Novel therapies for genetic disorders

Question 1

What is a nonsense mutation?

Answer 1

Premature stop

Question 2

What cellular response is induced by truncated proteins?

Answer 2

Degradation via ERAD

Question 3

What cellular responses are induced by misfolded proteins?

Answer 3

1. UPR: an effort to force correct folding and reduce translation
2. ubiquination and proteolytic degradation

Question 4

What cellular responses are induced by nonsense mutations?

Answer 4

1. nonsense mediated decay of mRNA

2. if translation does occur: ERAD

Question 5

What are the requirements of a protein used in gene product replacement?

Answer 5

• Stable in blood and intracellularly

- Targeted to correct cell type and organelle

Question 6

How can proteins be modified to improve their properties for gene product replacement?

Answer 6

• Modification of glycosylation pattern

- Fusion proteins

Question 7

What enzyme is used to treat Gaucher’s and what modifications are made to increase its uptake?

Answer 7

• Glucocerebrosidase = deficient enzyme
• Needs to be taken in by Kupffer cells (liver macrophages)
• The native carb side chain of glucocerebrosidase is cleaved to expose terminal mannose residues, which are recognized by the macrophage lectin receptor

Question 8

What is the genetic cause of achondroplasia?

Answer 8

• Normal: when bound by FGF, FGFR3 receptor inhibits bone growth
• Achondroplasia: constitutive activation of the receptor and thus constitutive inhibition of growth

Question 9

What pathway inhibits the activity of FGF/FGFR3?

Answer 9

• NPR-B receptor binds CNP => signaling cascade => inhibition of inhibition of bone growth

Question 10

Why was native CNP not a viable treatment for achondroplasia and how was it modified?

Answer 10

• Very short half life

- Added 2 N-ter residues and greatly increased 1/2 life

Question 11

What is the therapeutic benefit of creating a fusion protein?

Answer 11

• May improve uptake

- May increase stability

Question 12

What is hypophosphatasia?

Answer 12

• Alkaline phosphatase deficiency (PPi => Pi + Pi)

- Causes poor mineralization of bones

Question 13

How is hypophosphatasia treated?

Answer 13

Gene product replacement = fusion of alkaline phosphatase + Ig molecule + deca-asp tag to promote uptake

Question 14

Pharmacological chaperones

Answer 14

• Chemicals which bind to active site to help enzymes fold normally, even if primary sequence is changed
• Normally folded protein will be transported to lysozyme
• In lysozyme, chaperone will be released and enzyme can act on native substrate

Question 15

How can enzyme inhibition be used to treat enzymatic deficiency?

Answer 15

• Inhibit an earlier step to reduce accumulation of a product

Question 16

What is “read-through”?

Answer 16

• When a chemical prevents termination of translation when a stop codon is encountered
• A different AA is substituted for the stop but translation is continued, potentially preserving some protein function

Question 17

How can alternative splicing be used to treat an enzyme deficiency?

Answer 17

Spinal muscular atrophy:

• a pseudogene is very similar to the mutated gene in spinal muscular atrophy but lacks functionality due to an exon spliced out
• hnRNP modifies splicing to make pseudogene more active and able to replace the defective gene

Question 18

How can exon skipping be used to treat disease caused by mutant protein?

Answer 18

Duchenne’s muscular dystrophy:

• Antisense oligo masks splice site on pre-mRNA of gene with the problematic mutation
• Exon will be excluded, resulting in a less problematic protein

Question 19

What nucleic acid products are used for exon skipping?

Answer 19

Antisense oligos:

• 20-30 nt
• modified bases prevent nuclease digestion

Morpholinos:
- methylenemorpholine rings with phophorodiamidate linkages

Question 20

What are other potential novel therapies for genetic disorders?

Answer 20

• inhibitory mRNA to silence mutant alleles (Huntington’s)

- drug/protein/mRNA delivery in exosomes

Question 21

What is the blood-brain barrier?

Answer 21

• Highly selective permeability barrier that separates blood from brain fluid
• Formed by capillary endothelial cells joined by tight junctions
• Permits passage of: water, some gases, passive diffusion of lipid soluble molecules, selective transport of glucose, AAs, hormones

Question 22

What is a method that could be used to transport drugs across the BBB?

Answer 22

• Link drugs to molecules that are normally transported across the barrier with specific receptors
• ex: LRP1 transported in via an endosome