C9 miRNA therapies

Question 1

Molecular signature of cardiac stress genes in a diseased heart

Answer 1

• Anp: Released by heart in diseased states
• SERCA2a: Associated with cardiac contractile function

Question 2

miRNAs

Answer 2

• Short non-coding RNA molecules → no role in making protein
• Involved in range of biological process
• Binds to complementary 3’UTR (seed region)
  
  • Causes mRNA degradation → interfering with gene expression
  • Contributes to human diseases

Question 3

Targets of miRNA

Answer 3

• 1 miRNA can target hundreds of mRNA or many miRNA can target the same mRNA

Question 4

Are miRNAs critical for normal cardiac biology?

Answer 4

• Dicer creates miRNA
• Delete Dicer gene in mice
  
  • → dilated cardiomyopathy
  • → impaired cardiac function
    
    • Premature death

Question 5

Pathways regulating pathological/physiological cardiac hypertrophy

Answer 5

• Pathological cardiac hypertrophy pathway
  
  • Regulated by the GPCR pathway
• Physiological cardiac hypertrophy pathway
  
  • Regulated by the IGF1-PI3K-Akt pathway

Question 6

Role of PI3K

Answer 6

• cardioprotective
• Prevents excessive heart growth, dysfunction, fibrosis
• PI3K regulates miRNA

Question 7

What is the selection criteria for miRNAs to be identified?

Answer 7

• miRNA has to be Low [] in heathy and high [] in susceptible heart & in heart failure
• ∴able to use miRNA blockers to stop damaging effects of miRNA

Question 8

PI3K regulated miRNA

Answer 8

• miR-34
• miR-652
  
  • Both increased in disease heart and decreased in healthy heart

Question 9

LNA-anitmiR34

Answer 9

• High binding affinity → inhibits individual or families of miRNA
  
  • stable when delivered in vivo
  • no vesicle required
  • no toxicity

Question 10

Beneficial effect of LNA-antimiR34

Answer 10

• chronic knockdown of miR with only 3 injections
  
  • Improves heart function
  • Decrease heart growth, atrial and lung weight(markers of pathology)
  • ↑vinculin, Bcl6 gene response, Nolch 1
  • ↓Anp expression (releaed by heart in disease states)
  • Increase SERCA2a

Question 11

How do anti-miR work?

Answer 11

• Anti-miR binds to endogenous miR → ↓miRNA function → ↑target expression (i.e. vinculin) → ↑cardiac repair, electrical function

Question 12

Vinculin

Answer 12

• Role in cardiac and electrical functions
• Essential for cardiomyocyte stability and regulates expression of proteins residing at the intercalated disk
• ↓Expression of vinculin in diseased mice at intercalated disks, targeted by miR34

Question 13

Luciferase

Answer 13

• Enzyme produced by fireflies

Question 14

Target validation - luciferase assay

Answer 14

• Genetically engineer luciferase to check for gene activity in cells
• Mech:
  
  • Target plasmid + mRNA mimics
  • Using lipid substance → put plasmid and mimics into heart cells
  • No binding → luciferase emit light
  • Binding → decreased luciferase emitting light

Question 15

Luciferase assay of miR34 targeting vinculin

Answer 15

• Assay showed vinculin being a true target of miR-34

Question 16

Anti-miR652

Answer 16

• ↓ heart and cell size
• ↓ fibrosis
• ↓ cell death
• Improved cardiac function and preserve angiogensis
• ↑JAG1 (which has anti-hypertrophic effects on the heart)

Question 17

Experimental models of heart dysfunction used for the different anti-miRs

Answer 17

• MI model: miR34
• Pressure overload model: miR34 and miR652

Question 18

What is the limitation of LNA inhibitors?

Answer 18

• LNA inhibitors are not tissue specific
• Need to develop cardiac-specific miRNA-based gene therapy

Question 19

What are miRNA sponges

Answer 19

• Contains multiple binding sites (4-10 fully/partially complementary to miRNA target of interest)
  
  • Binding sites usually separated by 4 nucleotides → allows optimal binding
• Competitive inhibitors → soak up endogenous miRNA → prevent binding of miRNA → ↑expression of target

Question 20

Delivery of miRNA sponge

Answer 20

1. Using an AAV (For tissue selectivity)
   
   • AAV6 → preferential for heart and skeletal muscles with CMV promoter
2. Different promoters
   
   • CMV or cTNT
3. Inclusion of reporter gene
   
   • Determine tissue/cells that express AAV-miRNA
   • Monitor level and duration of AAV-miRNA sponge expression
   • Determine transduction efficiency