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1.
Front Immunol ; 12: 730825, 2021.
Article in English | MEDLINE | ID: mdl-34759919

ABSTRACT

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 high resolution. Using this platform, we have both 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. Transcriptomic analysis revealed that this neuronal bias is due mainly 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 transitory 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. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , RNA-Seq , Single-Cell Analysis , Transcription, Genetic , Viral Tropism , Animals , Astrocytes/metabolism , Astrocytes/virology , Databases, Genetic , Dependovirus/metabolism , Genetic Engineering , Genetic Vectors/metabolism , Host-Pathogen Interactions , In Situ Hybridization, Fluorescence , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Neurons/metabolism , Neurons/virology , Proof of Concept Study , Transduction, Genetic
2.
Sci Rep ; 10(1): 20540, 2020 11 25.
Article in English | MEDLINE | ID: mdl-33239690

ABSTRACT

An individual's immune system is driven by both genetic and environmental factors that vary over time. To better understand the temporal and inter-individual variability of gene expression within distinct immune cell types, we developed a platform that leverages multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to enable simplified, cost-effective profiling of the human immune system across people and time at single-cell resolution. Using the platform, we detect widespread differences in cell type-specific gene expression between subjects that are stable over multiple days.


Subject(s)
Blood/metabolism , Capillaries/metabolism , Gene Expression Profiling , Immunity/genetics , Single-Cell Analysis , Circadian Rhythm/genetics , Female , Gene Expression Regulation , Humans , Male
3.
Nat Commun ; 11(1): 2102, 2020 04 30.
Article in English | MEDLINE | ID: mdl-32355221

ABSTRACT

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.


Subject(s)
Brain/diagnostic imaging , Dependovirus/isolation & purification , Positron-Emission Tomography , Animals , Capsid , Chelating Agents/pharmacokinetics , Copper Radioisotopes/pharmacokinetics , Female , Genetic Vectors , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transduction, Genetic
4.
Nat Methods ; 17(5): 541-550, 2020 05.
Article in English | MEDLINE | ID: mdl-32313222

ABSTRACT

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood-brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.


Subject(s)
Brain/virology , Capsid Proteins/metabolism , Dependovirus/genetics , Gene Transfer Techniques , Genetic Engineering/methods , Genetic Vectors/genetics , Integrases/metabolism , Animals , Blood-Brain Barrier/metabolism , Capsid Proteins/genetics , Female , Genetic Therapy , Male , Mice , Mice, Inbred C57BL , Organ Specificity , Viral Tropism
5.
Front Syst Neurosci ; 12: 24, 2018.
Article in English | MEDLINE | ID: mdl-29915532

ABSTRACT

Sensory feedback is a critical aspect of motor control rehabilitation following paralysis or amputation. Current human studies have demonstrated the ability to deliver some of this sensory information via brain-machine interfaces, although further testing is needed to understand the stimulation parameters effect on sensation. Here, we report a systematic evaluation of somatosensory restoration in humans, using cortical stimulation with subdural mini-electrocorticography (mini-ECoG) grids. Nine epilepsy patients undergoing implantation of cortical electrodes for seizure localization were also implanted with a subdural 64-channel mini-ECoG grid over the hand area of the primary somatosensory cortex (S1). We mapped the somatotopic location and size of receptive fields evoked by stimulation of individual channels of the mini-ECoG grid. We determined the effects on perception by varying stimulus parameters of pulse width, current amplitude, and frequency. Finally, a target localization task was used to demonstrate the use of artificial sensation in a behavioral task. We found a replicable somatotopic representation of the hand on the mini-ECoG grid across most subjects during electrical stimulation. The stimulus-evoked sensations were usually of artificial quality, but in some cases were more natural and of a cutaneous or proprioceptive nature. Increases in pulse width, current strength and frequency generally produced similar quality sensations at the same somatotopic location, but with a perception of increased intensity. The subjects produced near perfect performance when using the evoked sensory information in target acquisition tasks. These findings indicate that electrical stimulation of somatosensory cortex through mini-ECoG grids has considerable potential for restoring useful sensation to patients with paralysis and amputation.

6.
Sci Transl Med ; 9(387)2017 04 26.
Article in English | MEDLINE | ID: mdl-28446689

ABSTRACT

Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed "Bone CLARITY," a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato-labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.


Subject(s)
Bone Marrow Cells/cytology , Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cells, Cultured , Mice , Mice, Transgenic , Osteoblasts/cytology , Osteogenesis/physiology , SOX9 Transcription Factor/metabolism , Stem Cells/metabolism
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