Elucidating brain transport pathways and cell type-dependent gene silencing of a durable lipid-siRNA conjugate administered into cerebrospinal fluid.

The clinical neurosciences are in the midst of a renaissance spurred by the development of new therapeutic modalities. Short interfering RNAs (siRNAs), in particular, are gaining interest for treating neurological diseases owing to their capacity to sustain inhibition of nearly any gene target. However, to be effective, siRNA therapies must achieve delivery and on-target gene silencing activity in specific sites and cells in the brain. To this end, we developed a lipid-siRNA conjugate (L2-siRNA) that transports effectively throughout the brain when injected into cerebrospinal fluid (CSF). We provide a detailed examination of regional bulk tissue gene silencing in mice, highlighting potent knockdown 5 months after a single injection without detectable toxicity. Intrathecal delivery of L2-siRNA in rats further illustrates effective transport and knockdown using a clinically relevant route of administration. Single-cell RNA sequencing was additionally performed in mice to generate an atlas of cell type-specific knockdown. Lastly, we benchmarked L2-siRNA gene silencing activity in different brain regions against antisense oligonucleotides, a related but different gene silencing modality. Collectively, this work examines properties of lipid-siRNA conjugates that facilitate CSF to brain delivery and supports L2-siRNA as a promising platform for silencing genes implicated in central nervous system disorders.