gene therapy dr. aws alshamsan & dr. nermin hassan department of pharmaceutics...
TRANSCRIPT
Objectives of this lecture
By the end of this lecture you will be able to:1. Define the term “Gene Therapy”2. Use the correct terminology for gene transfer3. Realize the significance of gene therapy research
Objectives of this lecture
By the end of this lecture you will be able to:1. Describe the different strategies for gene therapy2. Select the suitable strategy based on the clinical case3. Understand the complexity of clinical application of gene therapy4. Evaluate proposed strategies according to the therapeutic need
What is gene therapy?
What is gene therapy?
Introduction of new genetic material into a cell for therapeutic purposes
Genetic Materials:
DNA
RNA
Problem Example Solution
Defect in protein expression G6PD deficiency Introduce a correct
version of the gene
Expression of harmful proteins Cancer Inhibit or block the
harmful proteins
Advantages of gene therapy• Specificity• Can be either temporary or permanent• Localization• Low immunogenicity
Gene Therapy
• Germ-Line Gene Therapy:• Introduction of genes into germ cells (sperm and ovum)
• Somatic Gene Therapy:• Introduction of genes into somatic cells
Germ-Line Gene Therapy
• Hypothetically, germ-line therapy prevents transfer of defective genes to subsequent generation
• Due to ethical and safety reasons, it is not presently accepted for human application
• However, it is used for the transgenic laboratory animals production
Gene therapy
In vivo Ex vivo
Advantage DisadvantageSpecificity Time and labor consuming
High transfection efficiency Invasive
Contamination
Not every cell type is growable
Ex vivo gene therapy• Most widely used in clinical trials
• Some attempts in practice
Advantage Disadvantage
Low cost None specificity
None invasive Low transfection efficiency
Less contamination
Ballistic DNA Injection (gene guns)
Invented for DNA transfer to plant cells
Fully applicable to eukaryotic cells
plasmid DNA shown here
LiposomesNext level idea – why naked DNA?
Lets’ wrap it in something safe to increase transfection rate
Therapeutic drugs
Lipids – are an obvious idea !
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
In vivo gene therapy• Some organs are less suited for ex vivo e.g. (brain,
heart and lungs)
• More practical approach
Gene Therapy Considerations• What gene will be delivered or targeted?• What method will be used?
• Genetic material either for gene expression or downregulation
• Vector to carry the genetic material inside the cells
Elements for gene transfer
Expression Vector (Plasmid)
Viral VectorNon-Viral Vector
Gene Transfer• Transformation: introduction of genetic materials into
bacteria
• Transfection: introduction of genetic materials into eukaryotic cells (e.g. fungi, plant, or animal cells)
• Transduction: introduction of genetic materials using viruses
• Lipofection: introduction of genetic materials using liposomes
Stable vs. Transient Gene Transfer• Stable Gene Transfer: achieved by plasmid integration in the host
genome or episomal replication of the transferred plasmid.
• Transient Gene Transfer: the foreign DNA is usually not integrated into the nuclear genome and will be degraded or diluted through mitosis
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 long-term expression
Replacement strategy
Gene with defect Disease/Disorder
Adenosine deaminase (ADA) 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
Mucus in bronchi is thick, interfering with lung function
1 in 25 are carriers
One of the first disorders to be actively studied for gene therapy.
Most lethal autosomal recessive disorder in U.S.
Cystic Fibrosis
Based on: Harvard Family Health Guide, 1999
Sickle Cell Disease- recessive allele•Red blood cells are sickle shaped, issues with circulation causing anemia and pain
Hemophilia
• A disorder in which a person’s blood does not clot properly.
• It is a recessive sex-linked, X-chromosome disorder.
• 1 in 10,000 males born are afflicted.
“Royalty Disease”
ADA-deficient persons are affected by severe immunodeficiency, with recurrent infections that might be life-threatening.
First disease approved for gene therapy.
Autosomal recessive disorder.
The drug exists but is very expensive, needs to be injected in vein for life.
Adenoside Deaminase (ADA) Deficiency
Ashanti Disilva
Bubble Boy
David Phillip Vetter (September 21, 1971 – February 22, 1984)
Gene therapy trial
First successful 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 drug-sensitivity 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
Gene Therapy Strategies
•Replacement of a missing or defective gene
•Introduction of gene(s) to influence cellular process
•Interference with gene products
Interference strategy
Downregulation of gene expression at the mRNA level
Inhibition of mRNA translation
Interference nucleic acids
DNAAntisense oligodeoxynucleotide (ODN)DNAzyme
RNAAntisense RNARibozymeSmall interfering RNA (siRNA)Short hairpin RNA (shRNA)microRNA (miRNA)
RNA interference
(RNAi)
DNAzyme
DNAzyme binds target mRNA
mRNA cleavage and degradation
DNAzyme
AAAAAA….ATarget mRNA
protein
X
© American Society for Investigative Pathology
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 ODNAntisense ODN
mRNA
RNase H
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
RISC
siRNA is bound by RISC and unwound by RNA helicase
RISC
Sense RNA strand degraded
© American Society for Investigative Pathology
RISC
AAAAAA….A
Target mRNA
mRNA cleavage and degradation
RISC-bound antisense strand directed to target mRNA
AAAAAA….A
Target mRNARISC
© American Society for Investigative Pathology
siRNA
www.nature.com/focus/rnai/animations/rnai_revised_320x180.mov
Antisense ODN v.s. siRNA
Antisense ODN siRNA
Nucleotide sugar Deoxyribose Ribose
Structure Single stranded Double stranded
Length 16-30 bp 19-21 bp
Molecular weight ~ 6-9 kDa ~ 13-14 kDa
Precursor vailability No Yes
Site of action Cytoplasm / Nucleus Cytoplasm
mRNA cleavage RNase H RISC
Degradation upon activity Yes No
Effective concentration 50-400 nM 5-100 nM
shRNA
Plasmid DNA
miRNA
cytoplasmic processing
Dicer (RNase III)
pre-miRNA
miRNA
Interact with target mRNA
RISCribosome
mRNA A(n)
RISC RISC
RISC RNA-Induced Silencing Complex
© American Society for Investigative Pathology
miRNA
RNA-induced silencing complex
General mechanisms of siRNA and miRNA
siRNA v.s. miRNA
siRNA v.s. miRNA
Production and Regulation
Strict adherence to GLP and GMP principles
Effectiveness in an appropriate animal model
Safety in rodents and larger animals such as monkeys
Clinical Trials
Good Laboratory Practices
Good Manufacturing Processes
Gene Therapy Successes
approved by the FDA for sale, some diseases have been experimentally successful:
• Melanoma (skin cancer)• Severe Combined Immunodeficiencies• Hereditary Blindness• Sickle Cell Anemia
• 2006: Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients. This study constitutes one of the first demonstrations that gene therapy can be effective in treating cancer.
• 2007- 2011: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases.
Retinal disease Colour blindnessAdrenoleukodystrophy
• 2011: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has cured a man from HIV using gene therapy
Gene Therapy Disappointments
• In 1999 a boy died due to an immune response to an adenovirus gene therapy vector.
•Four children have developed cancer due to a retrovirus gene therapy vector
You are now able to: Define the term “Gene Therapy” Use the correct terminology for gene transfer Realize the significance of gene therapy research
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
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