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Flashcards in Gene therapy Deck (16)
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give examples of the current clinical applications of gene therapy

Only two licensed therapies worldwide
1) Gendicine (head and neck cancer, p53 adenovirus)
2) Glybera (rare enzyme deficiency)


what is Gene therapy?

Gene therapy can be defined as the treatment of human diseases by the transfer of genetic material into specific cells of the patient.


what is Recombinant DNA ?

1) Recombinant DNA is a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together;
2) Plasmids are extrachromosomal self-replicating circular forms of DNA present in most bacteria (pDNA) the therepeutic genes are inserted into plasmids


outline the different types of gene therapy

1) Gene-replacement therapy- supplies cells with healthy copies of a missing or flawed gene that encodes for production of a specific protein missing or under expressed in the cell (e.g. SCID bone marrow);
2) Suicide gene therapy- provides ‘suicide’ genes to target cancer cells for destruction (e.g. p53 gene that is lost in cancer cells);
3) Blocking gene therapy- provides a DNA gene inhibitor (e.g. siRNA) to block production of harmful proteins.


Summarise the Possible routes for administration of therapeutic genetic material

1) Conventional routes e.g. Inhalation (aerosols), Oral administration, Intramuscular injection, Intravenous injection
2) Ex vivo administration


what is Ex vivo gene therapy?

1) Ex vivo therapy involves removing cells from the patient’s blood or bone marrow and growing them in the laboratory
2) The desired gene is then inserted into the cells
3) The successfully altered patient cells are selected out, encouraged to multiply, and returned to the patient’s body via the blood stream
4) best suited for diseases in which the desired cells can be extracted from readily accessible locations such as the blood or liver.


discuss the Ideal gene delivery system

1) Should be specific for the targeted cells
2) Should be resistant to metabolic degradation and/or attack by the immune system
3) Should be biocompatible (non toxic, degradable)
4) Should have an ability to express the therapeutic gene efficiently
5) Should result in permanent incorporation of delivered gene including after cell replication


viruses are used as natural delivery vectors. Give examples of commonly used vectors

1) The most commonly used genetic vectors are retroviruses and adenoviruses;
2) Retroviruses are capable of creating DNA copies of their RNA genomes. These copies are integrated into the chromosomes of host cells.
3) Adenoviruses contain double-stranded DNA genomes that cause respiratory, intestinal and eye infections in humans.
- Viruses used for gene delivery must be unable to replicate and have no lytic activity


Outline the Advantages and disadvantages of viral vectors

1) advantage: High transfection efficiency
2) disadvantage: Viral toxicity
3) Host immune rejection
3) Cannot accommodate large DNA molecules
4) Difficulties in large scale manufacturing


list some Non-viral vectors

1) Lipid-based vectors
2) Peptide-based vectors
3) Polymer based-vectors
Main aim: package long negatively charged DNA molecule
- obstacle: entry and permanent delivery of DNA into nucleus


state what a cationic lipid consists of

1) hydrophobic lipid anchor group
2) A linker group
3) A positively charged headgroup


describe how Liposome-mediated DNA delivery works

1) Negatively charged DNA + Cationic lipids
2) this produces DNA/liposome complex (lipoplexes)
3) DNA/liposome complex is internalised by endocytosis. this forms an endosome
4) The complex is released from the endosome
and enters the nucleus


explain how Cationic polymers are used to deliver DNA into the nucleus

1) the polycation is attached to the ligand
2) DNA which is negatively charged complexes with the polycation ligand
3) the ligand binds to a receptor on the membrane and enters via endocytosis
4) the DNA is released from the endosome and enters the nucleus


discuss the structural parameters affecting transfection efficiency and toxicity of cationic polymers

1) Nature of amino-groups
2) Length of side chains
3) Presence of polar and non-polar functional groups
4) Molecular weight
5) Nature of backbones (linear or branched)
6) DNA/polymer ratio


compare viral- and non-viral based gene delivery systems:
1) Efficiency
2) Size of therapeutic DNA
3) Safety issues
4) Longevity of expression
5) Manufacturing

1) Efficiency: VIRAL - Highly efficient gene delivery
- NON VIRAL: Low efficiency of gene transfer
2) Size of therapeutic DNA:VIRAL- Limited by size that can be accommodated by viral genome
- NON VIRAL : Largely unlimited
3) Safety issues: Some viral vectors stimulate severe immune responses; concern regarding use of viral vectors based on HIV
- NON VIRAL : Vectors are often cytotoxic
4) Longevity of expression: Transient or long-term expression
- NON VIRAL: Transient expression
5) Manufacturing : VIRAL - High costs
- NON VIRAL: low costs


discuss the major barriers to gene therapy

1) For development: many steps needed that are not needed for ‘conventional’ drugs
2) Cost of API
3) Genetic modification regulation
4) Public perception
5) Fundamental delivery challenge: Overcoming ‘viral’ nature of gene therapy
6) Other biologic formulation and delivery problems