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Problems in Gene Therapy and Transgenesis
Prof. Dr. Oliver Kayser Technische Biochemie
TU Dortmund
16.03.2015 Chapter 5 - Transgenesis and Gene
Therapy
1
Producing transgenic animals
Purpose: Production of heterolog proteins in transgenic animals
Principle: Integration of heterolog DNA with selected genetic information in fertilised eggs
Methods: Mikroinjektion (-)
Retrovirus vectors (-)
Genetically modified
embryonic stem cells (++)
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Dolly
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Therapy
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Telomers
kla
us
urr
ele
va
nt
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Advantage of sheeps and goats as transgenic animals
• high milk production capacity (2-3 l/ day)
• short gestation period and quick maturation
• low capital investment
• easy handling and breeding
• high expression level of proteins (35g protein/ l milk)
• easy downstream processing • ongoing supply of product is guaranteed by breeding
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Drug safety
• Genetic stability from animal to anmial
• Variation in produktion und purity during lactation
• Microbial, varial, and mykoplasm contamination
• TSE, Scrapie
• Age and general conditions of animals
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Atryn
• First recombinant therapeutic protein approved
in 2009
• Anticoagulant for treatment of
antithrombin III (AT alpha)
deficiency in newborn
• 1 goat replaces 90,000 donators
16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 10 http://www.gtc-bio.com/
Blood Clotting
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Transgenic animals as disease
model
• Knock out animals with directed genetic
defect
• Testing of drugs and dosage forms
• Hypercholesterinemia
• Cancer
• Coronar diseases
• Onco-Mouse as first example
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Transgenic animals L
eh
rbuch
Mo
leku
lare
Zellb
iolo
gie
, K
ap
ite
l 1
0.5
Ageing-Mouse, Defect in DNA-Helcicase
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Transgenic salmon
• „Turbo-salmon“ AquAdvantage, AquaBounty Technologies
• Cloning of growth gene for improved growth (growth factors)
• Fish grows in 16 to 18 months instead of 3 years
• Triploid and not diploid (Biosafety)
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http://www.nature.com/news/transgenic-salmon-
nears-approval-1.12903
Kuru-Region in Papua-Neuguinea
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Prions
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Brain autopsy
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Somatic Gene Therapy
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Defective Genes
Disease Genetic defect
hemophilia A absence of clotting factor VIII
cystic fibrosis defective chloride channel protein
muscular dystrophy defective muscle protein (dystrophin)
sickle-cell disease defective beta globin
hemophilia B absence of clotting factor IX
severe combined
immunodeficiency
(SCID)
any one of several genes fail to make
a protein essential for T and B cell
function
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ADA und purine metabolim
IMP AMP ADP ATP dAMP dADP dATP
Adenosin 2-Deoxyadenosin
Inosin 2-Deoxyinosin
Adenosin-
Deaminase
Acumulation
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Basics of ADA-Defency
dAMP dADP dATP
2-Deoxyadenosin
2-Deoxyinosin
Adenosin-
Deaminase
Acumulation Inhibition of
Ribonucleotid-Reductase
Consequence:
No DNA-Replication
Apoptosis
Inhibition of T cell activation
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EX VIVO vs IN VIVO
Tumor cells
fibroblasts
Hemopathic
cells
Gene transfer in culture
Delivery to target
Vector
local
systemic
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Examples for local gene expression
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Biochemical vectors/Methods:
• viral vectors (Retro-, Adenovirus, AAV)
• ligands with polylysin
• 'Transferinfection'
Physical methods:
• Hydrodynamic pressure
• 'Biolistic Bombardment'
• electroporation
• direct tissue inclusion
Chemical vectors:
• cationic Liposomes
• polycations like DEAE-Dextran, Polyethylen-Imin
• poly-Lysin-polymers
Vectors in gene technology
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Retroviral vector system
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Summary
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Non-viral delivery
• The non-viral approaches provide a
safety (no virus) to gene therapy.
