Gene Therapy

  • Created by: LBCW0502
  • Created on: 02-03-19 13:59
Describe features of CF (1)
Defective CFTR channel (unable to transport Cl ions from inside cells to outside of cells, no Na efflux, leads to no movement of water out of cell/osmosis, mucus doesn't become thin, leading to coughing up thick mucus)
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Describe features of CF (2)
Due to G551D (gating defect - CFTR Cl channel gets to membrane but doesn't transport Cl ions). Delta/deleted F508 (expression/maturation defect, reduced expression of CFTR at cell surface)
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Describe features of CF (3)
Normal process - ATP binds to NBPs, hydrolysis of ATP, conformational change in protein, opening, Cl ion release, one way process (doesn't occur in CF due to no ATP hydrolysis to drive process)
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Outline how gene therapy works
Vector with nucleic acid enters cell, DNA needs to become part of genome in nucleus. Replace defective DNA with healthy DNA. Healthy DNA produces healthy protein (cure disease)
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Outline the timeline for gene therapy (1)
1990 (4 yr old girl treated for ADA-SCID). 1999 (18 yr old boy died from gene therapy treatment for ornithine transcarbamylase deficiency). 2003 (Gendicine Ad-p53 receives market approval in China). 2012 (Glynera approved by EMA)
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Outline the timeline for gene therapy (2)
2015 (CRISPR/Cas9 gene editing tools to genetically modify human embryos). 2018 (successful use of gene therapy to treat children suffering from early-onset Pompe's disease). 2018 (high titre AAV therapies shown to be fatal in animals)
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Describe features of gene therapy
Has the potential to sure disease rather than treat symptoms (as most medicines do). Can be applied to treat a wide range of diseases e.g. cancer, inherited disorders, infectious diseases, immune system disorders
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What is monogenic gene therapy?
Provides genes to encode for the production of specific proteins e.g. CF, muscular dystrophy, sickle cell disease, haemophilia
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What is suicide gene therapy?
Provide suicide genes to target cancer cells for destruction e.g. cancer
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What is antisense therapy?
Provides a single stranded gene in an antisense (backward) orientation to bind mRNA and block production of harmful proteins e.g. AIDS/HIV
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What is involved in gene therapy? (1)
DNA (and RNA). Highly negatively charged (phosphate groups). Unstable - degraded by enzymes. Not taken up by active mechanism. Active in cell nucleus. To be delivered by injection. Delivery vehicle must be simple/inexpensive
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What is involved in gene therapy? (2)
Non-toxic/non-immunogenic, ensure delivery to a specific cell type
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Describe features of in vivo gene delivery - viral vector
High gene expression, can transfect somatic cells, can integrate into host genome. Toxic side effects, strong immune response, limited cargo capacity, reversion to wild type, oncogenesis
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Describe features of in vivo gene delivery - non-viral vector
Low immunogenicity, high DNA loading, ease of manufacture and scale up. Toxicity of carrier vehicle, low transfection efficiency
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Give examples of viral gene delivery
Adenovirus (enters via CAF and alpha V integrin). Adreno-associated virus (enters via heparin sulfate proteoglycan). Retrovirus/Lentivirus (enters via CD4)
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Outline the process of non-viral gene delivery
Lipolex, endocytosis, endosome formed (normally joined to lysozome, to destroy contents), nucleic acid delivery into nucleus (barriers)
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Outline features of lipoplexes
Vesicle combined with DNA (ensure endosome is formed). Use of lipid which disrupts membranes. DOPE bursts endosome so DNA can escape before being destroyed by lysosome
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Outline features of polyplexes
Use of polymer (negative charges). Takes up protons, absorbs moisture, polymers swell, burst to release nucleic acid contents. Non-viral vector (forms endosome which needs to burst membrane of endosome to release DNA)
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Outline features of lipopolyplexes (1)
Target of non-viral vector to attach to required cells. Targeting moiety. Small peptide binds to receptor. Formation of adhesive bubble. Need PEG shielding, DNA binding, cleavable linker, spacer, cationic lipid component, integrin binding
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Outline features of lipopolyplexes (2)
Stabilised, cell targeting, degradable, gene delivery, vector, plasmid DNA (cationic interactions with DNA)
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What are the barriers to delivery?
Getting into systemic circulation and survive. Get to target cell. Get into target cell. Stay intact once inside target cell. Getting into nucleus. Transcription
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Outline the process of ex vivo gene delivery
Remove cells of required type. Grow cells in culture. Treat with wanted gene (use of gene gun/biolistics). Some cells die/some survive, DNA integrated in nucleus. Amplify corrected cells. Put genetically modified cells back into patient
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Outline features of successful gene therapy
Aim to correct a single gene defect. Transform cells that are readily accessible. Tackle conditions where low level transfection gives significant clinical effect. E.g. Leber's Congenital Amaurosis (deliver RPE65 to treat inherited vision loss)
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What is Gendicine?
Recombinant adenovirus carrying p53 (treat tumours)
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What is Glybera used to treat?
Lipoprotein lipase deficiency - rare inherited disorder associated with severe pancreatitis
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What are the limitations of gene therapy?
Very difficult to deliver DNA (siRNA?). Very expensive. Small scale clinical trials. Funding models (new ones required?). Ethical issues
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Outline features of gene editing
Exploit CRISPR/Cas9 DNA repair system (body's natural process). Replaces defective genes or correct their gene sequences (able to introduce this in the body to get system to remove defective gene and introduce healthy gene)
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Other cards in this set

Card 2

Front

Describe features of CF (2)

Back

Due to G551D (gating defect - CFTR Cl channel gets to membrane but doesn't transport Cl ions). Delta/deleted F508 (expression/maturation defect, reduced expression of CFTR at cell surface)

Card 3

Front

Describe features of CF (3)

Back

Preview of the front of card 3

Card 4

Front

Outline how gene therapy works

Back

Preview of the front of card 4

Card 5

Front

Outline the timeline for gene therapy (1)

Back

Preview of the front of card 5
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