Investigation of viral vector targeting in organotypic cerebellar slice culture Eric Garson, Helen Scott & James Uney. Regenerative Medicine Laboratory, School of Clinical Sciences, University of Bristol

Introduction • The cerebellum unconsciously controls movement and coordination. In addition to involvement in

development and cognition. These functions are determined by Purkinje neurons ,the principle

neuron of the cerebellum and Bergmann Glia ,the principle astrocytes of the cerebellum. Both are

found in the cerebellum cortex made up of three layers Granule, Purkinje Cell and Molecular layers.

Bergmann Glia cell bodies are found in the Purkinje cell layer. Purkinje neurons underpin the

cerebellum’s motor learning and coordination ability. While Bergmann Glia provide a scaffold and

adhesive for Granule cells to migrate into the Molecular layer.

• Numerous conditions have been associated with the cerebellum including Ataxia and Schizophrenia.

A form of human Ataxia caused by the TRPC3 (Transient Receptor Potential Cation Channel 3)

mutation found in Purkinje neurons leading to mutations in calcium and sodium channels. Whereas,

Schizophrenia causes malfunction of the DAO (D-Amino Acid Oxidase) enzyme found in Bergmann

Glia leading to the inability to metabolism the serine neurotransmitter. DAO activity is increased and

has been associated with symptoms and pathology of Schizophrenia.

• Gene therapy using viral vectors might overcome the barriers of targeting cerebellar cells and

compensate for TRPC-3 or DAO malfunction.

• In addition, it has been reported that Cathepsin K (a lysosomal enzyme) manipulates lentiviral vector

tropism towards Bergmann Glia. While its inhibition manipulates lentiviral vector tropism towards

Purkinje neurons both in vivo. Meanwhile, organotypic slice culturing provides a method to access

these factors because they retain the cyto-architecture and interactions of the cerebellum cortex,

permit immunostaining of Bergmann Glia and Purkinje neurons and permit assessment of viral vector

tropism and manipulation by Cathepsin K or Cathepsin K inhibitor.

Aims and Objectives The overall aim was to develop an optimised in vitro organotypic slice culture model for studying and

targeting cerebellar diseases. This would be achieved by using Cathepsin K or Cathepsin K Inhibitor to

manipulate the tropism of lentiviral vectors towards either Bergmann Glia astrocytes or Purkinje

neurons. Ultimately, these aims would realize a model for human cerebellar diseases, develop an

understanding of the underling disease processes involved and the prospect of developing novel

therapeutic treatments.

The specific objectives were to:

• Immunostain for Purkinje neurons and Bergmann Glia astrocytes.

• Manipulate lentiviral vector tropism towards Bergmann Glia astrocytes using Cathepsin K.

• Manipulate lentiviral vector tropism towards Purkinje neurons using Cathepsin K Inhibitors.

• Assess Cathepsin K or Cathepsin K Inhibitor addition 1 day before lentiviral vector addition.

• Access Adeno-Associated viral vector 2-9 (AAV2-9) transduction.

Methods

Results

-AraC

+AraC

Day 1 Day 8 Day 3 Day 2

Conclusions An optimised 8 day in vitro organotypic cerebellar slice culturing system achieved on tissue culture inserts permitted viral vector

expression beginning on day 5 in vitro with an optimum dilution/concentration at 5x105 particles/slice. Meanwhile, 1mM AraC

stopped Glia overgrowth for successful GFAP immunostaining. Successful immunostaining for Bergmann Glia and Purkinje

neurons using GFAP and Anti-Calbindin D-28k respectively was achieved. However manipulation of lentiviral vector tropism

towards Bergmann Glia using Cathepsin K and manipulation using Cathepsin K inhibitor towards Purkinje neurons was

unsuccessful because of no co-staining observation. AAV2-9 was successfully assessed using GFAP and Anti-Calbindin D28k.

References

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6. Burnet, P., et al. 2008. Mol Psychiatry 13: 658-660.

7. Becker, E. B. 2015. Cerebellum.

Figure 1: Optimisation and maintenance of cerebellar slice culture procedure

Figure 2: 1mM AraC Prevents Glial Overgrowth

Figure 3: Cathepsin K does not manipulate lentiviral vector tropism towards Bergmann Glia

Figure 4: Inhibition of Cathepsin K does not manipulate lentiviral vector tropism towards Purkinje neurons

Figure 5: Cathepsin K addition one day prior to lentiviral vector tropism addition does not manipulate lentiviral vectors towards Bergmann Glia

Figure 6: Cathepsin K Inhibitor addition one day prior to lentiviral vector addition does not manipulate lentiviral vectors towards Purkinje neurons

Figure 7: Assessment of AAV2-9 shows transduction in white matter

- Cathepsin K + Cathepsin K

- Cathepsin K Inhibitor + Cathepsin K Inhibitor

GFAP Overlay Anti-Calbinidn D28K Overlay

Anti-Calbindin D28 K Overlay

GFAP Overlay

500.000 μm

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500.000 μm 500.000 μm

500.000 μm

500.000 μm

Day 1 Day 3 Day 2 Day 4 Day 5 Day 8