Comparison of AAV9-driven motor neuron transduction following different CNS-directed delivery methods in mice

Mole, A. J.; Sander, C. F.; Parmar, A. R.; Williams, A. J.; Azzouz, M.; Hautbergue, G. M.; Shaw, P. J.; Ferraiuolo, L.; Mead, R. J.

doi:10.1101/2025.07.01.662642

bioRxiv · 10.1101/2025.07.01.662642

Comparison of AAV9-driven motor neuron transduction following different CNS-directed delivery methods in mice

Mole, A. J.·Sander, C. F.·Parmar, A. R.·Williams, A. J.·Azzouz, M.·Hautbergue, G. M.·Shaw, P. J.·Ferraiuolo, L.·Mead, R. J.

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Abstract

AbstractO_ST_ABSBackgroundC_ST_ABSGene therapies are promising for diseases previously considered incurable. Adeno-associated virus serotype 9 (AAV9) demonstrates remarkable tropism for motor neurons (MNs) and represents an exciting candidate to target genetic causes of motor neuron diseases like amyotrophic lateral sclerosis (ALS). However, systemic delivery risks immunogenicity and off-target effects, therefore localised delivery to the CNS is advantageous. New methodWe assessed MN transduction in wild-type mice using AAV9-controlled, cytomegalovirus-promoter driven, enhanced GFP expression. Intra-cisterna magna (ICM) and intra-cerebroventricular (ICV) methods were compared. Four weeks post-delivery, GFP positivity in MN and astrocytes were quantified via immunohistochemical approaches and viral genome copy number determined by qPCR. ResultsAll delivery methods achieved high MN transduction in lumbar spinal cord (>68%). Unilateral ICV delivery provided the highest and most consistent levels (89 {+/-} 3%), and minimal peripheral viral copies. ICV delivery resulted in higher astrocytic transduction, most notably in the cortex. Brainstem MN transduction was high with all methods (>55%). We failed to find evidence of neuronal transduction in motor cortex. Viral genome copies trended higher in spinal cord and brainstem with ICV approaches, however further work is required to understand how bilateral delivery leads to such profound increases. Comparison to existing methodsWhilst several routes of administration into cerebrospinal fluid exist, direct comparisons for targeting MNs in vivo remain limited. ConclusionsOverall, consideration of gene therapy delivery methods is critical to ensure that the most appropriate administration route is chosen to reach MNs effectively, selectively, and at high levels to exact biological effects. HighlightsO_LICSF delivery of AAV9 targets >68% of lumbar spinal cord (SC) motor neurons C_LIO_LIUnilateral ICV yields high and consistent lumbar SC motor neuron transduction (>88%) C_LIO_LIUnilateral ICV results in the lowest number of peripheral viral copies C_LIO_LIICV targets significantly more astrocytes, particularly in cortex C_LIO_LIBilateral ICV leads to significantly higher viral copies in the liver C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/662642v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bd83a5org.highwire.dtl.DTLVardef@df63aorg.highwire.dtl.DTLVardef@a09142org.highwire.dtl.DTLVardef@1a0887a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Mole, A. J., Sander, C. F., Parmar, A. R., Williams, A. J., Azzouz, M., Hautbergue, G. M., Shaw, P. J., Ferraiuolo, L., Mead, R. J.. 2025-07-03. Comparison of AAV9-driven motor neuron transduction following different CNS-directed delivery methods in mice. https://doi.org/10.1101/2025.07.01.662642

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