Problems in Gene Therapy and Transgenesis Bio...Producing transgenic animals Purpose: Production of heterolog proteins in transgenic animals Principle: Integration of heterolog DNA

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 (++)

16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 2


Dolly


Therapy

4

Telomers

kla

us

urr

ele

va

nt


Therapy

5



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


Therapy

8

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


Therapy

9

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


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


Transgenic animals L

eh

rbuch

Mo

leku

lare

Zellb

iolo

gie

, K

ap

ite

l 1

0.5

Ageing-Mouse, Defect in DNA-Helcicase

16.03.2015 13 Chapter 5 - Transgenesis and Gene Therapy

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)


http://www.nature.com/news/transgenic-salmon-

nears-approval-1.12903

Kuru-Region in Papua-Neuguinea


Prions


Therapy

16

Brain autopsy


Somatic Gene Therapy


Therapy

18


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


Therapy

20

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Clotting.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Muscles.html




http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Clotting.html

ADA und purine metabolim

IMP AMP ADP ATP dAMP dADP dATP

Adenosin 2-Deoxyadenosin

Inosin 2-Deoxyinosin

Adenosin-

Deaminase

Acumulation


Therapy

21

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


Therapy

22

EX VIVO vs IN VIVO

Tumor cells

fibroblasts

Hemopathic

cells

Gene transfer in culture

Delivery to target

Vector

local

systemic


Therapy

23

Examples for local gene expression


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


Therapy

25

Retroviral vector system


Therapy

26

Summary



Therapy

28

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


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


Therapy

30

Electroporation

Physical Principle

Gehl J (2003) Acta Physiol Scand 177:437

CHO-cells, 20 ms, 1 kV cm-1


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


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


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


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.


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


DNA Condensation


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


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


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


Summary


Problems in Gene Therapy and Transgenesis Bio...Producing transgenic animals Purpose: Production of heterolog proteins in transgenic animals Principle: Integration of heterolog DNA

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