7. gene therapy teacher
DESCRIPTION
PPT gen terapiTRANSCRIPT
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GENE THERAPY
Bagian Biokimia Fakultas Kedokteran Universitas Hasanuddin Makassar
Dr. Marhaen Hardjo, M.Biomed, PhD
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DNA sequence
↓
mRNA sequence
↓
Polypeptide
Gene mutations which affect only one gene
Transcription
Translation
© 2010 Paul Billiet ODWS
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DNA (antisense strand)
mRNA
Polypeptide
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
The antisense strand is the DNA strand which acts as the template for mRNA transcription
© 2010 Paul Billiet ODWS
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Mutations: Substitutions
Substitution mutation
GGTCACCTCACGCCA
↓
CCAGUGGAGUGCGGU
↓
Pro-Arg-Glu-Cys-Gly
Substitutions will only affect a single codonTheir effects may not be serious unless they affect an amino acid that is essential for the structure and function of the finished protein molecule (e.g. sickle cell anaemia)
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
© 2010 Paul Billiet ODWS
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The genetic code is degenerate
A mutation to have no effect on the phenotype
Changes in the third base of a codon often have no effect.
© 2010 Paul Billiet ODWS
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No change
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTTCTCACGCCA
↓
CCAGAAGAGUGCGGU
↓
Pro-Glu-Glu-Cys-Gly
© 2010 Paul Billiet ODWS
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Disaster
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTCCTCACTCCA
↓
CCAGAAGAGUGAGGU
↓
Pro-Glu-Glu-STOP
© 2010 Paul Billiet ODWS
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Mutations: Inversion
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Inversion mutation
GGTCCTCTCACGCCA
↓
CCAGGAGAGUGCGGU
↓
Pro-Gly-Glu-Cys-Gly
Inversion mutations, also, only affect a small part of the gene
© 2010 Paul Billiet ODWS
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Mutations: Additions
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Addition mutation
GGTGCTCCTCACGCCA
↓
CCACGAGGAGUGCGGU
↓
Pro-Arg-Gly-Val-Arg
A frame shift mutation
© 2010 Paul Billiet ODWS
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Mutations: Deletions
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Deletion mutation
GGTC/CCTCACGCCA
↓
CCAGGGAGUGCGGU
↓
Pro-Gly-Ser-Ala-Val
A frame shift mutation
© 2010 Paul Billiet ODWS
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Mutations of haemoglobin
• Haemoglobin is a tetramer = 2 and 2 -chains• The genes for these polypeptides are found on
different chromosomes• The -chain gene is found on chromosome 11• The -chain gene is found on chromosome 16• The nucleotide sequences have been worked
out• Several inherited diseases occur on the -chain,
which contains 146 amino acids.
© 2010 Paul Billiet ODWS
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haemoglobin sense strand cDNA sequence
• cDNA (complementary DNA) is obtained by back-transcribing the mRNA used to translate the polypeptide
• So cDNA has no introns
• This is done using reverse transcriptase enzyme.
© 2010 Paul Billiet ODWS
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ATG GTG CAT CTG ACT CCT GAG GAG AAG TCT GCC GTT ACT GCC CTG TGG GGC AAG GTG AAC GTG GAT GAA GTT GGT GGT GAG GCC CTG GGC AGG CTG CTG GTG GTC TAC CCT TGG ACC CAG AGG TTC TTT GAG TCC TTT GGG GAT CTG TCC ACT CCT GAT GCT GTT ATG GGC AAC CCT AAG GTG AAG GCT CAT GGC AAG AAA GTG CTC GGT GCC TTT AGT GAT GGC CTG GCT CAC CTG GAC AAC CTC AAG GGC ACC TTT GCC ACA CTG AGT GAG CTG CAC TGT GAC AAG CTG CAC GTG GAT CCT GAG AAC TTC AGG CTC CTG GGC AAC GTG CTG GTC TGT GTG CTG GCC CAT CAC TTT GGC AAA GAA TTC ACC CCA CCA GTG CAG GCT GCC TAT CAG AAA GTG GTG GCT GGT GTG GCT AAT GCC CTG GCC CAC AAG TAT CAC TAA
Methionine initiator
Nonsense terminator© 2010 Paul Billiet ODWS
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Mutation Codon Change to DNA sense strand
Change in Amino Acid
S (sickle cell anaemia)
6 GAG to GTG Glu to Val
C (cooley’s syndrome)
6 GAG to AAG Glu to Lys
GSan Jose 7 GAG to GGG Glu to Gly
E 26 GAG to AAG Glu to Lys
MSaskatoon 63 CAT to TAT His to Tyr
MMilwauki 67 GTG to GAG Val to Glu
OArabia 121 GAA to GTA Glu to Val
© 2010 Paul Billiet ODWS
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Sickle Cell Anaemia
Blood smear (normal)Image Credit: http://lifesci.rutgers.edu/~babiarz/
Sickle cell anemiaImage Credit: http://explore.ecb.org/
