Potential therapies for Neuromuscular disorders

Question 1

Most current therapies for neuromuscular disorders include?

Answer 1

• steroids
• physiotherapy
• orthopaedic input
• cardiac surveillance
• ventilatory support

All not due to genetic information and not cures

Question 2

What are the challenges of designing a therapy?

Answer 2

• muscles make up 40% of a person’s body mass an injection will only transduce cells within a couple centimetres of injection site. Multiple injections required (not realistic)
• Many of the defective genes are very large (larger than the size limit for viral vectors)
• Many different genes and many different mutations, thus there is no “one size fits all”

Question 3

What do we need to start with for neuromuscular therapies?

Answer 3

• knowledge of the disease-causing gene mutation
• understanding the pathway, pathobiology, dominant or recessive?
• appropriate model for screening/testing putative therapies
• knowledge of different therapeutic strategies

Question 4

What are the potential therapeutic therapies?

Answer 4

• gene replacement therapies
• antisense oligonucleotides (exon skipping)
• upregulation therapy
• CRISPR/Cas9

Question 5

What is gene replacement therapy?

Answer 5

Viral delivery of a functional copy of the gene.

• using adenoviruses, retroviruses and lentivirus
• packaging limit

Question 6

Why is adenoviral vectors in gene therapy not used?

Answer 6

• was popular due to large coding capacity

- however unexpected death of patient, after AV delivery of ornithine carbamylase (possibly toxic shock)

Question 7

What is the vector of choice in gene replacement therapy?

Answer 7

Adeno-associated virus (AAV) as it’s not associated with human disease and cannot replicate by itself.
- can only accommodate 4.7kb

Question 8

What are the pros of gene replacement therapy?

Answer 8

• can persist as episomes for a long time in the nucleus or can integrate into the genome (either one-off or infrequent treatments)
• can be delivered systemically

Question 9

What are the cons of gene replacement therapy?

Answer 9

• can immune response (either against previously missing protein or viral proteins)
• difficult to produce large quantities of high [ ] at reasonable cost
• safety?
• where integrated in genome (small chance it could randomly integrate)
• can’t incorporate large genes

Question 10

What is the erase and replace method in gene therapy?

Answer 10

Gene therapy by knock down and replacement with a single AAV vector using shRNA

Question 11

What is exon skipping?

Answer 11

Mutations which introduce a premature stop codon in repetitive regions of large genes (e.g. regions that are redundant or dispensable for protein function). Introduces an antisense oligonucleotide, to cause exon with premature stop codon, to be skipped.

• not applicable to most genes

Question 12

Why isn’t precise correction possible in CRISPR/Cas9 editing?

Answer 12

• immunogenicity of Cas9 (a bacterial protein) not tested long-term
• NHEJ much more frequent than HDR (=errors)
• ethical considerations
• off target effects difficult to screen for

Question 13

What is Actin aggregate myopathy and what is it caused by?

Answer 13

A disease that results in the accumulation of excess thin actin filaments.

Caused by ACTA1 dominant mutations (90%). Dominant disease believed to be caused by the poison protein effect

Question 14

What is upregulation?

Answer 14

Upregulate the expression of a similar gene which could perform the same function. E.g. For the recessive ACTA1 disease use cardiac actin (ACTC1).

Cas9 tethered to gene activation domain could be used to upregulate expression of ACTC1? - currently limited due to unknown longevity of Cas9 upregulation and potential immune reaction