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Jonathan L. Kirschman

Bio: Jonathan L. Kirschman is an academic researcher from Georgia Institute of Technology. The author has contributed to research in topics: Interferometry & Tiltmeter. The author has an hindex of 12, co-authored 15 publications receiving 664 citations. Previous affiliations of Jonathan L. Kirschman include Lawrence Berkeley National Laboratory.

Papers
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Journal ArticleDOI
TL;DR: This work established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.
Abstract: DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

176 citations

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TL;DR: A new facility for microdiffraction strain measurements and microfluorescence mapping has been built at the advanced light source of the Lawrence Berkeley National Laboratory and allows a variety of experiments, which have in common the need of spatial resolution.
Abstract: A new facility for microdiffraction strain measurements and microfluorescence mapping has been built on beamline 12.3.2 at the advanced light source of the Lawrence Berkeley National Laboratory. This beamline benefits from the hard x-radiation generated by a 6 T superconducting bending magnet (superbend). This provides a hard x-ray spectrum from 5 to 22 keV and a flux within a 1 microm spot of approximately 5x10(9) photons/s (0.1% bandwidth at 8 keV). The radiation is relayed from the superbend source to a focus in the experimental hutch by a toroidal mirror. The focus spot is tailored by two pairs of adjustable slits, which serve as secondary source point. Inside the lead hutch, a pair of Kirkpatrick-Baez (KB) mirrors placed in a vacuum tank refocuses the secondary slit source onto the sample position. A new KB-bending mechanism with active temperature stabilization allows for more reproducible and stable mirror bending and thus mirror focusing. Focus spots around 1 microm are routinely achieved and allow a variety of experiments, which have in common the need of spatial resolution. The effective spatial resolution (approximately 0.2 microm) is limited by a convolution of beam size, scan-stage resolution, and stage stability. A four-bounce monochromator consisting of two channel-cut Si(111) crystals placed between the secondary source and KB-mirrors allows for easy changes between white-beam and monochromatic experiments while maintaining a fixed beam position. High resolution stage scans are performed while recording a fluorescence emission signal or an x-ray diffraction signal coming from either a monochromatic or a white focused beam. The former allows for elemental mapping, whereas the latter is used to produce two-dimensional maps of crystal-phases, -orientation, -texture, and -strain/stress. Typically achieved strain resolution is in the order of 5x10(-5) strain units. Accurate sample positioning in the x-ray focus spot is achieved with a commercial laser-triangulation unit. A Si-drift detector serves as a high-energy-resolution (approximately 150 eV full width at half maximum) fluorescence detector. Fluorescence scans can be collected in continuous scan mode with up to 300 pixels/s scan speed. A charge coupled device area detector is utilized as diffraction detector. Diffraction can be performed in reflecting or transmitting geometry. Diffraction data are processed using XMAS, an in-house written software package for Laue and monochromatic microdiffraction analysis.

172 citations

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TL;DR: A high-throughput method to measure how >100 nanoparticles delivered mRNA that was translated into functional protein in vivo and identify 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells.
Abstract: Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

164 citations

Journal ArticleDOI
TL;DR: The potential of a cell‐penetrating peptide containing the amphipathic RALA motif to deliver antigen‐encoding mRNA to the immune system to elicit potent cytolytic T cell responses against the antigenic mRNA cargo is described.
Abstract: To date, the mRNA delivery field has been heavily dominated by lipid-based systems. Reports on the use of nonlipid carriers for mRNA delivery in contrast are rare in the context of mRNA vaccination. This paper describes the potential of a cell-penetrating peptide containing the amphipathic RALA motif to deliver antigen-encoding mRNA to the immune system. RALA condenses mRNA into nanocomplexes that display acidic pH-dependent membrane disruptive properties. RALA mRNA nanocomplexes enable mRNA escape from endosomes and thereby allow expression of mRNA inside the dendritic cell cytosol. Strikingly, RALA mRNA nanocomplexes containing pseudouridine and 5-methylcytidine modified mRNA elicit potent cytolytic T cell responses against the antigenic mRNA cargo and show superior efficacy in doing so when compared to RALA mRNA nanocomplexes containing unmodified mRNA. RALA's unique sequence and structural organization are vital to act as mRNA vaccine vehicle, as arginine-rich peptide variants that lack the RALA motif show reduced mRNA complexation, impaired cellular uptake and lose the ability to transfect dendritic cells in vitro and to evoke T cell immunity in vivo.

93 citations

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TL;DR: The data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.
Abstract: The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.

