Gene therapy

Question 1

What is the aim of gene therapy?

Answer 1

To cure genetic disease by replacing the mutated gene with a non-mutated gene

Question 2

How does the replacement gene get to the cell?

Answer 2

Via vector sequence or lyzosome

Question 3

What is an example of a vector sequence?

Answer 3

AAV
A virus which is very good at penetrating into tissue

Question 4

What are the types of genetic disorders?

Answer 4

Monogenic/Mendelian
Polygenic/Complex

Question 5

What are the types of monogenic disorders?

Answer 5

Dominant
Recessive
X-linked
Mitochondrial

Question 6

Give an example of a monogenic disorder

Answer 6

Marfan’s syndrome
Retinitis Pigmentosa
Leber’s Congenital Amaurosis

Question 7

Give an example of a polygenic disorder

Answer 7

Cataracts
Glaucoma
Refractive error

Question 8

Why is a polygenic disorder harder to ‘cure’ than a monogenic disorder?

Answer 8

Polygenic caused by multiple gene mutations, monogenic caused by one. Polygenic also affected by other environmental factors.

Question 9

What are the difficulties of replacing non-functional genes?

Answer 9

Delivering gene to the correct tissue
Achieving the desired level of gene expression (generally only need a few %)
Preventing the vector from being degraded (needs to stay present, while not integrating into host cell DNA - so could be detected as virus)
Unwanted side effects (occur if vector integrates into host cell DNA)

Question 10

What are the types of dominant disorders?

Answer 10

Haploinsufficiency
Dominant-negative effect

Question 11

What is haploinsufficiency?

Answer 11

50% functional protein is present, which is not enough for normal function

Question 12

How can gene therapy help haploinsufficiency? What does it need to achieve to work?

Answer 12

Replacement of dysfunctional genes
Expression needs to be brought up to 100% for normal function

Question 13

What is the dominant-negative effect?

Answer 13

Abnormal protein has an adverse effect, preventing the function of the normal protein

Question 14

What does gene therapy need to achieve to help the dominant-negative effect?

Answer 14

Block expression of abnormal protein but allow expression of normal protein

Question 15

How is the abnormal protein blocked in treating the dominant negative effect?

Answer 15

Translation of dominant negative mRNA blocked by nucleic acid drugs which bind to the mutant mRNA and cleave it in two.
Delivered via ribozymes, antisense nucleic acids, siRNA or CRISPR-Cas

Question 16

Give an example of a haploinsufficiency

Answer 16

Stickler syndrome - vitreous malfunction which causes strands to be seen on slit lamp

Question 17

Give an example of the dominant-negative effect

Answer 17

Marfan’s syndrome - fibrillin gene mutation prevents triple helix formation so elastic fibres are not formed (e.g. if zonules of zinn dysfunctional, lens may dislocate)

Question 18

What are the difficulties of blocking the expression of an abnormal protein?

Answer 18

Achieving specificity for the mutant mRNA without degrading non-mutant mRNA
Delivering the gene to the correct tissue
Achieving desired level of gene expression
Preventing the delivery agent from being degraded

Question 19

How is CRISPR-Cas used for blood disorders?

Answer 19

Blood removed from px
CRISPR-Cas delivered to cell culture in lab (genes edited and cultured further)
Edited cells are returned to the px

Question 20

Why is the ability to edit genes outside of the body good?

Answer 20

Risk can be assessed before returning cells to the px
Less side effects
More controlled environment
Less clinic time for px and practitioner?

Question 21

What makes gene therapy difficult?

Answer 21

Mutation heterogeneity - mutation can occur anywhere on gene and usually varies between families
Non-mendelian diseases (polygenic) - difficult to correct multiple mutations
Ethics