Introduction to Gene Therapy

Introduction to Gene Therapy:

Introduction to Gene Therapy Aws Alshamsan, RPh

Nucleus – Chromosome - DNA:

Nucleus – Chromosome - DNA

Gene Structure:

Gene Structure

Exon v.s. Intron:

Exon v.s. Intron

Protein Synthesis:

Protein Synthesis

Protein is Disease?:

Protein is Disease? Problems: Defect in protein expression Expression of harmful proteins Solutions: Inhibit or block the harmful proteins Introduce a correct version of the gene Gene Therapy

Gene Therapy:

Gene Therapy Introduction of new genetic material into cells for therapeutic purposes Advantages: Specific Permanent No immunogenicity

Strategies for Gene Therapy:

Strategies for Gene Therapy

Ex Vivo:

Ex Vivo Most widely used in clinical trials Advantages: Specific High transfection efficiency Disadvantages: Time consuming Expensive Invasive Contamination

In Vivo:

In Vivo Some organs are less suited for ex vivo e.g. (brain, heart and lungs) Advantages: Lower cost tan in vivo Non-invasive Less contamination Disadvantages: Non-specific Low transfection efficiency

Potential Targets:

Potential Targets Inherited Diseases: Problem: Defect in a gene Strategies: Integration of therapeutic gene into host Using episomal expression vector

Potential Targets:

Potential Targets Cancer: Problem: Abnormal expression of growth genes Strategies: Cytokine genes Suicide genes Tumor suppressor genes Protection of hematopoietic stem cells

Cytokine Genes:

Cytokine Genes Introduction of genes encoding for proinflammatory cytokines e.g. IL-12 and IFN- g : Induce local inflammation Tumor destruction Induction of cell mediated immunity (T cells)

Suicide Genes:

Suicide Genes HSV-tk Phosphorylation of Ganciclovir Bystander effect

Tumor Suppressor Gene:

Tumor Suppressor Gene Introduction of p53 gene: Usually mutated in most cancers Antioncogene

Protection of HSC:

Protection of HSC Introduction of MDR-I gene in HSC: Increases HSC tolerance to chemotherapy Allows more aggressive treatment

Elements for Gene Transfer:

Elements for Gene Transfer Gene Vector

Gene:

Gene Mammalian Expression Vector

Methods of Transfection:

Methods of Transfection

Non-viral “Chemical”:

Non-viral “Chemical” DNA

Non-viral “Physical”:

Non-viral “Physical”

Viral Vectors:

Viral Vectors General requirements: Must be replication defective to prevent uncontrolled spreading in vivo Should not process undesirable properties The viral genome should be able to accommodate the therapeutic gene

Viral Vectors:

Viral Vectors Adenovirus Retrovirus Adeno-associated virus HSV

Summary:

Summary Retrovirus Adenovirus AAV Naked DNA Liposome Genome transfer RNA DNA DNA DNA RNA or DNA Purification Difficult Yes Yes Yes Yes Max. Size of gene 8kb 7.5kb 5kb At least 50kb At least 50kb In vivo use No (but the producer cells are used) Yes Yes Yes Yes Integration Yes No Yes Low Low Efficiency High Very high Moderate Moderate Low Non-dividing cells No Yes Yes Probably Probably Limitation Cell division needed Transient correction Production is difficult Low efficiency Low efficiency