L47 Gene Therapy Flashcards
What is somatic gene therapy?
Replacing an individual’s ‘bad’ genes with ‘good’ ones.
Somatic gene therapy means this will only have an impact on the individual receiving the treatment, not future generations.
What is germline gene therapy?
Replacing heritable ‘bad’ genes with ‘good’ ones.
Germline gene therapy influences not only the individual receiving it, bu also future generations, who inherit the altered genes.
What is a transgene?
Gene or genetic material that has been transferred (naturally or artificially) from one organism to another.
Transgenes have the potential to change the phenotype of an organism.
What is the difference between ‘in vivo’ and ‘ex vivo’ in gene therapy?
In vivo: Transgene is delivered directly to target tissue in situ, in the body
Ex vivo: Target tissue is removed from body prior to transgene delivery
Are transgenes normally inserted as an addition or a replacement?
Addition
Ideally, gene replacement would be done. But this is very difficult to achieve.
Current gene therapy treatments utilise gene addition.
In gene therapy, transgenes that do not integrate exist as __?__
Episomes
What delivery vectors are available for gene therapy?
- Viruses (most common)
- Lipoplexes (lipid-based vectors)
Which type of virus copies its own RNA into DNA before integrating into the host’s DNA?
Retrovirus
In gene therapy, which delivery vector has specific target tissues (tropism)?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
b) Herpesvirus
In gene therapy, which delivery vector is highly immunogenic?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
a) Adenovirus
In gene therapy, which delivery vector cannot infect non-dividing cells?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
c) Retrovirus
In gene therapy, which delivery vectors have no genetic integration?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
a) Adenovirus; and,
b) Herpesvirus
In gene therapy, which delivery vector can activate oncogenes?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
c) Retrovirus
In gene therapy, which delivery vector can carry very large transgenes?
a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes
b) Herpesvirus
True or false: Lipoplexes can be easily prepared in a lab, but a poor at delivering transgenes
True
What is the Weismann Barrier?
The line between somatic and germline gene therapy, protecting the testes. Therapies that breach the Weismann barrier have the potential to modify the germline.
Doing so is illegal in most countries.
What is X-linked severe combined immunodeficiency (X-SCID) and how is it treated?
Immunogenic disorder leading to catastrophic failure of the immune system.
Loss of interleukin function, T cell and B cell failure (B cells don’t produce IgG).
Treated by Ig supplementation, bone marrow transplant.
Graft v Host disorder common.
Gene therapy is being trialled.
What problem was encountered in gene therapy trials for X-SCID?
Some of the patients developed leukaemia because retroviral integration was too close to LMO2 gene - a proto-oncogene that causes ALL (acute lymphoblastic leukaemia) when activated
What is an oncolytic virus?
A virus that specifically targets cancer cells.
Potential benefit for gene therapy in cancer.
e.g. Onyx-15 (modified adenovirus) induced necrosis in tumour cells
How is ganciclovir used in conjunction with gene therapy?
Kills brain tumours.
Ganciclovir is a prodrug that is activated by HSV kinase to form toxic ganciclovir triphosphate.
Retrovirus delivers HSV-tyrosine kinase transgene to brain tumour and then the patient is given ganciclovir. The drug is activated inside the tumour and kills the tumour.
Not 100% efficient due to transduction.
How can is genetic engineering used to combat melanoma?
MART-1 antigen expressed by melanoma.
Dendritic cells, genetically engineered (ex vivo) by adenovirus to express MART-1, are put back in body. Activates cytotoxic T cells against MART-1 - immune system targets melanoma.
What causes cystic fibrosis?
Deletion mutation in CFTR gene (cystic fibrosis transmembrane conductance regulator) breaks the chloride channels in cells –> inactivates cilia.
F508del. (phenylalanine replaced by stop codon)
Mucus gathers in the lungs and digestive system as it isn’t shifted by cilia.
What are some of the symptoms of cystic fibrosis?
- Thickened mucous membranes
- Infertility due to mucus-clogged vas deferens
- Pancreatic insufficiency due to mucus-clogged pancreatic ducts
How are liposomes delivered to CF patients undergoing gene therapy?
Nebuliser
What viral delivery vector(s) is/are used to treat cystic fibrosis?
- Adenovirus
- Adeno-associated virus
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
How does CRISPR-Cas9 work?
Cas9 protein severs DNA at a specific point (identified by guide RNA (gRNA)) and either removes faulty genes or overwrites them. The cell’s natural repair mechanisms ‘stitch it back together’.
What is different about more recent versions of CRISPR?
Instead of cutting the genome, the new method inserts a code attached to a protein (Cas-9) that latches onto the DNA and boosts expression.
Give 2 examples of diseases that may be treated by CRISPR.
Muscular dystrophy: CRISPR boosts activity of gene that produces utrophin to compensate for absence of dystrophin. Mice recovered muscle growth and strength.
AKI: target klotho and IL-10 genes. Klotho protects from renal damage. IL10 ameliorates (makes better) renal injury.
