10th March - Therapies, mRNA export

Question 1

What is morpholino?

Answer 1

A treatment for duchenne’s muscular dystrophy.
It is an oligonucleotide, in which the DNA bases are attached to a backbone of methylenemorpholine rings, which promotes skipping of exon 51

Question 2

How can pro-apoptotic isoforms of Bcl-X and Mcl-1 be promoted?

Answer 2

Using oligonucleotides which block the anti-apoptotic splice site and enhance apoptosis,

Question 3

What is the problem with using oligonucleotides in treatment in early tumours?

Answer 3

Delivery as there is no vascular network

Question 4

What causes spinal muscular dystrophy?

Answer 4

Mutation in SMN1

Question 5

Why does a mutation in SMN1 cause spinal muscular dystrophy, when everyone carries another copy of SMN, SMN2?

Answer 5

1 base change in SM2 means that 90% of transcripts have exon 7 spliced out, making the resultant protein useless

Question 6

How could splicing be restored of SMN2 exon 7?

Answer 6

Solution 1.

• -An oligonucleotide that would anneal to the splice site with an RS tail that would bind SRSF1.
• -This worked in vitro
• -However such a long nucleic acid sequence would be difficult to deliver to the tissue

Solution 2.

• -Block a silencer using oligonucleotides
• -Approves as Spinraza in 2017 but had to stop phase II trail early due to failure of placebo
• -Injected oligos into CNS, lasted for about 6 months

Question 7

Outline Palacino’s 2015 experiment

Answer 7

Screened 1.4 million compounds on SMN2 using a reporter system
1st assay = Exon inclusion –> luciferase expression
2nd assay = Exon exclusion –> luciferase expression
Used two assays to rule out a global increase in expression
–>Found NVS SM1
-Prolonged SMA model mouse life >35 days
- Interacts with U1snRNA and stabilised interaction with 5’ss of exon 7

Question 8

What does mutant SF3B1 in cancer promote?

Answer 8

Use of alternative branchpoints

Question 9

What is the function of SF3B1?

Answer 9

Holds the branch site 50A away from the active site

Question 10

How can mutant SF3B1 be inhibited in cancer?

Answer 10

Use of certain anti-biotics e.g. sudemycins, pladienolide B

Question 11

Why do SF3B1 inhibitors work on tumours which do not carry a mutation is SF3B1?

Answer 11

Believed that other oncogenic mutations causes the same changes that are seen in mutant SF3B1

Question 12

What are the therapeutic benefits of inhibiting SR protein kinase 1?

Answer 12

Prevent SRSF1 activity therefore inhibit angiogenesis

Question 13

How is the mRNA exported from the nucleus?

Answer 13

The EJC helps to recruit proteins necessary for export from the nucleus.

• -P15 binds to RNA
• -TAP binds to nucleoporin, holding the mRNA at the pore
• -Then is exported in a ‘ratcheting’ fashion, similar to a zip tie, in which it accidentally moves forward through natural oscillation and then can’t move back