Nanobody-Functionalized AAV achieves Promoter-independent Neuronal Targeting in the CNS
- 1University of Minnesota
Abstract
Adeno-associated virus (AAV) vectors are widely used for gene delivery to the central nervous system, but natural capsid tropism is broad and cell-type restriction is typically imposed transcriptionally using promoters and enhancers that consume packaging capacity and often drive weak expression. Here, we engineer neuronal targeting directly into the AAV-DJ capsid by ablating its endogenous heparan sulfate proteoglycan (HSPG) affinity and genetically displaying a nanobody against the Group 1 metabotropic glutamate receptor mGluR5 within the VP1 subunit at a permissive capsid loop (T456), generating AAV-m5. Western blot confirmed incorporation of the nanobody-VP1 fusion into assembled capsids. In primary hippocampal neuron cultures, ablating HSPG binding abolished infectivity and nanobody display rescued transduction while restricting GFP expression almost exclusively to mGluR5-positive neurons. Heparin competition assays showed that, unlike wild-type AAV-DJ, AAV-m5 transduction was unaffected by exogenous heparin, confirming that entry occurs independently of HSPG binding. Packaged with a strong constitutive promoter (CAG), AAV-m5 achieved neuron-restricted expression comparable to or exceeding that of wild-type AAV-DJ driven by a neuron-specific promoter (hSyn) and produced negligible expression under an astrocyte-specific promoter (GFAP) despite promoter activity in glia, demonstrating that capsid-level targeting can substitute, or complement, transcriptional restriction. Following stereotactic injection into the mouse hippocampus, AAV-m5 achieved an eight-fold higher proportion of transduced neurons than wild-type AAV-DJ at equivalent titers. Delivery to Grm5-null hippocampus reduced both the intensity and the spatial extent of transduction, confirming that the broad hippocampal transduction achieved by AAV-m5 is mGluR5-dependent. Together, these results establish nanobody-functionalized AAV-DJ as a modular, single-component platform for precision CNS gene delivery that circumvents the packaging and expression trade-offs of promoter-based cell-type restriction, with potential for retargeting to additional CNS cell types and disease-relevant receptors.
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BibTeX
@misc{extross2026nanobodyfunctionalized,
title = {{Nanobody-Functionalized AAV achieves Promoter-independent Neuronal Targeting in the CNS}},
author = {A. Extross and E. M. Fernandez de Velasco and Y. He and K. Wickman and D. Schmidt},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.752817},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.752817v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Nanobody-Functionalized AAV achieves Promoter-independent Neuronal Targeting in the CNS
AU - A. Extross
AU - E. M. Fernandez de Velasco
AU - Y. He
AU - K. Wickman
AU - D. Schmidt
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.752817
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.752817v1
AB - Adeno-associated virus (AAV) vectors are widely used for gene delivery to the central nervous system, but natural capsid tropism is broad and cell-type restriction is typically imposed transcriptionally using promoters and enhancers that consume packaging capacity and often drive weak expression. Here, we engineer neuronal targeting directly into the AAV-DJ capsid by ablating its endogenous heparan sulfate proteoglycan (HSPG) affinity and genetically displaying a nanobody against the Group 1 metabotropic glutamate receptor mGluR5 within the VP1 subunit at a permissive capsid loop (T456), generating AAV-m5. Western blot confirmed incorporation of the nanobody-VP1 fusion into assembled capsids. In primary hippocampal neuron cultures, ablating HSPG binding abolished infectivity and nanobody display rescued transduction while restricting GFP expression almost exclusively to mGluR5-positive neurons. Heparin competition assays showed that, unlike wild-type AAV-DJ, AAV-m5 transduction was unaffected by exogenous heparin, confirming that entry occurs independently of HSPG binding. Packaged with a strong constitutive promoter (CAG), AAV-m5 achieved neuron-restricted expression comparable to or exceeding that of wild-type AAV-DJ driven by a neuron-specific promoter (hSyn) and produced negligible expression under an astrocyte-specific promoter (GFAP) despite promoter activity in glia, demonstrating that capsid-level targeting can substitute, or complement, transcriptional restriction. Following stereotactic injection into the mouse hippocampus, AAV-m5 achieved an eight-fold higher proportion of transduced neurons than wild-type AAV-DJ at equivalent titers. Delivery to Grm5-null hippocampus reduced both the intensity and the spatial extent of transduction, confirming that the broad hippocampal transduction achieved by AAV-m5 is mGluR5-dependent. Together, these results establish nanobody-functionalized AAV-DJ as a modular, single-component platform for precision CNS gene delivery that circumvents the packaging and expression trade-offs of promoter-based cell-type restriction, with potential for retargeting to additional CNS cell types and disease-relevant receptors.
ER -