emerging treatments

Question 1

• By what mechanism are inborn errors in metabolism generally caused by?
• How are these treated?

Answer 1

Lack of enzyme or too much substrate

Replacement- replace the enzyme lost

Question 2

• In what case would a pharmacological chaperone be necessary and what does it do?

Answer 2

When there is a fault in the protein folding due to a mutation, leading it down a degradation pathway

allows accurate folding of protein, stabilising protein and forcing it into correct shape

Question 3

• What does a pharmacological modulator do?

Answer 3

Receptor agonist/antagonist

Ion channel activators/blockers

Can be designed to have these effects on mutant receptors or channels

Question 4

• Explain the combination therapy used in Cystic Fibrosis

Answer 4

In CF there can be both a mutation leading to a misfolded, inactive channel and a defective chloride channel

Treated with both chaperone (Lumacaftor) and activator (Ivacaftor)

Question 5

• What is a non-sense mutation?

- How does a Non sense suppressor work?

Answer 5

Mutation that introduces a stop codon prematurely in the protein sequence

The drug forces DNA polymerase to keep going and not stop at premature codon
This is a stop codon readthrough

Question 6

• How does gene therapy work with recessive diseases?

- How does gene therapy work with dominant diseases?

Answer 6

Replace the defective gene

Delete the defective gene

Question 7

• What does ‘in vivo’ mean?
• What does ‘in vitro’ mean?
• What is the most well known in vitro gene therapy?

Answer 7

In the living

In glass

Mitochondrially inherited disease therapy

Question 8

• Explain the steps of Maternal spindle transfer

Answer 8

Chromosomes are removed from an unfertilised egg that has mutated mitochondrial DNA

These are added to an unfertilised donor egg with normal mitochondria, that has had its nucleus removed

This fused egg is fertilised in vitro

The egg develops normally to form an embryo

Question 9

• Explain the steps of pronuclear transfer

Answer 9

An egg with mutated mitochondrial DNA is fertilised in vitro

Resulting pronucleus is removed

This is transferred to a fertilised donor egg that has had its pronucleus removed

The fused egg develops normally

Question 10

• What is the overall process of virus gene therapy?

Answer 10

Removing DNA from virus

Engineering the DNA so that it has a new gene that would benefit the host

Place the DNA back into the virus

Allow it to enter the host cells so that host cells can start transcribing this new DNA, to make proteins that body is lacking

Question 11

• What is the virus choice for viral gene therapy largely dependent on?
• What is meant by viral tropism?

Answer 11

The target tissue that you want to treat

The affinity for infecting certain cell types that a virus has

Question 12

• What else limits the amount of DNA injected in viral gene therapy?
• What types of cells are viral gene therapy most commonly used to treat?

Answer 12

Size of the virus

Haemopoietic stem cells
They are easy to reach

Question 13

• How does in vitro gene therapy for ADA-SCID work?

Answer 13

Take the bone marrow from the patient

Isolate the embryonic haemopoietic stem cells (specifically CD 34 positive ones)

Transfect them with lentiviral virus that encodes ADA

Treat patient with busulfan to kill their own haemopoietic stem cells so they can be infused with the transformed cells

These are therefore able to replace the cells lacking ADA and replace them with cells that are healthy

Question 14

• Where can local injections of viruses carrying functional genes be injected in vivo?

Answer 14

Eye, Spine, Brain

Question 15

• Describe what is meant by an antisense oligonucleotide

Answer 15

Short modified nucleic acid complementary to target

Modification prevents degradation and allows entry to cell

Binds to target → Blocks translation → Can also alter splicing

Question 16

• How does exon skipping occur?

- What is exon skipping used for?

Answer 16

Antisense oligonucleotide will bind to the acceptor site of exon to prevent it from acting as an exon
Excluding this exon from the protein

To put RNA back in reading-frame (dividing the sequence of nucleotides into a set of consecutive, non-overlapping triplets)

Question 17

• What are the requirements for exon skipping to occur?

Answer 17

The exon being skipped must not be vital for proteins function

Therefore mainly used for larger proteins where a single exon does not largely affect the protein

Question 18

• In DMD, what does exon skipping achieve?

Answer 18

Results in production of partially active dystrophin instead of fully inactive- results in condition BMD which is less severe than DMD

Question 19

• How does CRISPR-Cas9 work?

- What are the 2 methods of repair of the genome after CRISPR-Cas9 has acted on it?

Answer 19

Targets specific regions (CRISPR regions) of genome using the gRNA and cleaves them using the Cas9 protein

Non-homologous end joining
Homology directed repair

Question 20

what virus are used for gene therapy

Answer 20

AVA
lentivirus
vaccinia
adenovirus

Question 21

what is an example of in vivo viral gene therapy

Answer 21

supplement

for recessive diseases

virus insert a working copy of the gene into cell- working gene will produce enough normal protein to survive

can inject systemically

or, can inject locally through eyes, spine or brain

Question 22

when is gene silencing used

Answer 22

usually for dominant diseases- gain of function

or recessive disease with downstream effects

Question 23

how does RNAi work

Answer 23

dsRNA is complementary to target
modification prevents degradation allowing entry into the cell
activated via dicer or risc pathway
targeted RNA is cleaved

Question 24

can gene editing correct large changes eg deletion or triplet expansion

example of gene editing?

Answer 24

gene editing corrects small errors
may have off target effects
same problem as other methods eg targeting/ getting into cell

CRISPR-Cas9 edits genes by precisely cutting DNA and then letting natural DNA repair processes to take over
bacteria use CRISPR-Cas9 to disable bacteriophage
CAS-9 is an bacterial enzyme that recognises and cleaves DNA CRISPR hybrids