Magnetically guided virus stamping for the targeted infection of single cells or groups of cells

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette—loaded with magnetic nanoparticles to which viral particles are bound—is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette—loaded with magnetic nanoparticles to which viral particles are bound—is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo

CRISPR-clear imaging of melanin-rich B16-derived solid tumors

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature

Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion, and neurite outgrowth using atomic force microscopy-based single-cell force spectroscopy

Teneurins are evolutionarily conserved transmembrane receptors that function as axon guidance and target selection molecules in the developing nervous system. How teneurins recognize each other, whether they establish neuronal adhesion, and which teneurin specific interactions guide neurons remains to be determined. To reveal insight into these pertinent questions we combine atomic force microscopy-based single-cell force spectroscopy with genetic engineering and quantify the interactions teneurins establish between animal cells. Using a combinatorial approach of deletions and swaps of teneurin-1 and teneurin-2 domains, we unravel that teneurins use their NHL (NCL-1, HT2A, and Lin-41) domain to select homophilic teneurins from adjacent cells. This homophilic recognition of teneurins initiates cell–cell adhesion that, dependent on the intracellular domain, strengthens over time. Neurite outgrowth assays show that establishing and strengthening of teneurin-mediated homophilic cell–cell adhesion is required to stop outgrowth. On the basis of the results, we introduce a molecular model of teneurin domains that specify cellular recognition, adhesion strengthening, and neuronal pathfinding. The combined force spectroscopy and genetic approach can be applied to quantitatively decipher the contribution of any neuronal receptor domain and more generally of a given cell surface receptor domain to cell–cell recognition and adhesion.ISSN:1530-6984ISSN:1530-699

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells

ISSN:1750-2799ISSN:1745-2481ISSN:1754-218

Deciphering Teneurin Domains That Facilitate Cellular Recognition, Cell–Cell Adhesion, and Neurite Outgrowth Using Atomic Force Microscopy-Based Single-Cell Force Spectroscopy

Teneurins are evolutionarily conserved transmembrane receptors that function as axon guidance and target selection molecules in the developing nervous system. How teneurins recognize each other, whether they establish neuronal adhesion, and which teneurin specific interactions guide neurons remains to be determined. To reveal insight into these pertinent questions we combine atomic force microscopy-based single-cell force spectroscopy with genetic engineering and quantify the interactions teneurins establish between animal cells. Using a combinatorial approach of deletions and swaps of teneurin-1 and teneurin-2 domains, we unravel that teneurins use their NHL (NCL-1, HT2A, and Lin-41) domain to select homophilic teneurins from adjacent cells. This homophilic recognition of teneurins initiates cell–cell adhesion that, dependent on the intracellular domain, strengthens over time. Neurite outgrowth assays show that establishing and strengthening of teneurin-mediated homophilic cell–cell adhesion is required to stop outgrowth. On the basis of the results, we introduce a molecular model of teneurin domains that specify cellular recognition, adhesion strengthening, and neuronal pathfinding. The combined force spectroscopy and genetic approach can be applied to quantitatively decipher the contribution of any neuronal receptor domain and more generally of a given cell surface receptor domain to cell–cell recognition and adhesion

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows