Gene Therapy

Gene Therapy
Jianjun Wang
[email protected]
Content

the basic principles of GT
some examples of gene vectors & delivery systems
main practical problems
Parkinson’s disease
GT for PD (our work)
Gene Therapy: What is it?
The application of genetic principles in the treatment of human disease
By introduction of genetic material into target cells in order to
counteract the effect of a disease gene or
introduce a new function
Somatic and Germline approaches possible
What can gene therapy
be applied to?
Cancers
Inherited disorders
Infectious diseases (viral or bacterial)
Immune system disorders
Vaccination
History of Gene Therapy
1930’s “genetic engineering” - plant/animal breeding
60’s first ideas of using genes therapeutically
50’s-70’s gene transfer developed
70’s-80’s recombinant DNA technology
1990 first GT in humans (ADA deficiency)
2001 596 GT clinical trials (3464 patients)

1) Understanding of the disease process
2) Structure/function of gene to be introduced
3) Efficient delivery of gene
4) Control of gene expression
5) Prevention/control of immune responses
6) Animal model and assessment of function
7) Clinical trial
What is required for
Gene Therapy to be possible?
Gene Therapy Strategies
Gene replacement
Gene Augmentation Therapy (GAT)
Gene Correction (Chimeraplasty)
Targeted killing of specific cells
Targeted inhibition of gene expression
(Gene ablation)
Gene Augmentation
For diseases caused by loss of gene function
More copies of normal gene
raise levels of gene product
restore normal phenotype
Apply to: Monogenic recessive diseases eg. cystic fibrosis, haemophilia, muscular dystrophy
Targeted inhibition
Ribozymes
can cleave (or repair) mRNA
Triple helix oligonucleotides
block gene transcription
Antisense oligos
block mRNA translation
Ribozyme
Cleavage site
Ribozyme mechanism
Correcting strand
3’
5’
X
X
: : : : : :
: : : : : : : : : : : : : : : : : : : : : : : : :
Me-RNA
Me-RNA
DNA
Targeting strand
Mutator base
Gene Correction - Chimeraplasty
viral vector
thymidine kinase
gene
tumour cell
ganciclovir phosphate
ganciclovir
tk
Targeted Killing -
Genetic Pro-drug Activation Therapy
Ganciclovir phosphate
inhibits DNA polymerase
Cell death –
Including bystander cells
All gene therapy strategies depend

on getting the gene or genetic material

into the target cells
= Transduction
Gene Transfer:
getting genes into cells

Two main routes:

In vivo: i.v. or i.m. injectable; or
non-invasive (eg “sniffable”)

Ex vivo: hepatocytes, skin fibroblasts
haematopoietic cells
“bioreactors”


Gene delivery approaches
Physical methods

Non-viral vectors

Viral vectors
Physical methods
Not limited by size or number of genes
Inefficient
Advantage Disadvantage
Injection of naked DNA
Electroporation in vivo or ex vivo
Ballistic technology – the “gene gun”
Effect of Electric Field on Cell Membrane
cells obtained from
skin biopsy
gene introduced
into cells by
electroporation
genetically engineered
cells are propagated
cells are
reinjected
Ex vivo electroporation
Non-Viral methods
Liposomes

Gene pill

Receptor-mediated delivery

Non-Viral methods
Liposomes
Not limited by size or number of genes

Safe (relatively)

Easy to produce

Short-term expression usually
DNA
liposome
complexes
Liposomes
liposome

nucleus
DNA
endocytosis
complex
membrane fusion
Liposome-mediated Gene Transfer
Oral route of gene delivery
- gene pill
“Viruses are highly evolved
natural vectors
for the transfer of foreign genetic information
into cells”

Kay et al 2001
But to improve safety,
they need to be replication defective
Compared to naked
DNA, virus particles
provide a relatively
efficient means of
transporting DNA into cells, for expression in the nucleus as
recombinant genes
(example = adenovirus).

[figure from Flint et al. Principles of Virology,
ASM Press, 2000]
Viral vectors
Retroviruses
eg Moloney murine leukaemia virus
(Mo-MuLV)
Lentiviruses (eg HIV, SIV)
Adenoviruses
Herpes simplex
Adeno-associated viruses (AAV)
Engineering a Virus into a Vector
wildtype virus
Based on Kay et al 2001
structural
proteins
Packaging cell
Y
vector
Vector uncoating
Therapeutic mRNA
and protein
Episomal vector
Integrated expression
cassette
Target cell
Gene Transfer
Based on Kay et al 2001
Retrovirus Vectors
Adenovirus
Advantages
High transduction efficiency
Insert size up to 8kbHigh viral titer (1010-1013)
Infects both replicating and differentiated cells

Disadvantages
Expression is transient (viral DNA does not integrate)
Viral protiens can be expressed in host following vector administration
In vivo delivery hampered by host immune response
Herpes Simplex Virus
Advantages
Large insert size
Could provide long- term CNS gene expression
High titer
Disadvantages
System currently under development
Current vectors provide transient expression
Low transduction efficiency
AAV Vectors
Advantages
Permanent Expression
Without Immune Response
Infects both replicating and differentiated cells
Disadvantages
Insert size up to 4.5 kb
Low transduction efficiency
Low viral titer
Applications: Target tissues
Haematopoietic cells – easiest
Muscle, Liver - good targets
Eye retina – accessible but fragile
Brain (neurons) – v. difficult
Tumours – access to interior may be difficult
Problems with GT
Safety:
Toxicity
Immune response
Integration
Malignant transformation
Germline integration
Viral replication through recombination
Efficacy:
Target cell uptake
Control of gene expression
Summary
Gene Therapy is still in its infancy
Early promise and hope not fulfilled
Risks, including death, seen as a major issue
But, recent trials look more promising