddononline.org/image/virus2.JPG
Slide 5: . . _ _ _ .
Slide 7: http://adam.about.com/reports/Parkinson-s-disease.htm
Slide 8: http://blog.lib.umn.edu/trite001/studyinghumananatomyandphysiology/dopamine_recep.jpg
Slide 11:
https://reader009.{domain}/reader009/html5/0509/5af2209a476ad/5af220a9ccaae.jpg Slide 12:
http://www.slimfilms.com/graphics/6adenovirus.jpeg \
Slide 13: https://reader009.{domain}/reader009/html5/0509/5af2209a476ad/5af220aa3f0fe.jpg
Slide 15: http://www.osc.edu/education/si/projects/flu_virus/flu-3d.gif
Slide 16: http://upload.wikimedia.org/wikipedia/commons/1/12/Hemagglutinin_in_action.png
Slide 18: http://upload.wikimedia.org/wikipedia/commons/a/a0/Antibody_je2.png
Slide 21: http://www.neb.com/nebecomm/ManualFiles/manualE8100.pdf
Slide 23: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/ferguson/plasmidinsert.gif
Slide 24: http://microbiology.muohio.edu/people/images/GFP.GIF