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Published April 28, 2020 | public
Journal Article

Targeting the Rodent Peripheral Nervous System Efficiently and with Greater Specificity through Intravenous Delivery of AAV Capsids Evolved by Multiplexed-CREATE


The peripheral nervous system (PNS) is critical in regulating end-organ function and feedback signal transmission to the central nervous system (CNS). The sensory and enteric nervous systems (SNS and ENS), key components of the PNS, are of increasing interest to both scientific and clinical communities. With the proper gene delivery vectors, SNS gene therapy could be applied to neurological disorders such as neuropathic pain whereas ENS gene therapy could address the gastrointestinal symptoms, including dysregulated digestion and transit, common to many disorders including Parkinson's. To achieve precise genetic manipulation within the PNS however, more efficient and targeted gene delivery vehicles are needed. Adeno-associated viruses (AAVs) have become the vector of choice for gene delivery in vivo but non-invasive systemic administration of natural serotypes targets the PNS with mixed efficiency and specificity. Attempts to circumvent these limitations by direct administration of AAV are often either surgically challenging (e.g. for dorsal root ganglia (DRG)) or have incomplete coverage (e.g. for ENS). In prior work we have shown that modification of the AAV9 capsid by directed evolution generates AAV variants that target the CNS or selected PNS regions by systemic delivery (Chan et al, 2017). To further explore the potential of targeting PNS with engineered-capsid, we utilized the advanced selection platform Multiplexed-CREATE (Kumar et al, Nature Methods, 2020), that builds upon our prior method CREATE (Deverman et al, Nature Biotechnology, 2016). M-CREATE is a high-confidence AAV selection platform that uses next generation sequencing (NGS) and Cre-transgenic mouse lines (e.g. neuronspecific) to perform positive selection in vivo on PNS areas of interest (including DRG, small and large intestine), and a post-hoc negative selection against off-targets (including liver). After 2 rounds of in vivo M-CREATE selection in C57BL/6J adult mice, we recovered several variants that were positively enriched in the PNS areas of interest compared to their parent capsid AAV9. Preliminary NGS analysis revealed two novel AAV variants: AAV-PNS1, with the greatest relative enrichment between the PNS and off-target tissues, and AAV-PNS2 with the greatest overall enrichment in the PNS compared to AAV9. In vivo validation in C57BL/6J adult mice by intravenous delivery of the variants packaging nuclear-localized EGFP under a strong ubiquitous CAG promoter (ssAAV-CAG-NLS-EGFP) showed enhanced EGFP expression in neurons of the nodose ganglia and DRG. Consistent with NGS enrichment data, AAV-PNS1 showed greater specificity to the nodose ganglia and DRG by exhibiting lower overall transduction in peripheral organs including liver (an otherwise strong AAV9 target), while AAV-PNS2 showed higher overall efficiency in targeting both the ENS and SNS. The preliminary outcomes described here demonstrate the potential of M-CREATE to identify designer AAVs with greater efficiency and specificity towards the PNS in adult mice and thereby enabling basic and pre-clinical research.

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© 2020 American Society of Gene & Cell Therapy. Available online 28 April 2020.

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