M4 Gene therapy: Non-viral strategies

Question 1

3 main strategies of development of therapeutics agents for treatment of DMD

Answer 1

1. Prevention of the secondary consequences of the phenotypic defect (generally pharmacological therapies)
2. Correction of genetic defect – replace the defective gene or protein (gene therapy)
3. Cell therapy (myoblast transfer therapy)

Combining therapy = most efficacious

Question 2

1) Prevention of secondary consequences of the phenotypic defect

Answer 2

• Aimed at preserving healthy muscle tissue
• Delaying disease progression
• Reduce muscle fibrosis

Question 3

2) Correction of genetic defect – replace the defective gene or protein (gene therapy)

Answer 3

• Approaches to restore or replace the defective genes
• Most useful in treatment of genetic causes of muscle wasting (i.e. muscular dystrophy)

Question 4

3) Cell therapy( Myoblast transfer therapy)

Answer 4

• Regenerate or replace lost muscle tissue’

Question 5

What is gene therapy?

Answer 5

• experimental technique that uses genes to treat or prevent disease
• Replacing mutated gene that causes disease with a healthy copy
• Altering a mutated gene to restore functionality
• Introducing a new gene into body to help fight a disease

Question 6

Tool: Vector (mode of delivery)

Answer 6

• A deliver vehicle that carry a engineered genetic construct (carries gene of interest into the cell)
  
  • Influence delivery method, expression stability, and safety
  • E.g.:
    
    • Naked DNA/RNA fragment
    • Protein/lipid complex
    • Virion
    • Whole cell

Question 7

Tool: gene expression therapy (What you are delivering)

Answer 7

• 3 elements:

1. Promoter (non-coding DNA that controls expression of gene of interest)
   
   • Constitutive (turns gene on when cells get into gene and stay on - high level of expression)
   • Tissue specific (only turned on in the cells that elicit benefit)
   • Inducible (Control length of time that gene is expressed for)
2. Gene of interest (Downstream of promoter)
   
   • Native or modified
   • Able to code for protein or regulatory RNA
   • Able to add tags for easier identification
3. Transcript stabilising element
   
   • Enhances lifespan of the transcript

Question 8

Criteria for gene therapy tool

Answer 8

1. Enter skeletal muscle cells
2. Enter heart muscles
   
   • DMD also affects heart cells
3. Specific to heart and muscles cells
4. Not cause harm
5. Correct genetic impairment

Question 9

What percentage is muscle is needed to be treated before seeing improvements in quality of life?

Answer 9

• Only 20% of the skeletal muscles + heart needs to be treated → to see improvements in quality of life

Question 10

Considerations for therapy

Answer 10

1. Route of delivery
   
   • Systemic/muscle to muscle?
2. Carrying capacity
   
   • Is there a vector that can deliver entire fragment
3. Timing of delivery
4. Patient-specific vs generic therapy
5. Immune response
   
   • Potentially to vector or to dystrophin itself

Question 11

Non-viral gene therapy for DMD

Answer 11

1. Unencapsulated plasmid delivery
   
   • Delivery of naked DNA/RNA
2. Dystrophin restoration
   
   • Stop codong read-through
   • Exon skipping

Question 12

What is unencapsulated plasmid delivery

Answer 12

• Injection of muscles with naked plasmid DNA
  
  • Safe, simple, cost effective
  • BUT low efficacy (1% of fibres expressed reporter gene)

Question 13

How can unencapsulated plasmid delivery’s efficacy be improved?

Answer 13

• Protect from DNA degradation
• Combine with carrier molecule
  
  • Put inside lipid complex → better transport through lipid membrane
• Ultrasound → increase membrane permeability
• Electroporation
  
  • Electrical pulse across muscle to assist DNA to pass cell membrane
  • But may result in damage to muscle
  • Good efficacy with hyaluronidase (a compound which breaks down the ECM)

Question 14

Adv/Dadv of unencapsulated plasmid delivery

Answer 14

• ADV:
  
  • Able to deliver full length
  • Safe, simple, cost-effective
• Dadv
  
  • Low efficacy in absence of adjunctive therapy
  • Localised delivery only
  • Plasmid DNA molecules decrease overtime

Question 15

Dystrophin restoration

Answer 15

• stop codon read-through
• 10-15% from non-sense mutation in DMD
• Allows transcription machinery to read through stop codon and restore dystrophin expression

Question 16

Gentamicin

Answer 16

• Interacts with 40s ribosomal subunit
• Recognise stop-codon → desensitise ribosomal machinery to allow transcription machinery to read through and continue with translation
• Restoration in dystrophin expression (variable 0-15%) - not very efficacious

Question 17

Ataluren

Answer 17

• Overcome DNA non-sense mutation responsible for 10-15% cases of DMD
• Oral medication
• Aims to increase expression of full-length dystrophin protein

Question 18

Mech of ataluren

Answer 18

• Binds to 60s ribosomal subunit and read through stop codon
• Efficacy similar to gentamicin but at MUCH lower doses
  
  • Good safety profile
  • Improve functional parameters in long-term treatment

Question 19

Dystrophin restoration: Exon skipping

Answer 19

• Restore mRNA reading frame by skipping over exon in mutated area → allowing exons to join together and create a functional protein
• Need to know which exon to skip (79 exons in the dystrophin gene)

Question 20

2MeAON

Answer 20

• Oligonucleotide:
• Not broken down in vivo
• Binds to RNA with high afffinity
  
  • Able to skip exons (skips exon 23)
  • Restoration of dystrophin 15-21% (good time frame, able to be delivered at any age)

Question 21

Drug used in clinical trial of 2MeAON

Answer 21

• Drisapersen
  
  • Skips exon 51 (prevalent in 13% of DMD pop)
  • Improvement in 6minute walk test

Question 22

PMO

Answer 22

• Morpholino:
• Alternative exon skipping molecules
• Able to be delivered systemically
• 10-50% dystrphin restoration -> more efficacious than oligonucleotide

Question 23

Drug used in clinical trials of PMO

Answer 23

• Eteplirsen
  
  • Safe and efficacious in clinical trials
  • Improvements in ambulation and respiration
  • Significant improvement in dystrophin

Question 24

Do 2MeAONs and PMO treat dystrophic hearts?

Answer 24

• However, 2MeAONs & PMO does not treat dystrophic heart

Question 25

PPMO

Answer 25

• Peptide-conjugated PMO
• More efficiently transfer morpholino across cell membrane -> allows dystrophin expression in the heart
• Subsequent studies demonstrated some toxicity

Question 26

Adv/Dadv of dystrophin restoration

Answer 26

• ADV of dystrophin restoration:
  
  • Systemic delivery (all muscle and heart treated)
  • Safe and efficacious
  • Three drugs close to approval
• Dadv:
  
  • Patient-specific therapies
  • Repeated administration required for therapeutic benefits (only targeting dystrophic muscle fibres at the time of injection)

Question 27

Summary of treatments

Answer 27