However, the route to nucleus for stable
expression is not completely understood
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Biochemical vectors/Methods:
• viral vectors (Retro-, Adenovirus, AAV)
• ligands with polylysin
• 'Transferinfection'
Physical methods:
• Hydrodynamic pressure
• 'Biolistic Bombardment'
• electroporation
• direct tissue inclusion
Chemical vectors:
• cationic Liposomes
• polycations like DEAE-Dextran, Polyethylen-Imin
• poly-Lysin-polymers
Vectors in gene technology
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Therapy
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Electroporation
Physical Principle
Gehl J (2003) Acta Physiol Scand 177:437
CHO-cells, 20 ms, 1 kV cm-1
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Electroporation
Examples Pros and Cons
+ High safety
+ High compliance
+ No risk of infection
Target cells / organs
• liver
• skin
• muscel
Mainly:
Transdermal applications
– low transfection
– no tissue specifity
– transient expression K562 cells with phGFPS65T transfection
Beta-Galactosidase transfection
in vivo in muscel cells
Mir et al. (1999) Proc Natl Acad Sci USA 96:4262
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Biobalistics
Principle
Gold partikel Skin Gene Gun
Expression
Examples Pros and Cons
• Application of vaccines
• Arilvax®, Hepagene®
• Powderject Inc: Accell®
• Biorad Inc: Helios®
• Transdermal Application
+ easy to use
+ using already existing technology
- high costs
- not only transdermal Application
- low efficancy
1-3 µm
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Powderject Inc.: Accell®
Maa et al. (2000) Curr Pharm Biotechnol 3:283
Yang et al. (1999) in Nonviral vectors for gene therapy, Academic Press, 171
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Liposomes
• This technique relies upon the negative charge
of DNA-phosphate, cationic lipids, and cell
surfaces (negative charge, sialic acid).
• Two classes of cationic lipids have been used:
– two alkyl chain in each cationic lipid molecules
(DOTMA)
– cholesterol as a backbone.
• Dioleoylphosphoatidylethanolamine, a helper
lipid is usually included to improve efficiency.
• Expression is transient for several days only.
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O
O
H
O
H
H
HH
P OO
O
H
O
H
H
HH
P OO
O
-
-
BASE
BASE
O
ON
+
O
O
O
ON
+
O
O
O
ON
+
O
O
O
ON
+
O
O
Complexing DNA (+/- -ratio)
O
ON
+
O
O
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DNA Condensation
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Cellular uptake and migration
Wattiaux et al. (2000) ADDR 41:201
active uptake
into nucleus
DNA degradation
by nucleases
pH 4,5-6
Long persistence
DNA Hydrolysis
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Polyethylenimine (PEI)
Wightman et al. (2001) J. Gene Med 3:362
Gene transfer complex Uptake Migration / Transfection
High stability
Half life time: 30-60 min
High toxicity (40-100 µg in mice)
50-100 nm
Linear structure high transfection
High branching low transfection
Loading up to 800 kB
Coupling with ligands (Transferrin, Monoclonal
antibodies, sugar, folate)
High stability
„Sponge effect“
Nuclease degradation
transient
expression
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Nucleus
Proton sponge
PEI-Gene transfer complex
+
- - -
- +
+ + +
+ +
+
+
+ +
+ -
- - -
- - - -
- - -
+
+
+ +
+
+
+
+ +
- - -
- +
+ + +
+ +
+
+
+ +
+ -
- - -
- - - -
- - -
+
+
+ +
+
+
+
+ Endocytosis
+
- - -
- +
+ + +
+ +
+
+
+ +
+ -
- - -
- - - -
- - -
+
+
+ +
+
+
+
+
+
-
+
+ +
+ +
+
+
- - -
+ +
+
+
+
+
- - -
- +
+ + +
+ +
+
+
+ +
+ -
- - -
- - - -
- - -
+ +
+
+
+
+
H+
H+
+
- - -
- +
+ + +
+ +
+
+
+ +
+ -
- - -
- - - -
- - -
+ +
+
+
+
+
Cl-
Cl- H2O
H2O
+
+ - - +
Inner tertiar amines neutralise low pH in
endosome swelling breaking
„Proton sponge“ Behr (1997) Chimia 51:34
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Summary
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