bio03 gene theraphy

8/3/2019 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

bio03 gene theraphy

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