Defects in CLN6 neuron cell cultures can be corrected using media from normal control cells. Using mass spectrometry to analyze the media, researchers found additional lysosomal proteins secreted from CLN6 cells that are not present in normal cell media and may contribute to disease. These proteins are being investigated further for their potential as disease biomarkers and roles in CLN6 disease pathogenesis. While CLN6 mRNA has been found in extracellular vesicles, sequences aimed at enhancing extracellular trafficking of mRNAs from gene therapy vectors have worked in some cell types but not neurons. Researchers are now searching for new sequences that can enhance CLN6 gene therapy delivery in the brain.

1. CLN6
Secretion of proteins and extracellular vesicles in CLN6 Batten disease
Stephanie M Hughes, Hannah L Best, Hollie E Wicky, Alison J Clare, Isaiah Cheong, Harry AM Biggs, Chris Brown. Neurodegenerative Disease
Lab, Department of Biochemistry, University of Otago, New Zealand
Introduction
• Mutations in CLN6 predominantly cause late infantile
Batten disease.
• We found that there were factors secreted from normal
cells that protected CLN6 cells and have been working to
identify what these factors are.
Conclusions and future directions:
•We have identified an abnormal release of lysosomal proteins
from CLN6 mouse neuronal cultures. We are currently working
out what role these factors have in disease processes and how
normal control media corrects deficits in affected cells.
•CLN6 mRNA can be secreted from cells and this might still be a
way of mediating correction between cells.
•We can use a short exosome targeting sequence or sequences
from CLN6 to enhance extracellular trafficking of mRNAs from
gene therapy vectors, however these sequences don’t work in
neurons.
•We are now searching for new extracellular targeting sequences
that can enhance CLN6 gene therapy.
Acknowledgements:
We thank David Palmer and Nadia Mitchell for sheep samples and the BSDRA, Cure Kids NZ, Gray Foundation – Cure Batten and the Neurological Foundation of New Zealand for support. HB is supported by a University of Otago
PhD scholarship and Roche Hanns Möhler scholarship. For further details please contact Steph Hughes stephanie.hughes@otago.ac.nz
What is in normal media that corrects CLN6 cells?
Using mass spectrometry – a method of
identifying protein components in
samples, we compared media from
neuron cultures of mouse CLN6 and
controls. Interestingly, there was nothing
extra in normal media, but there
were additional proteins found in media
from CLN6 cells which might
contribute to disease.
These lysosomal proteins are now being
investigated for their potential
as disease biomarkers and their role in
CLN6 disease.
Could the factor correcting CLN6 cells be CLN6?
CLN6 is a membrane protein, so each cell that requires it must produce it.
MAYBE NOT??
CLN6 mRNA has been found in extracellular vesicles – can we enhance this
process for gene therapy?
Figure 3. Cross-correction of a
membrane protein deficiency using gene
therapy and exosomes (our idea). A. An
exosome localisation tag (red) added to a
membrane protein-encoding cDNA (e.g.
CLN6) will be delivered to the mouse
brain using a viral vector. B. The exosome
tag will allow incorporation of the tagged
RNA into exosomes. C. Exosomes will be
secreted from donor cells and will D.
cross-correct CLN6 protein deficiency in
recipient cells throughout the brain.
Defects in CLN6 neuron cell cultures
can be corrected using media from normal
control cells
Figure 1. CLN6-/- Batten disease ovine neural cultures show a
restoration in lysosomal activity after incubation with conditioned
medium from healthy CLN6+/+ ovine neural cultures. a. Quantification
of average LysoTracker Red fluorescent intensity per cell, in affected
culture, after incubation with ‘healthy” media. b-d. Representative
images of LysoTracker (red) and DAPI (blue), indicating lysosomal
function, in b. healthy CLN6+/+ culture, c. affected CLN6-/- culture and
d. affected CLN6-/- after incubation with media from healthy cells.
’
This suggests “something” in the normal media corrects
defects in CLN6 cells.
We thank Prof. Dave Palmer and Dr. Nadia Mitchell for the sheep
CLN6 and control samples used in these experiments.
Figure 2. Right: A representation of proteins
that were uniquely detected in CLN6 mouse
neuronal media. The red box shows a group of
lysosomal proteins.
Adding sequences from CLN6 mRNA to gene therapy vectors
enhances extracellular trafficking from gene therapy vectors in some
cell types such as kidney cells – but not in brain cells.
Kidney cells Brain cells
Figure 3. Sequences added
to gene therapy vectors (.25
and UTR) enhance transfer
of RNA to extracellular
vesicles released from kidney
cells, but not brain cells.
Here we used GFP as a
marker of RNA trafficking.
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