L47 Gene Therapy

Question 1

What is somatic gene therapy?

Answer 1

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.

Question 2

What is germline gene therapy?

Answer 2

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.

Question 3

What is a transgene?

Answer 3

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.

Question 4

What is the difference between ‘in vivo’ and ‘ex vivo’ in gene therapy?

Answer 4

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

Question 5

Are transgenes normally inserted as an addition or a replacement?

Answer 5

Addition

Ideally, gene replacement would be done. But this is very difficult to achieve.

Current gene therapy treatments utilise gene addition.

Question 6

In gene therapy, transgenes that do not integrate exist as __?__

Answer 6

Episomes

Question 7

What delivery vectors are available for gene therapy?

Answer 7

• Viruses (most common)

- Lipoplexes (lipid-based vectors)

Question 8

Which type of virus copies its own RNA into DNA before integrating into the host’s DNA?

Answer 8

Retrovirus

Question 9

In gene therapy, which delivery vector has specific target tissues (tropism)?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 9

b) Herpesvirus

Question 10

In gene therapy, which delivery vector is highly immunogenic?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 10

a) Adenovirus

Question 11

In gene therapy, which delivery vector cannot infect non-dividing cells?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 11

c) Retrovirus

Question 12

In gene therapy, which delivery vectors have no genetic integration?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 12

a) Adenovirus; and,

b) Herpesvirus

Question 13

In gene therapy, which delivery vector can activate oncogenes?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 13

c) Retrovirus

Question 14

In gene therapy, which delivery vector can carry very large transgenes?

a) Adenovirus
b) Herpesvirus
c) Retrovirus
d) Lipoplexes

Answer 14

b) Herpesvirus

Question 15

True or false: Lipoplexes can be easily prepared in a lab, but a poor at delivering transgenes

Answer 15

True

Question 16

What is the Weismann Barrier?

Answer 16

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.

Question 17

What is X-linked severe combined immunodeficiency (X-SCID) and how is it treated?

Answer 17

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.

Question 18

What problem was encountered in gene therapy trials for X-SCID?

Answer 18

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

Question 19

What is an oncolytic virus?

Answer 19

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

Question 20

How is ganciclovir used in conjunction with gene therapy?

Answer 20

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.

Question 21

How can is genetic engineering used to combat melanoma?

Answer 21

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.

Question 22

What causes cystic fibrosis?

Answer 22

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.

Question 23

What are some of the symptoms of cystic fibrosis?

Answer 23

• Thickened mucous membranes
• Infertility due to mucus-clogged vas deferens
• Pancreatic insufficiency due to mucus-clogged pancreatic ducts

Question 24

How are liposomes delivered to CF patients undergoing gene therapy?

Answer 24

Nebuliser

Question 25

What viral delivery vector(s) is/are used to treat cystic fibrosis?

Answer 25

• Adenovirus

- Adeno-associated virus

Question 26

What does CRISPR stand for?

Answer 26

Clustered regularly interspaced short palindromic repeats

Question 27

How does CRISPR-Cas9 work?

Answer 27

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’.

Question 28

What is different about more recent versions of CRISPR?

Answer 28

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.

Question 29

Give 2 examples of diseases that may be treated by CRISPR.

Answer 29

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.