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Pizza Club, 22nd February 2017Gemma Gómez Giró

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Neuronal Ceroid Lipofuscinoses (NCL)

o Group of severe autosomal recessive neurodegenerative disorders, affectingchildren and young adults.

o Lysosome storage disease: accumulation of autofluorescent, electron dense material in cells.

NCL foundation

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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.

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Most common CLN3 JNCL causing mutation

Del exons 7-8: c.461-280_677+382del966,p.[Gly154Alafs*29,Val155_Gly264del]

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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. Serotypewith CNS tropism, but not exclusively.

Gene delivery approach using scAAV9

Chickenβ-actin promoter-GFP

MouseMethyl-CpG-binding protein 2-GFP

Because CLN3 regulatory elements are not defined

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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 neuronaland non-neuronal cells.

o Mice sacrificed after 5 months.

Gene delivery approach using scAAV9

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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.

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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 as1 month after injection- whereas AAV9/β-actin-hCLN3 is ineffective.

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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.

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Results

o Similar results obtained while evaluating reactive astrocytes.

o Only AAV9/MeCP2-hCLN3 reduces astrocyte activation.

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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 trendtowards lower levels of SCMAS (mice are injected when inclusions arealready present).

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Thank you!