Maitreyee Mittal- Gene Therapy for Parkinson’s Disease

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

Maitreyee Mittal- Gene Therapy for Parkinson’s Disease

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