Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids

Flytzanis, Nicholas C.; Goeden, Nick; Goertsen, David; Cummins, Alexander; Pickel, James; Gradinaru, Viviana

doi:10.1101/2020.06.16.152975

Cited by 12 publications

(15 citation statements)

References 54 publications

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“…To produce viruses carrying in trans constructs, we followed established protocols for the production of rAAVs (Challis et al, 2019). In short, HEK293T cells were triple transfected using polyethylenimine (PEI) with three plasmids: pAAV (see Plasmids), pUCmini-iCAP-PHP.eB (Chan et al, 2017), pUCmini-iCAP-CAP-B10 (Flytzanis et al, 2020), or pUCmini-iCAP-PHP.V1 (Ravindra Kumar et al, 2020), and pHelper. After 120 h, virus was harvested and purified using an iodixanol gradient (Optiprep, Sigma).…”

Section: Viral Productionmentioning

confidence: 99%

“…Here, we introduce an experimental and bioinformatics workflow capable of profiling the viral tropism and response of multiple barcoded AAV variants in a single animal across numerous complex cell types by taking advantage of the transcriptomic resolution of scRNA-seq techniques (Figure 1 A). For this proof-of-concept study, we profile the tropism of previously-characterized AAV variants that emerged from directed evolution with the CREATE (AAV-PHP.B, AAV-PHP.eB) (Chan et al, 2017;Deverman et al, 2016) or M-CREATE (AAV-PHP.C1, AAV-PHP.C2, AAV-PHP.V1, AAV.CAP-B10) (Flytzanis et al, 2020;Ravindra Kumar et al, 2020) platforms. We selected the AAV variants based on their unique CNS tropism following intravenous injection.…”

Section: Introductionmentioning

confidence: 99%

“…AAV-PHP.C1 and AAV-PHP.C2 have both demonstrated enhanced bloodbrain barrier (BBB) crossing relative to AAV9 across two mouse strains (C57BL/6J and BALB/cJ). Finally, AAV.CAP-B10 is a recently-developed variant with a bias toward neurons compared to AAV-PHP.eB (Flytzanis et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Brown

Altermatt

Dobreva

et al. 2021

Preprint

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Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.

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Section: Viral Productionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Brown

Altermatt

Dobreva

et al. 2021

Preprint

Self Cite

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“…Continuously evolving viral vector genetic technologies provide a unique opportunity to manipulate specific projections and/or populations of brain cells. Researchers are developing viral vector technologies to optimize delivery across the blood brain barrier (Flytzanis et al, 2020), and to specifically infect specific populations of cells. For example, (Dimidschstein et al, 2016) developed a GABAergic-specific infection strategy by inserting the GABA-neuron-specific mDlx enhancer in an AAV.…”

Section: Fdg and Gene Therapymentioning

confidence: 99%

Combining Brain Perturbation and Neuroimaging in Non-human Primates

Klink¹,

jean-francois.aubry@espci.fr²,

Ferrera³

et al. 2020

Preprint

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Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the combination of brain perturbation methods and neural measurement techniques is inherently challenging, research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological lesions. Non-human primates have been indispensable as a neurobiological system that is highly similar to humans while simultaneously being more experimentally tractable, allowing visualization of the functional and structural impact of systematic brain perturbation. This review considers the state-of-the-art in non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference methods. Method-specific considerations from the research and development community are offered to facilitate research in this field and support further innovations. We conclude by identifying novel avenues for further research and innovation and by highlighting the clinical translational potential of the methods.

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“…Despite these limitations, the XINTRINSIC system developed in our study could provide data to guide subsequent electrophysiology recordings, two photon imaging, or perturbation experiments that require skull opening. It may yet prove powerful in concert with non-invasive viral gene delivery in primates 47 for potential through-skull calcium imaging 2, 3 as well as minimally invasive optogenetic perturbations 48 where targeting of causal manipulations could benefit from our non-invasive imaging approach. We provide open access to our imaging setup design and software code for straightforward duplication (https://x-song-x.github.io/XINTRINSIC/).…”

Section: Discussionmentioning

confidence: 99%

Functional maps of the primate cortex revealed by through-skull wide-field optical imaging

Song

Guo

et al. 2020

Preprint

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The primate cerebral cortex is organized into specialized areas representing different functional modalities (e.g., vision, audition, touch) and their associations along a continuous surface. The functional maps of these areas, however, are often investigated in a single modality at a time. Here, we developed and applied to awake primates a polarization-enhanced wide-field optical imaging method for measuring cortical hemodynamics through the intact skull. Adjacent somatosensory, auditory, and visual cortices were noninvasively localized and rapidly parcellated in awake marmosets (Callithrix jacchus), a primate model featuring a smooth cortex. Detailed somatotopy, tonotopy, and retinotopy were also mapped out on an individual-subject basis, with a new pure-tone-responsive tonotopic gradient discovered outside the auditory core. Moreover, the motion-sensitive extent surrounding the primate-specific MT/V5 and the location of a face-sensitive patch were both revealed with respect to retinotopy. This approach provides a powerful tool for mapping the functional landscape across modalities in a single non-human primate subject, and thus opens new opportunities for probing how primate cortical system is organized to enable real-world behaviors.

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Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids

Cited by 12 publications

References 54 publications

Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Combining Brain Perturbation and Neuroimaging in Non-human Primates

Functional maps of the primate cortex revealed by through-skull wide-field optical imaging

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