Advances in AAV technology for delivering genetically encoded cargo to the nonhuman primate nervous system.

Modern neuroscience approaches including optogenetics, calcium imaging, and other genetic manipulations have facilitated our ability to dissect specific circuits in rodent models to study their role in neurological disease. These approaches regularly use viral vectors to deliver genetic cargo (e.g., opsins) to specific tissues and genetically-engineered rodents to achieve cell-type specificity. However, the translatability of these rodent models, cross-species validation of identified targets, and translational efficacy of potential therapeutics in larger animal models like nonhuman primates remains difficult due to the lack of efficient primate viral vectors. A refined understanding of the nonhuman primate nervous system promises to deliver insights that can guide the development of treatments for neurological and neurodegenerative diseases. Here, we outline recent advances in the development of adeno-associated viral vectors for optimized use in nonhuman primates. These tools promise to help open new avenues for study in translational neuroscience and further our understanding of the primate brain.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

Spatial transcriptomics for profiling the tropism of viral vectors in tissues.

A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Carbon monofilament electrodes for unit recording and functional MRI in same subjects.

Extracellular electrophysiology and functional MRI are complementary techniques that provide information about cellular and network-level neural activity, respectively. However, electrodes for electrophysiology are typically made from metals, which cause significant susceptibility artifacts on MR images. Previous work has demonstrated that insulated carbon fiber bundle electrodes reduce the volume of magnetic susceptibility artifacts and can be used to record local field potentials (LFP), but the relatively large diameter of the probes make them unsuitable for multi- and single-unit recordings. Although single carbon fiber electrodes have recently been used to record single-unit activity, these probes require modifications in order to aid insertion and the use of these probes in fMRI has yet to be validated. Therefore, there is a need for a single-carbon fiber electrode design that (1) minimizes the volume of the susceptibility artifact, (2) can record from a wide frequency band that includes LFP and multi- and single-unit recording, and (3) is practical to insert without additional modifications. Here, we demonstrate that carbon-fiber electrodes made from single carbon monofilaments (35 µm in diameter) meet all of these criteria. Carbon monofilament electrodes modified with the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) have lower impedances and higher signal-to-noise ratio recordings than platinum-iridium electrodes, a current gold standard for chronic single-unit recording. Furthermore, these probes distort a significantly smaller volume of voxels compared to tungsten and platinum-iridium electrodes in agarose phantom and in vivo MR images, leading to higher contrast-to-noise ratio in regions proximal to the electrode implantation site during fMRI. Collectively, this work establishes that carbon monofilaments are a practical choice for combined electrophysiology-fMRI experiments.

MRI compatible optrodes for simultaneous LFP and optogenetic fMRI investigation of seizure-like afterdischarges.

In preclinical studies, implanted electrodes can cause severe degradation of MRI images and hence are seldom used for chronic studies employing functional magnetic resonance imaging. In this study, we developed carbon fiber optrodes (optical fiber and electrode hybrid devices), which can be utilised in chronic longitudinal studies aiming to take advantage of emerging optogenetic technologies, and compared them with the more widely used tungsten optrodes. We find that optrodes constructed using small diameter (~130 µm) carbon fiber electrodes cause significantly reduced artifact on functional MRI images compared to those made with 50 µm diameter tungsten wire and at the same time the carbon electrodes have lower impedance, which leads to higher quality LFP recordings. In order to validate this approach, we use these devices to study optogenetically-induced seizure-like afterdischarges in rats sedated with dexmedetomidine and compare these to sub (seizure) threshold stimulations in the same animals. The results indicate that seizure-like afterdischarges involve several extrahippocampal brain regions that are not recruited by subthreshold optogenetic stimulation of the hippocampus at 20 Hz. Subthreshold stimulation led to activation of the entire ipsilateral hippocampus and septum, whereas afterdischarges additionally produced activations in the contralateral hippocampal formation, neocortex, cerebellum, nucleus accumbens, and thalamus. Although we demonstrate just one application, given the ease of fabrication, we anticipate that carbon fiber optrodes could be utilised in a variety of studies that could benefit from longitudinal optogenetic functional magnetic resonance imaging.

The mid-fusiform sulcus: a landmark identifying both cytoarchitectonic and functional divisions of human ventral temporal cortex.

Human ventral temporal cortex (VTC) plays a pivotal role in high-level vision. An under-studied macroanatomical feature of VTC is the mid-fusiform sulcus (MFS), a shallow longitudinal sulcus separating the lateral and medial fusiform gyrus (FG). Here, we quantified the morphological features of the MFS in 69 subjects (ages 7-40), and investigated its relationship to both cytoarchitectonic and functional divisions of VTC with four main findings. First, despite being a minor sulcus, we found that the MFS is a stable macroanatomical structure present in all 138 hemispheres with morphological characteristics developed by age 7. Second, the MFS is the locus of a lateral-medial cytoarchitectonic transition within the posterior FG serving as the boundary between cytoarchitectonic regions FG1 and FG2. Third, the MFS predicts a lateral-medial functional transition in eccentricity bias representations in children, adolescents, and adults. Fourth, the anterior tip of the MFS predicts the location of a face-selective region, mFus-faces/FFA-2. These findings are the first to illustrate that a macroanatomical landmark identifies both cytoarchitectonic and functional divisions of high-level sensory cortex in humans and have important implications for understanding functional and structural organization in the human brain.