M5 Gene therapy: Viral-mediated strategies

Question 1

3 different types of virus ultilised to treat DMD

Answer 1

1. Hd-Adenovirus
2. Lentivirus
3. Adeno-associated virus

Question 2

Adenovirus

Answer 2

• Cause mild diseases in humans
• Medium size and non-eveloped
• Large carrying capacity (has a large genome)
• Capable of transducing both proliferating and non-proliferating cells
• Needed for utilising vectors into viable tools

Question 3

1st Generation vectors

Answer 3

• Made by substituting E1 and E3 bit of genome
• Genes adenoviruses use to invade host-cell and proliferate
• Carrying capacity only 8kb (dystrophin is 14kb)
• Elicit a significant immune response (due to de novo syn of viral proteins)
  
  • When injected iv → leads to sepsis (need to limit it to local injections only)

Question 4

2nd generation vectors

Answer 4

• Deletion of additional viral genes and increase carrying capacity of 10kb (E2B gene deleted)
• Viral genes still expressed on vector

Question 5

3rd generation vectors

Answer 5

• AKA Helper dependent viral vector/Gutted adenoviral vector
• Lacks all the viral gene, increasing carrying capacity up to 30kb
  
  • Therefore has a lower immune response
• Functional improvement
• Needs to be delivered at high doses
  
  • Which still elicits immune responses

Question 6

Adv/Dadv of adenoviruses

Answer 6

• Adv
  
  • Able to deliver full-length dystrophin gene
• Dadv
  
  • Not suitable for systemic delivery due to immune response

Question 7

Lentivirus

Answer 7

• Envelop retrovirus
• Cause more severe disease (HIV and associated viruses..)
• Has RNA genome
• Carrying capacity = 8kb
• Able to integrate into the genome of the host cells

Question 8

Generation of lentivirus

Answer 8

• Subsequent retroviral vectors have been generated based on human and feline lentivirus
  
  • Efficiently transduce non-cycling cells including post-mitotic neurons, hepatocytes and muscle fibres
• Significant improvements when injected in newborns but not much in adults

Question 9

Adv/Dadv of lentivirus

Answer 9

• Adv
  
  • Able to transduce muscle stem cells
• Dadv
  
  • Able to infect both proliferating myoblasts and differentiated myotube in vitro BUT Limited capacity in vivo
  • Insertional mutagenesis limits possibility of systemic delivery

Question 10

Adeno associated virus (AAV)

Answer 10

• Small parvovirus -> Doesn’t cause virus
• 80% of population already infected by virus (we are natural host)
• Requires a second virus to allow proliferation (improves safety as it cannot proliferate itself)
• Carry capacity < 5kb

Question 11

Recombinant AAV (rAAV)

Answer 11

• Gene of interest flanked by viral Inverted Terminal Repeat (ITR) -> gene of interest into viral capsid
  
  • Requires presence of viral helper genes
  • Different tropism (where they will go and transduce)
    
    • rAAV 1,6,8,9 -> transduce into heart and skeletal muscles

Question 12

Effectiveness of AAV in end stage diseases

Answer 12

• Efficient transduction of cardiomyocytes in aged dystrophic mice
• No improvement in heart fibrosis
• Small improvements in heart function
• Efficacy of treatment limited by disease pathology?

Question 13

Immunosuppression during vector delivery

Answer 13

Efficient delivery following i.m/i.v

Question 14

Adv/Dadv of AAV

Answer 14

• Adv
  
  • Capable for systemic delivery
• Dadv
  
  • Not able to delivery full-length dystrophin gene
  • No transduction of muscle stem cells (needs to be delivered more than once in a lifetime)

Question 15

Utrophin

Answer 15

• alternative to dystrophin
• Utrophin is a homologue of dystrophin
• Normally expressed in NMJ (expressed in the SL)
  
  • May be upregulated to compensate for the loss of dystrophin
• Over-expressed in mdx mouse

Question 16

AAV to restore dystrophin

Answer 16

• Deliver 2 dystrophin constructs that recombine in vivo to form a functional mini-dystrophin
• Potentially for delivery of full-length dystrophin

Question 17

Overall summary

Answer 17

Upregulating other genes is more beneficial