82 citations


Cited by
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Journal ArticleDOI
TL;DR: This Review discusses key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clinical pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.
Abstract: The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA molecules can efficiently trigger RNAi silencing of specific genes, but their therapeutic use has faced numerous challenges involving safety and potency. However, August 2018 marked a new era for the field, with the US Food and Drug Administration approving patisiran, the first RNAi-based drug. In this Review, we discuss key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clinical pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.

779 citations

Journal Article
TL;DR: In this article, the authors investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K(+) depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC-poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene.
Abstract: We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K(+) depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC-poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene. Uptake of the complexes was assayed by fluorescence-activated cell sorting. Lipoplex internalization was inhibited by chlorpromazine and K(+) depletion but unaffected by filipin and genistein. In contrast, polyplex internalization was inhibited by all four inhibitors. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while polyplexes are taken up by two mechanisms, one involving caveolae and the other clathrin-coated pits. Transfection by lipoplexes was entirely abolished by blocking clathrin-mediated endocytosis, whereas inhibition of the caveolae pathway had no effect. By contrast, transfection mediated by polyplexes was completely blocked by genistein and filipin but was unaffected by inhibitors of clathrin-mediated endocytosis. Fluorescence colocalization studies with a lysosomal marker, AlexaFluor-dextran, revealed that polyplexes taken up by clathrin-mediated endocytosis are targeted to the lysosomal compartment for degradation, while the polyplexes internalized via caveolae escape this compartment, permitting efficient transfection.

692 citations

Journal ArticleDOI
TL;DR: Selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule is reported.
Abstract: CRISPR–Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues. The addition of selective organ targeting molecules to nanoparticles allows the specific targeting of extrahepatic tissues, enabling gene editing of distinct cell populations outside the liver.

643 citations

Journal ArticleDOI
TL;DR: The origin of DNA nanotechnology is reviewed, followed by summarizing state-of-the-art strategies for the construction of DNAnanostructures and drug payloads delivered by DNA nanovehicles and challenges and opportunities for DNA nanostructure-based drug delivery.
Abstract: Over the past decade, we have seen rapid advances in applying nanotechnology in biomedical areas including bioimaging, biodetection, and drug delivery. As an emerging field, DNA nanotechnology offers simple yet powerful design techniques for self-assembly of nanostructures with unique advantages and high potential in enhancing drug targeting and reducing drug toxicity. Various sequence programming and optimization approaches have been developed to design DNA nanostructures with precisely engineered, controllable size, shape, surface chemistry, and function. Potent anticancer drug molecules, including Doxorubicin and CpG oligonucleotides, have been successfully loaded on DNA nanostructures to increase their cell uptake efficiency. These advances have implicated the bright future of DNA nanotechnology-enabled nanomedicine. In this review, we begin with the origin of DNA nanotechnology, followed by summarizing state-of-the-art strategies for the construction of DNA nanostructures and drug payloads delivered ...

628 citations

Journal Article
TL;DR: It is found that two distinct types of blinking are possible: conventional (A-type) blinking due to charging and discharging of the nanocrystal core, in which lower photoluminescence intensities correlate with shorter photolumscence lifetimes; and a second sort (B-type), in which large changes in the emission intensity are not accompanied by significant changes in emission dynamics.
Abstract: Photoluminescence blinking—random switching between states of high (ON) and low (OFF) emissivities—is a universal property of molecular emitters found in dyes, polymers, biological molecules and artificial nanostructures such as nanocrystal quantum dots, carbon nanotubes and nanowires. For the past 15 years, colloidal nanocrystals have been used as a model system to study this phenomenon. The occurrence of OFF periods in nanocrystal emission has been commonly attributed to the presence of an additional charge, which leads to photoluminescence quenching by non-radiative recombination (the Auger mechanism). However, this ‘charging’ model was recently challenged in several reports. Here we report time-resolved photoluminescence studies of individual nanocrystal quantum dots performed while electrochemically controlling the degree of their charging, with the goal of clarifying the role of charging in blinking. We find that two distinct types of blinking are possible: conventional (A-type) blinking due to charging and discharging of the nanocrystal core, in which lower photoluminescence intensities correlate with shorter photoluminescence lifetimes; and a second sort (B-type), in which large changes in the emission intensity are not accompanied by significant changes in emission dynamics. We attribute B-type blinking to charge fluctuations in the electron-accepting surface sites. When unoccupied, these sites intercept ‘hot’ electrons before they relax into emitting core states. Both blinking mechanisms can be electrochemically controlled and completely suppressed by application of an appropriate potential.

590 citations