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Diseases for applying gene therapy
Disease Defect Target cell
Severe combined Bone marrow cells or
immunodeficiency T-lymphocytes
Hemophilia Liver, muscle
Cystic fibrosis Lung Cells
Cancer Many cell types
Neurological diseases Parkinson’s/ Alzheimers Nerve Cells
Infectious diseases AIDS, hepatitis B White Blood Cells
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Gene therapy could be very different for different diseases
• Gene transplantation (to patient with gene deletion)
• Gene correction (To revert specific mutation in the gene of interest)
• Gene augmentation (to enhance expression of gene of interest)
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Gene therapy
In vivo Ex vivo
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in vivo and ex vivo schemes
http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm
IN VIVO
EX VIVO
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1. The genetic material is transferred directly into the body of the patient
2. More or less random process; small ability to control; less manipulations
3. Only available option for tissues that can not be grown in vitro;
or if grown cells can not be transferred back
In vivo gene therapy
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1. The genetic material is first transferred into the cells grown in vitro
2. Controlled process; Genetically altered cells are selected and
expanded; more manipulations
3. Cells are then returned back to the patient
Ex vivo gene therapy
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Injections of naked DNA
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Current attempts with naked DNA vaccination in infectious diseases
HIV HepatitisInfluenza
Tuberculosis,
Lyme disease
Malaria
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Ballistic DNA Injection (gene guns)
Invented for DNA transfer to plant cells
Fully applicable to eukaryotic cells
plasmid DNA shown here
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LiposomesNext level idea – why naked DNA?
Lets’ wrap it in something safe to increase transfection rate
Therapeutic drugs
Lipids – are an obvious idea !
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DNA delivery of genes by liposomes
Cheaper than viruses
No immune response
Especially good for in-lung delivery (cystic fibrosis)
100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector
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Cystic fibrosismost common lethal genetic disorder in Caucasian populations (1 in 2000 live births.) . Among African and Asian is really rare
a defect in the CFTR gene
Lungs create thick mucus secretion(prone to infections,
constant cough, leading cause of death)
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Lungs in cystic fibrosisNormal lung CF lungs
dilated crypts filled with mucus and bacteria.
Normal alveolar appearance
CF lungs filled with mucus lung did not collapse when it was removed postmortem
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Cystic fibrosis lungs are prone to infections
The battle between WBC’s and bacteria leads ultimately to lung fibrosis and damage
"Hyperinflammation" and WBC’s unable to eradicate bugs, instead damage lung tissue.
Mucus protects bugs
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MOST COMMON VIRAL VECTORS
Retroviruses
Herpes simplex viruses
can create double-stranded DNA copies of their RNA genomes. Can integrate into genome. HIV for example
dsDNA viruses that infect a neurons. Cold sores virus
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Retroviral vectors are able to infect dividing cells only
Good for cancer gene therapy
So, retroviruses are most often used vectors for common disease gene therapy
In dividing cells nuclear membranes are broken down, so viral genome can enter and integrate into the chromosome
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Real treatments performed with retroviral system
Severe Combined Immunodoficiency (SCID)Mutation on Chromosome 20 is often to blame
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What is Severe Combined Immunodoficiency (SCID)?
> 8 new ear infections per year
> 2 serious sinus infections per year
> 2 month on antibiotics with little effect
> 2 pneumonias per year
-- failure to gain weight and grow
-- recurrent deep skin and organ abscesses
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SCID treatments
Life in germ-free envinronment
Bone-marrow transplantations
Enzyme replacement therapy VERY expensive; not a cure; temporary effect
GENE THERAPY