Gene Therapy Flashcards
What is gene therapy?
Use of nucleic acid polymers as a drug to treat disease by therapeutic delivery into the cells of a patient
When does reverse transcription occur?
Used by virus
RNA virus
Specialised enzymes convert back to DNA
What are the gene therapy approaches?
Replace non-functional gene
Swap abnormal gene
Selective reverse mutation
Gene regulation
What are the methods of vector delivery?
In vivo
Ex vivo
What is in vivo?
Directly into the body
What is ex vivo?
Cell type taken out + modified then put back into the body
What are the choices of gene delivery vectors?
Viral
Non-viral
What are examples of viral vectors?
Retrovirus
Adenovirus
Adeno-associated virus
Herpes simplex virus
What are examples of non-viral vectors?
Liposome
DNA-polymer conjugates
Naked DNA
What characteristics would an ideal vector system have?
Undetectable by immune system
Non-inflammatory
Safe
Efficiency sufficient
Long duration of expression
What is the objective of non-viral gene therapy?
Insertion of normal gene to replace abnormal
Describe the non-viral approach: direct delivery
Direct introduction of DNA into target cells
Restriction to certain tissues
Large amount of DNA required
How is non-viral direct approach done?
Electroporation = electric charge across membranes = open up
Sonoporation = sound waves
Magnetofection = magnets change the charge
What are the pro of non-viral gene therapy?
Possible to work with large pieces of DNA
Non-toxic
No immune response
What are the cons of non-viral gene therapy?
Inefficient
Limited to accessible cells
What is a liposome?
Non-viral vectors
Artificial lipid sphere with aqueous core, carrying therapeutic DNA
= can pass through membrane + deliver gene
What is receptor-mediated endocytosis?
Non-viral vectors
DNA molecule encapsulated in delivery device
Then endocytosed into cell
Describe the viral approach of gene therapy
Carrier molecule (vector) used to deliver DNA
Viruses
What happens to the virus in viral approach of gene therapy?
Virus genome modified to deliver therapeutic gene
What are the requirements for viral vector?
Cell-specific
Immune evasion
Non-immunogenic
Safe
Express for long as required
Describe retroviral vectors
Creates DNA via reverse transcription
DNA integrates into host chromosome via integrase
What are the advantages of retroviral vectors?
Long term expression
Low toxicity
High capacity
Low immunity allowing repeat administration
What are the problems of retroviral vectors?
Lack of cell specificity
Only infect dividing cells
What is an example of retroviral vectors?
Treat CF by injections directly into bronchial lining
What are X-SCID patients?
Immuno deficiency
What happens to patients with X-SCID?
Cannot make T lymphocytes their B lymphocytes fail to make essential Abs for fighting infections
What do X-SCID patients need?
Bone marrow transfer
What is X-SCID caused by?
Mutations in X-linked gene
Describe gene therapy of X-SCID patients
Stem cells taken out
Genetically manipulated
Put back into patient
= express T + B cells
Gene therapy via injection of retrovirally transduced stem cells
Describe adenoviral vectors
Viruses with dsDNA genome = double stranded
Remains in episome in cells
DNA transcribed
Why are adenoviral vectors widely used in gene therapy?
High transfection efficiencies
Large quantities possible
What are the cons of adenoviral vectors?
More likely to be attacked by immune system
Trigger inflammatory response = NO repeat administration
Describe adeno-associated vectors (AAVs)
ssDNA = single stranded
DNA integrates into single site
Infect many cell types
What are the pros of AAVs?
Non-toxic
Don’t trigger immune response
NO inflammation
What are the disadvantages of AVVs?
Small = carry only 2 genes
Difficult to make in large quantities
Describe herpes simplex viral (HSV) vectors
Gene transfer in nervous system
What are the advantages of HSV vectors?
Large genome = larger genes
Infect wide range of tissues
What are the disadvantages of HSV vectors?
Relatively untested
Stigma due to “herpes”
What are the disadvantages of gene therapy?
Short-lived nature
Immune response
Viral vectors = toxicity, immune response
Multi-gene disorders = difficult to treat
Insertional mutagenesis
What is insertional mutagenesis?
Virus may target wrong cells
What is Glybera?
Treatment for lipoprotein lipase deficiency
Introduce normal, healthy LPL gene
What is CF?
Autosomal recessive disease
Why is CF an ideal candidate for gene therapy?
Single gene defect
Recessive condition
Main pathology = lung = accessible
Progressive = at birth normal phenotype
What does gene delivery for CF involve?
Large vol of vector-containing fluid to lung via the nose
What is the problem with gene delivery for CF?
Safety problems = aspiration
Large vol = enhanced alveolar exposure
What is another way for gene delivery for CF?
Oral inhalation of corticosteroid
What is the problem with oral inhalation for gene delivery for CF?
Small particles enhance delivery to peripheral airways
= undesirable for gene vector delivery
= adverse effects
What is success of CF gene delivery restricted by?
Airways well defended
How does the covid-19 vaccine work?
Raise immune response to spike protein
mRNA in liposome then injected into patient
= encode for spike protein
mRNA degraded = NOT long lasting in genome
