Download Gene Therapy (I)

Gene Therapy (IV)
“Strategies and Applications”
Dr. Aws Alshamsan
Department of Pharmaceutics
Office: AA87
Tel: 4677363
[email protected]
Objectives of this lecture
By the end of this lecture you will be able to:
1. Describe the different strategies for gene
therapy
2. Select the suitable strategy based on the
clinical case
3. Understand the complexity of clinical
application of gene therapy
4. Evaluate proposed strategies according to the
therapeutic need
Gene Therapy Strategies
•Replacement of a missing or defective gene
•Introduction of gene(s) to influence cellular
process
•Interference with gene products
Replacement strategy

Applies to diseases caused by single gene
defects

Transfer of a functional copy of the
defective or missing gene

Examples: enzyme deficiencies
Replacement strategy

To apply this strategy, three
requirements must be met:
1. The specific gene defect must be known
2. A functional copy of the gene must be
available
3. Target cells must be available and amenable
to transfection methods resulting in longterm expression
Replacement strategy
Gene with defect
Adenosine deaminase (ADA)
Disease/Disorder
SCID
a-1-antitrypsin
Emphysema
CF transmembrane regulator
Cystic fibrosis
Clotting factor VIII
Hemophilia A
Clotting factor IX
Hemophilia B
b-chain of hemoglobin
Sickle cell anemia
Bubble Boy
David Phillip Vetter (September 21, 1971 – February 22, 1984)
Gene therapy trial

First clinical trial in gene therapy was
initiated in September 14, 1990

Hematopoietic stem cells were isolated
from the patient (4 y/o girl) and
transduced with retroviral vector
containing ADA gene

25% recovery of normal ADA in patient
T cells
Why was ADA suitable?

Single gene defect

Gene was isolated and cloned in 1983

HSC are easy to obtain and maintain in
vitro
Influence strategy

Applies to complex disorders were more
than one gene is involved

Based on in vitro cloning of human genes
that were derived from human tissue

Examples: cancer
Areas of investigation

Enhancement of anti-tumor response

Introduction of drug-resistance genes

Introduction of drug-sensitivity genes

Replacement of tumor suppressor genes
Introduction of drugsensitivity genes

Suicide gene therapy

Gene that converts non-toxic
prodrug into a toxic
metabolite

Bystander effect

Gancyclovir triphosphate

Problem: it can transfect
normal cells too
Interference strategy

Downregulation of gene expression at the
mRNA level
Inhibition of
mRNA translation

Interference nucleic acids

DNA
 Antisense oligodeoxynucleotide (ODN)
 DNAzyme

RNA
 Antisense RNA
 Ribozyme
 Small interfering RNA (siRNA)
 Short hairpin RNA (shRNA)
 microRNA (miRNA)
RNA
interference
(RNAi)
DNAzyme
Ribozyme
Antisense ODN
• Sequence-selective
oligonucleotide that
can bind to a target
mRNA to inhibit
gene expression i.e.
to inhibit translation
Antisense ODN
Antisense ODN
Antisense
ODN
mRNA
RNase H
Disadvantages of Conventional
Therapy
• Requires screening of thousands of
compounds to find an active molecule.
• Lacks specificity of action.
We don’t have antisense ODN for every disease
• The main barrier to antisense strategy is optimal
delivery in sufficient quantities to the correct
target and for the desired time frame to achieve
the desired level of gene inhibition
• ODNs are polyanionic macromolecule (large and
charge)
• Stability issues in vivo
Designing Biologically Stable ODNs
Designing Biologically Stable ODNs
Fomivirsen Sodium (Vitravene)®
• FDA-approved for the
local treatment of
CMV retinitis in AIDS
patients
Fomivirsen Sodium (Vitravene)®
• Dose 150-330 μg intravitreal injection
• Every other week for 2 doses
• Cleared locally by exonucleases 1-2 hr after
injection
Antisense RNA
What is RNAi?
• Post-transcriptional phenomenon that was initially discovered
in plants
• Mediated by double-stranded RNA
siRNA
Antisense ODN v.s. siRNA
Antisense ODN
Nucleotide sugar
Structure
siRNA
Deoxyribose
Single stranded
Ribose
Double stranded
Length
16-30 bp
19-21 bp
Molecular weight
~ 6-9 kDa
~ 13-14 kDa
Precursor vailability
Site of action
mRNA cleavage
Degradation upon activity
Effective concentration
No
Cytoplasm / Nucleus
RNase H
Yes
50-400 nM
Yes
Cytoplasm
RISC
No
5-100 nM
shRNA
Plasmid DNA
miRNA
miRNA
siRNA v.s. miRNA
siRNA v.s. miRNA
Now you are able to:
 Describe the different strategies for gene
therapy
 Select the suitable strategy based on the
clinical case
 Understand the complexity of clinical
application of gene therapy
 Evaluate proposed strategies according to the
therapeutic need
Next Lecture
Protein pharmaceuticals