Key result
Unilateral intra-cerebroventricular delivery of AAV9 in neonatal mice achieved the highest and most consistent lumbar spinal cord motor neuron transduction (89 ± 3%) compared to intra-cisterna magna delivery.
Why the study?
Systemic AAV9 delivery risks immunogenicity and off-target effects, and direct comparisons of CNS-directed cerebrospinal fluid delivery routes for targeting motor neurons in vivo remain limited.
Absolute Event Rate: 89% vs 68.4%
Unilateral intra-cerebroventricular delivery of AAV9 in neonatal mice yields the highest and most consistent motor neuron transduction in the lumbar spinal cord, informing optimal delivery routes for gene therapies targeting motor neuron diseases.
Should not alter clinical practice; leaves open AAV9 route optimization for motor neuron targeting in larger models.
Background Gene 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 method We 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. Results All 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 methods Whilst several routes of administration into cerebrospinal fluid exist, direct comparisons for targeting MNs in vivo remain limited. Conclusions Overall, 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. Highlights CSF delivery of AAV9 targets >68% of lumbar spinal cord (SC) motor neurons Unilateral ICV yields high and consistent lumbar SC motor neuron transduction (>88%) Unilateral ICV results in the lowest number of peripheral viral copies ICV targets significantly more astrocytes, particularly in cortex Bilateral ICV leads to significantly higher viral copies in the liver Graphical Abstract
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Mole et al. (2025) studied Motor neuron targeting (wild-type mice model) (n=18). AAV9-CMV-eGFP via unilateral intra-cerebroventricular (ICV) delivery vs. Intra-cisterna magna (ICM) delivery and vehicle control was evaluated on Percentage of transduced motor neurons (eGFP+/ChAT+) in the lumbar spinal cord. Unilateral intra-cerebroventricular delivery of AAV9 in neonatal mice achieved the highest and most consistent lumbar spinal cord motor neuron transduction (89 ± 3%) compared to intra-cisterna magna delivery.
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