Muscle and Exercise Lecture 2.1 : Gene therapy for DMD: Non - viral strategies

Question 1

Name 6 muscle wasting conditions.

Answer 1

-Disuse atrophy, when not in use (limb casting/best rest).
-Denervation - spinal cord injury
-Sepsis - systemic infection (in blood)
-Sarcopenia - age related wasting
-Cancer cachexia - cancer related wasting
Muscular dystropy - genetic cause, genetic theraphy most helpful.

Question 2

What is the best therapy for a disease with genetic causes?

Answer 2

Genetic therapy is best.

Pharmacological treatment prevents secondary consequences only.

Question 3

What does genetic therapy do (name 3)?

Answer 3

Restores or replaces the defective gene. Can also introduce new gene to help fight diease.

Question 4

What is used in cell therapy?

Answer 4

Myoblast transfer.

Question 5

What is non-viral mediated therapy best for?

Answer 5

Altering a gene

Question 6

What is viral mediated best for? What is needed to carry it out?

Answer 6

Replacing a defective gene. A vector is needed to carry it out.

Question 7

What is a common vector?

Answer 7

Retrovirus.

Question 8

What is a gene expression construct? What is it made up of (3)?

Answer 8

It is the intervention body.

It needs a promotor, the gene of interest and a stabilising element.

Question 9

What is the promotor in a gene expression construct?

Answer 9

Promotor is used to induce high levels of expression, or make it cell specific by making it inducible in only certain cells.

Question 10

In making a promotor for DMD, it is best to make it specific to which tissue?

Answer 10

Muscle tissue (including cardiac).

Question 11

Aside from inducing high expression levels, what else can a promotor do?

Answer 11

Allows you to induce manually, such that one can induce when needed and turn off when uneeded.

Question 12

How can the gene of interest in a gene expression construct be easily identified?

Answer 12

Adding a tag.

Question 13

What is the purpose of a stabilising element in a gene expression construct?

Answer 13

Prolongs lifespan of the construct, allowing it to enter the cell.

Question 14

How does cell based therapy differ to genetic therapy?

Answer 14

The cell is already modified to express the new gene, is injected into the affected area to replace defective cells.

Question 15

In treating DMD, what must the treatment used have to do, and what percentage of muscles must be restored to begin improving function?

Answer 15

Must be able to enter both skeletal and cardiac cells, must be specific to them, and must be safe.
Only need 20% restoration to improve quality of life.

Question 16

Whats special about the dystrophin gene? Why is this an issue? How can it be resolved?

Answer 16

Its the largest gene in humans. Hard to deliver the entire corrected gene. Can cut out large parts of the gene and still have a functional protein, is truncated.

Question 17

What are the two main non-viral therapy techniques?

Answer 17

Unencapsulated plasmid delivery

Dystrophin restoration.

Question 18

What is unencapsulated plasmid delivery, and give a pro/con.

Answer 18

Form a plasmid vector with the corrected gene, then inject directly to muscles.
Is safe and simple, but very low efficiency, only 1% of localised cells (injection site, not the whole muscle) epress the gene.

Question 19

How can unencapsulated plasmid delivery be improved (5)?

Answer 19

Can combine with other therapies, add a carrier molecule (lipid) and add a DNA stabiliser.
Can use ultrasound to increase permeability.
Also electroporation, but damaging to cells.

Question 20

How can hyaluronidase be used to improve unencapsulated plasmid delivery?

Answer 20

It breaks down the extracellular matrix, and improves efficiency when paired with electroporation, not localised to injection site, but the whole muscle.

Question 21

Can unencapsulated plasmid delivery transfer the entire dystrophin gene? Is it permanent?

Answer 21

Yes, it can. It is not permanent, is only local to injection site, and repeat injections are needed.

Question 22

Name 2 dystrophin restoration techniques.

Answer 22

Stop codon read through

Exon skipping

Question 23

What is stop codon read through? What mutation does it work with?

Answer 23

Only works with nonsense mutation, 10-15% of DMD cases.
Is a premature stop codon, truncating the protein.
Stop codon read through uses a drug to allow ribosome to bypass the stop codon and keep reading.

Question 24

What are two drugs in stop codon read through, and what do they interact with? Are they effective?

Answer 24

Gentimycin - ribosomal subunit 140s, drops CK levels too, moderately effective.
Ataluren - ribosomal subunit 60s

Question 25

What is exon skipping? What type of mutation does it target? What is a consequence of this?

Answer 25

Targets patients with internal deletions.
Restores the mRNA reading frame by skipping an exon downstream of the mutated exon.
ie exon 50 deleted, exon 51 skipped. Will have a missing section, but still gives a usable form of dystrophin.

Question 26

What 3 drugs are used in exon skipping? Are they effective?

Answer 26

2MeAON - not very effective
Drisapersen - being reviewed
PMO - most efficient

Question 27

What is a problem with PMO? What was done to resolve it? What new issue arose?

Answer 27

It didnt affect the heart much.

PPMO was developed, is PMO with a cell permeable peptide added. Is toxicity associated with it however.

Question 28

Which therapies are patient specific?

Answer 28

Stop codon read through

Exon skipping

Question 29

Does exon skipping require repeat injections or is it permanent?

Answer 29

Repeat injections needed.

Question 30

Collectively, what percentage of DMD cases can exon skipping and stop codon read through treat and why?

Answer 30

Only 30% at best due to the nature of their mutation targets.