Adeno-associated viruses (AAVs) have found extensive use as carriers for transferring genes to the nervous system. This enables activities such as gene expression and knockdown, gene editing, circuit modulation, in vivo imaging, disease model development, and evaluating potential therapies for neurological diseases. AAVs are an excellent choice for these applications because they provide safe, long-term expression in the nervous system. Typically, researchers achieve this by locally injecting AAVs into the adult brain, bypassing the blood-brain barrier, and restricting transgene expression to specific regions.
Targeted Injections for Peripheral Neurons
Targeted AAV injections have also proven useful for delivering genes to peripheral neurons. Researchers have employed this method to test strategies for treating chronic pain and to trace, monitor, and modulate specific subpopulations of vagal neurons. However, accessing certain peripheral neuron populations via surgery, such as dorsal root ganglia (DRG), nodose ganglia, sympathetic chain ganglia, and cardiac ganglia, can be challenging. In addition, widely distributed populations like the enteric nervous system present limitations when it comes to genetic manipulation. Similarly, in the central nervous system (CNS), single localized injections may not suffice for studying circuits in larger species or testing gene therapies for diseases involving the entire nervous system or widely distributed cell populations (such as Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, Alzheimer’s, spinal muscular atrophy, Friedreich’s ataxia, and numerous lysosomal storage diseases).
Systemic Delivery as an Alternative
Systemic AAV delivery offers a non-invasive alternative for delivering genes to the nervous system as a whole. However, the high viral load required and relatively low transduction efficiency have hindered widespread adoption of this method. Nevertheless, several groups have developed AAVs capable of enhancing gene transfer to the CNS after intravenous delivery. For example, the AAV-AS capsid, which incorporates a polyalanine N-terminal extension to the AAV9.4719 VP2 capsid protein, shows improved neuronal transduction, particularly in the striatum, making it a potential candidate for treating Huntington’s disease. Similarly, the AAV-BR1 capsid, based on AAV2, may prove useful for applications requiring more efficient and selective transduction of brain endothelial cells. Recently, employing a cell type-specific capsid selection method called CREATE (Cre REcombinase-based AAV Targeted Evolution), researchers discovered AAV-PHP.B. This capsid transduces the majority of neurons and astrocytes across various regions of the adult mouse brain and spinal cord after intravenous injection. However, the efficiency of AAV-PHP.B necessitates a substantial dose of vector, such as 1 × 1012 vg per adult mouse or higher.
Evolution of AAV-PHP.B
Using CREATE, researchers further evolved the AAV-PHP.B capsid to achieve more efficient neuronal transduction throughout the adult mouse brain and spinal cord. The result is a novel enhanced variant called AAV-PHP.eB, which lowers the required viral load to transduce the majority of CNS neurons. Additionally, researchers characterized a second capsid variant, AAV-PHP.S, which exhibits improved tropism towards peripheral neurons, including those in the DRG, cardiac ganglia, and enteric nervous system.
AAVs for Neuronal Connectivity Studies
AAVs are also widely employed for studying the anatomical connectivity and morphology of neurons as a whole. Furthermore, they play a crucial role in multi-viral strategies that trace the relationships between bulk inputs and outputs. At the single-cell level, researchers have developed AAV-based multicolor labeling systems to enhance tracing efforts. However, challenges persist in terms of controlling labeling density and achieving uniform color diversity. To address these issues, a two-component viral vector system has been developed to stochastically label cells with a wide range of hues while independently controlling the fraction of labeled cells. Moreover, by utilizing the newly reported capsids, researchers have successfully expressed various fluorescent reporters under different cell type-specific promoters. This supports the potential use of these vectors for population-wide genetic manipulations of the nervous system in Cre transgenic or wild-type mice.
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