Engineering Vectors for Non-Invasive Gene Delivery to the Central Nervous System using Multiplexed-CREATE

Viruses are widely modified and used as gene delivery vectors for various applications in science and therapeutics. To this end, my thesis focuses on modifying the recombinant adeno-associated viral (rAAV) vectors that are identified as a safer choice for cargo delivery compared to other known viral vectors. They are widely used in the scientific communities, have seen promising outcomes in gene therapy clinical trials, and as of today have three products approved to use in humans. However, the natural repertoire of rAAVs have broad tropism when delivered systemically, and there is room for further improvement on the efficiency and specificity, especially for gene delivery in the central nervous system (CNS). The prior work done in Dr. Gradinaru lab addresses the issue by using a directed evolution approach called CREATE, Cre recombination-based AAV targeted evolution, to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). CREATE selects for functional lox-flipped viral DNA that crosses the blood-brain barrier (BBB) and successfully transduces a specific nerve cell-type expressing Cre, thereby applying a strong selection pressure. However, the method is limited by its ability to identify a handful of enriched variants, and may also be prone to false positives resulting from experimental biases. The effort to fully understand the selection landscape, and to select for capsids that are not just efficient towards a cell-type but also specific towards it, led to the development of Multiplexed-CREATE (M-CREATE). M-CREATE allows parallel positive selections across different cell-types of interest, enables post-hoc negative selections across off-targets using a next-generation sequencing (NGS) based capsid recovery, and retains the principles of Cre-dependent functional recovery from CREATE. The method has a synthetic library generation approach to minimize biases within selection rounds, a variant replicate feature to identify the signal versus noise within a biological system, and an analysis pipeline to group families of enriched variants based on amino acid motifs, all of which together increases the confidence in the outcome and the throughput from a single experiment. Selections across brain endothelial cells, neurons, and astrocytes yielded several AAV-PHP.B-like variants that broadly transduce the CNS, AAV-PHP.V variants that can efficiently transduce the vascular cells forming the BBB, a AAV-PHP.N variant that transduces neurons with greater specificity, and AAV-PHP.C variants that cross the BBB without murine strain specificity across tested strains. The AAV-PHP.C variants have different amino acid motifs compared to the AAV-PHP.Bs that have been previously shown to have limited CNS transduction across some mouse strains due to its interaction with the strain specific host cell surface receptor, ly6a, a homolog of which is not found in humans. (Hordeaux et al, 2018, Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) Therefore AAV-PHP.Cs offer some hope towards translation across other species. In summary, the M-CREATE methodology turns out to be a high-confidence, robust selection platform to yield several novel viral capsids for use in neuroscience and potential gene therapy related applications.</p

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

The mammalian microbiome has many important roles in health and disease1,2, and genetic engineering is enabling the development of microbial therapeutics and diagnostics3,4,5,6,7. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism8,9. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers10,11,12. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution13,14,15. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy16,17. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 μm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo

Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering

Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., and Achromobacter xylosoxidans, we demonstrate a spectrum of aggregation states: from mostly single cells (A. xylosoxidans), to medium-sized clusters (S. aureus), to a mixture of single cells and large aggregates (P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum

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

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

The mammalian microbiome has many important roles in health and disease1,2, and genetic engineering is enabling the development of microbial therapeutics and diagnostics3,4,5,6,7. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism8,9. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers10,11,12. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution13,14,15. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy16,17. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 μm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents

Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types

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