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Pizza club - February 2017 - Gemma


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Self-Complementary AAV9 Gene Delivery Partially Corrects Pathology Associated with Juvenile Neuronal Ceroid Lipofuscinosis (CLN3), Bosch et al, Journal of Neuroscience, 2016

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Pizza club - February 2017 - Gemma

  1. 1. 1 Pizza Club, 22nd February 2017 Gemma Gómez Giró
  2. 2. 2 Neuronal Ceroid Lipofuscinoses (NCL) o Group of severe autosomal recessive neurodegenerative disorders, affecting children and young adults. o Lysosome storage disease: accumulation of autofluorescent, electron dense material in cells. NCL foundation
  3. 3. 3 Juvenile NCL (JNCL) or Batten Disease o Most common type of NCL. o Appearing age 6-8 years, life expectancy between 20s-30s. o Lysosomal storage material: subunit c of mitochondrial ATP synthase F0. o Gene affected: CLN3 (16p12.1). o 438-aa transmembrane protein (Mw=48kDa) of unknown function and ubiquitous localization.
  4. 4. 4 Most common CLN3 JNCL causing mutation Del exons 7-8: c.461- 280_677+382del966, p.[Gly154Alafs*29, Val155_Gly264del]
  5. 5. 5 o Objective: correct CLN3 deficiency. o Hypothesis: Low CLN3 levels are required for cellular homeostasis. o Method: Use of adenovirus (non-integrative, dsDNA, transducing replicative and non-replicative cells) to introduce the transient expression of CLN3. Serotype with CNS tropism, but not exclusively. Gene delivery approach using scAAV9 Chicken β-actin promoter-GFP Mouse Methyl-CpG-binding protein 2-GFP Because CLN3 regulatory elements are not defined
  6. 6. 6 o Cln3Δex7/8 o 1-month-old mice: • To more accurately depict human age of onset based on mouse-human age equivalent estimates. • When disease manifest (although phenotype is proven very modest in mice before). o Systemic (intravenous) delivery (1X) to enhance virus biodistribution. AAV9 crosses BBB and transduces neuronal and non-neuronal cells. o Mice sacrificed after 5 months. Gene delivery approach using scAAV9
  7. 7. 7 Results o Widespread GFP expression with both constructs detected throughout the brain, eye and spinal cord. o MeCP2 primarily drove expression in neurons and β-actin in astrocytes.
  8. 8. 8 Results o To them, Cln3Δex7/8 developed robust and persistent motor deficits beginning with 2 months of age. o Rotarod test: o AAV9/MeCP2-hCLN3 reverses motor deficits in Cln3Δex7/8 mice -as early as 1 month after injection- whereas AAV9/β-actin-hCLN3 is ineffective.
  9. 9. 9 Results o In a previous study they showed that microglia in Cln3Δex7/8 mice are primed to be proinflammatory. o Only AAV9/MeCP2-hCLN3 can significantly reduce microglia reactivity.
  10. 10. 10 Results o Similar results obtained while evaluating reactive astrocytes. o Only AAV9/MeCP2-hCLN3 reduces astrocyte activation.
  11. 11. 11 Results o Effect of AAv9/hCLN3 transduction on lysosomal pathology by immunostaning for LAMP-1 and subunit c of mitochondrial ATP synthase (SCMAS). o Only AAV9/MeCP2-hCLN3 reduces LAMP-1 levels and there is a trend towards lower levels of SCMAS (mice are injected when inclusions are already present).
  12. 12. 12 Thank you!