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Flashcards in Muscle 4 Deck (26):

What is gene therapy most useful in treating?

Genetic causes of muscle wasting


What are the 3 stages for developing therapeutic agents to treat DMD?

Prevention of secondary consequences of phenotype, correction of genetic defect, cell therapy


What are the mechanisms of gene therapy?

Replace a mutated gene with a healthy copy, alter a mutated gene to restore functionality, introduce new gene into the body to fight disease


What is a vector?

A delivery vehicle carrying an engineered genetic construct. Attributed of the vector influence construct delivery, expression stability, and safety in the target cells


What are the types of vectors?

- Naked DNA/RNA fragment
- Protein or lipid complex
- A virus derived virion
- A whole cell


What is the transcription promoter?

Upstream of the gene - can use this to control expression


What is the gene of interest for DMD?



What is the transcription stabilising element?

A polyadenylation sequence to enhance transcription lifespan


What are the criteria for an ideal gene therapy tool to treat muscle disease?

1. Must be able to enter skeletal muscle cells
2. Must be able to enter heart muscle cells
3. Must be specific to heart and skeletal muscle cells (if you only treat skeletal muscle then the heart muscle gets worse)
4. Must not cause harm
5. Must be able to correct the genetic impairment


How much skeletal muscle (including heart) must be corrected to observe an improvement in function and quality of life in DMD patients?



What is the efficacy of delivering unencapsulated plasmid DNA?

Low - 1% of fibres expressing reporter gene


How might delivering unencapsulated plasmid DNA be improved?

Deliver with something that protects DNA, improve transport across membranes (ultrasound or electroporation)


What is the consequence of electroporation?

May result in local damage to skeletal muscle


What are the advantages & disadvantages of delivery of unencapsulated plasmid DNA?

Advantages: able to deliver full length dystrophin gene, safe, simple protocol, cost-effective
Disadvantages: low efficacy in absence of adjunctive therapy, localized delivery only, plasmid DNA molecules decrease over time


What do 10-15% of DMD cases arise from?

Non-sense mutations - early stop codon


What are the features of gentamicin?

Aminoglycoside antibiotic with the ability to interact with the 40S ribosomal subunit when it recognises a stop codon, allows for the introduction of an aa at the stop codon, induced up to 20% dystrophin positive fibres in treated mdx mice


What are the features of ataluren (translarna)?

Designed to overcome DNA nonsense mutations which cause 10-15% of all cases of DMD, oral mediation, aims to increase production of full length dystrophin protein, binds to 60S ribosomal subunit and works in a similar manner to gentamicin, 20-25% dystrophin positive fibres achieved in treated mdx mice


What is exon skipping?

Restoration of mRNA reading frame, correct reading frame by inducing skipping of mutation containing exons during pre-mRNA splicing using antisense oligonucleotides that interfere with the splicing of targeted exons


What are the features of 2MeAON?

Resistant to endonuclease and RnaseH, binds to RNA with high affinity, skips exon 23 mutation in mdx mice resulting in a functional product


What are the features of drisaperson?

Skips exon 51 mutations (13 % of DMD population), dystrophin expression is observed following intramuscular injection


What are the features of PMO?

Alternative exon skipping molecule, demonstrated efficacy in mdx mice following trials, 10-50% dystrophin restoration levels achieved in skeletal muscles


What are the features of eteplirsen?

Skipping of exon 51 mutations, safe and efficacious for long term treatment, improvements in ambulation and respiration compared to control group, significant improvement in dystrophin restoration following 48 weeks of continuous treatment


What are the advantages and disadvantaged of exon skipping?

Advantages: systemic delivery, shown to be safe and efficacious in clinical trials, three drugs close to approval

Disadvantages: patient specific therapies (only targeting 13% of population), repeated administration required for therapeutic benefit


How many exons in dystrophin gene?



What is PPMO?

Conjugation of a morpholine to a cell penetrating peptide


What is the administration method for exon skipping and what age of animals is it efficacious in?

Systemic - efficacy shown in young and old mice