Download - BIO03 Gene Theraphy
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Any procedure intended to treat or alleviatedisease by genetically modifying the cells of a
patient(Human Molecular Genetics)
Gene therapy involves delivery of genes that: Replace the defective genes causing the disease
Drives the cells with the defective geneticstructure to death
Genes, gene segments or oligonucleotides
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Genetic disorders
Cancer (oncogenes, tumor-suppressor genes,
apoptosis genes) Infectious diseases (viral or bacterial)
Immune system disorders (allergies,
inflammations and also autoimmunediseases)
Cardiovascular diseases
Neurological diseases
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In vivo gene therapy
Direct delivery ofgenes
http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture25/Lecture25.html
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In vitro gene therapy(Exvivo gene therapy)
Removal of patients cells,transfection of cells with thespecific gene, re-introductionof the cells to the body
http://library.thinkquest.org/28000/load_image.php3?id=50
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Gene augmentation therapy (GAT)
Loss-of-function diseases autosomal recessive
Additional copies of wildtype gene Enhanced gene product
Even slight expression make a considerabledifference no requirement for high expression
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Example;
Hemophilia
http://www.nwabr.org/studentbiotech/winners/studentwork/2007/WB_BA_TRONGTHAM/9_developingcures.htm
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Direct killing of the disease cells
Transgene expression cause cell death
Suicide genes:encode lethal toxins
Prodrug genes:
confer sensitivity to asubsequent drugtreatment
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or Indirect killing by provoking immune
response against target cells
Targeted killing ofdiseased cells ispopular with cancertreatments
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Targeted Mutation Correction
Gain-of-function diseases where GAT has nohelp
At gene level: via HR
At mRNA level: therapeutic ribozymes & RNA Editing
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Targeted inhibition of gene expression
Novel or inappropriate gene expression indiseased cells
Block expression at either mRNA or protein level
ODN, oligodeoxynucleotide;TFO, triplex-forming oligonucleotide
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ClassicalGTaims the introduction of genes that;
expresses a product that the patient lacks
kill diseased cells directly by expressing lethal toxins activate cells of the immune system to attack
diseased cells
Non-classicalGTaims
to inhibit the expression of genes related topathogenesis
to restore normal gene expression via correction ofthe mutation
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High expression is usually desired
Thus, cDNA with flanking regulatory sites are
introduced Upon transfer, the inserted genes may
integrate into the chromosomes of the cell
remain as extrachromosomal genetic elements --called episomes
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Long-term stable expression
In each celldivison, theinserted gene
will bereplicated too !
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Also has drawbacks!
Integration is almost alway random
No expression of the inserted gene if integrationoccurs on heterochromatin
Insertional inactivation of an essential gene resulting in cell death
Activation of oncogenes cancer development
Inactivation of tumor-suppressors or apoptosisgenes cancer development
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What can you do then?
Exvivo delivery screen the cells before after thegenome integration
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For treatment of actively dividing cells, long-
term expression and stable transmission to
progeny cells may be problematic Repeated application
Thus, episomal expression is usuallyemployed for targeted killing of the diseasecells once theyre dead, you dont need the
therapeutic anymore
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Gene Therapy
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Reverse-transcriptase activity & genome
integration
up to 8 kb of exogenous DNA Nucleus excludespreintegration complexThus, only actively
dividing cells are targets Certain blood cells
Cells lining the
gastrointestinal tract
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Then, whats the use of retroviruses if target
cells are limited?
Ideal for cancerous cells of otherwise non-dividingcells targeted killing approach
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Replicationincompetent
Transduction(infection +integration) capable
http://www.genetherapyreview.com/gene-therapy-education/gene-transfer-vectors/1-viral-vectors/8-retrovirus.html
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DNA viruses of the upper respiratory tract
infections
Receptor-mediated endocytosis High transduction efficiency
Can infect non-dividing cells
Large viruses accept large inserts,
Up to ~35 kb
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Episomal expression transient, may require
repeated application
Infects both dividing and non-dividing cells
not suitable for targeted killing strategy
Virtually non-specific
May trigger serious inflammatory responses!
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ssDNA viruses Requires co-infection by a helper virus to replicate
In the absence of such a helper virus, AAVs integrate intothe host genome
Any subsequent infection by a helper virus can activate its
reproduction
Safe - 96% of all AAV genes can be removed Long-term expression Up to ~4.5 kb
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Complex retroviruses inc Capable of transducing non-dividing cells
For HIV, preintegration complex contains nuclearlocalization signal
Thus, HIV is actively transported through nuclearpores during interphase
Neurons, macrophages, lymphocytes, hematopoieticstem cells, retinal photoreceptors, muscle and liver cells
They do not trigger immune responses
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Gene Therapy
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Enclosed circle of synthetic lipid bilayers
Endocytosis by the plasma membrane
No size limit for transgenes, easy to prepare
Pitfalls
Episomal expression
transient
Low delivery
efficiency
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Injection by a syringe into a specific tissue
Early mouse model: intramuscular injection of a
dystrophin minigene for DMD Biolistics Naked DNA-coated metal
particles
Relatively safe
Low efficiency
Low level of integration
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Reversible attachment of
DNA to a targeting molecule
Interaction with specific
receptor for the targetingmolecule
Lysosomic degradation is a
threat!
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AkhtarS,2006,Non-viral cancer gene therapy:Beyond delivery
http://www.nature.com/gt/journal/v13/n9/full/3302692a.html
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http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture25/Lecture25.html
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Which routeto take?
http://journals.cambridge.org/fulltext_content/ERM/ERM1_11/S1462399499000691sup007.pdf
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GENE THERAPY APPLICATIONS
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http://www.nature.com/nrg/journal/v4/n5/fig_tab/nrg1066_F2.html
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Serious Combined Immunodeficiency 25% of cases are autosomal
associated with chromosome 20, ADA mutation
X-linked SCID mutation in a receptor gene
Polygenic
Treatment
Isolation from all pathogens Bone-marrow transplant Enzyme replacement GT
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1990, 1st trial
ADA adenosine deaminase
ADA mutants: Adenosine compounds areaccumulated induce cell death in immunesystem cells
ADA gene is small and T-cells are easy to culture
Exvivo GT
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1990, Ashanthi DeSilva
MoMLV (Retrovirus)
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Simultaneously,
enzyme replacement
was continued.
Thus, there is a little
controversy concerningthe actual action of GT
2003
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Bubble Boy David Vetter (1971-1984)
Lived in a sterile plastic bubble for 12 years!
No B-cells, T-cells, NKs
Sadly, David died following bone
marrow transplant from his sister- His sister was later revealed
to be + for EBV
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SCID development
http://www.nature.com/ni/journal/v11/n6/pdf/ni0610-457.pdf
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Alain Fischer, 2000
10 affected babies 1-9 months IL-2 receptor- K gene is defected
Kc cytokine receptor subunit is impaired
Retroviral application for 3 days
Kc cDNA insert
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2 out of 10
babies
developedleukemia!
1 died
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Ornithine transcarbamylase
important enzyme in urea metabolism
OTC deficiency leads tohyperammonia in bloodstream
Affected people should
avoid high protein diets
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1999, Jesse Gelsinger Mosaic individual forOTC deficiency
Relatively mild symptoms
Volunteered for a clinical trial
Single shot on hepatic artery using Adenoviral vectors
Fewer & sudden decrease in platelet count in 12 h Death in 3 days as a result ofSystemic Inflammatory
ResponseSyndrome
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For more information: Human Molecular Genetics 2http://www.ncbi.nlm.nih.gov/books/NBK7569/#A2865