Patent Grant
12054764
The subject matter disclosed herein is generally directed to methods, devices, and reagents for high-throughput biological function screens for cellular and acellular samples.
Natural products are an important class of molecules due to their functions as antimicrobials, antifungals, immunosuppressants, and antitumor agents. However, their discovery remains a challenging problem. Progress has been made in identifying the capacity of microorganisms to produce these compounds, but the reality is that many environmental bacteria are difficult to work with under laboratory conditions. Since their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, and cancer therapy.
Antibiotics are, for the vast majority, low molecular weight (<1000 Da) secondary products of microbial, fungal or plant metabolism. Several lines of evidence suggest that these or similar molecules may have been around since the pre-biotic era and may have played an important role as modulators or effectors of the primeval RNA molecules from which “modern” biological structures like the ribosomes and riboswitches have evolved. Indeed, the fact that several antibiotics may have coevolved with RNA is suggested by the fact that they bind select RNA targets and that some of them, like the aminoglycosides, can equally influence ribosomal decoding and inhibit the second step of Group I T4 phage-derived td intron splicing, two activities possibly having a common origin in the “RNA World.”
Since their discovery and their first therapeutic applications, antibiotics can be credited with having saved millions of human lives. Nevertheless, after an initial period in which the detection, biological, pharmacological and clinical characterization as well as marketing of new antibiotics have flourished, the discovery of new anti-infective agents has slowed down considerably. This has resulted in a long “innovation gap” which extends from 1962, when quinolones and streptogramin were first applied in human therapy, until 2000, when oxazolinidones were introduced.
In one example embodiment, a method for screening biological functions of microscale biological systems comprises segregating each microscale biological system from a set of microscale biological systems to be screened into individual discrete volumes, the individual discrete volume comprising a first polymer. The first polymer is then forced or allowed to polymerize, depending on the polymer used, to form a set of polymerized beads that encapsulate an individual microscale biological system. The polymerized beads are then further encapsulated in a second droplet comprising a second polymer and one or more reporter elements. The reporter elements are configured to produce a readout upon detecting the absence or presence of a biological function to be screened. The second polymer is then forced or allowed to polymerize to form an outer capsule around each individual bead thereby forming a set of encapsulated beads. One or more biological functions of the double-encapsulated microscale biological system are then identified by detecting the readout of the one or more reporter elements.
The microscale biological systems comprise naturally occurring cells. The naturally occurring cells may be prokaryotic, eukaryotic, or a mixture thereof. The naturally occurring cells may be derived from clinical isolates or environmental samples. In certain example embodiments, the naturally occurring cells may be prokaryotic cells to be screened for antibiotic production. In certain other example embodiments, the microscale biological system may comprise engineered cells. The engineered cells may comprise one or more genetic perturbations. In certain example embodiments, the microscale biological systems may comprise cell free systems. The cell free systems may comprise one or more nucleic acid constructs encoding one or more gene expression products or gene expression regulator elements.
The polymer used to form the bead and outer capsule may be the same or different. In certain example embodiments the polymer used to form the bead, the outer capsule, or both is a hydrogel polymer. The hydrogel polymer may comprise a polysaccharide, a synthetic polymer, a natural polymer, and/or a block copolymer. The polysaccharide may comprise hydroxyethyl cellulose.
In certain example embodiments, the method may further comprise sorting the encapsulated gel beads based at least in part on the detected readout of the one or more reporter elements, isolating the sorted encapsulated beads identified as having one or more target biological functions. In certain example embodiments, one or more chemical or biological agents produced by the microscale biological system may be isolated and analyzed to determine one of more properties of said chemical or biological agents. The chemical or biological agents may be an expressed RNA, a protein, a lipid, a polysaccharide, a small molecule, a metabolite, or a combination thereof. In certain example embodiments, the method may further comprise introducing one or more capture agents for the one or more chemical or biological agents into the bead, outer capsule, or both to facilitate isolation of the chemical or biological agent.
In certain example embodiments, the method may further comprise sequencing the microscale biological systems. In order to identify the sequence of a microscale biological system as originating from a particular bead, the microscale biological system, or its transcripts, may be labeled with an origin-specific barcode prior to sequencing. In the context of cell free microscale biological systems comprising one or more nucleic acid constructs, sequencing may comprise sequencing of the one or more nucleic acid constructs. In the context of a cellular microscale biological system, sequencing may comprise sequencing a genome of the cell, RNA transcripts of the cell, or an entire transcriptome of the cell.
In certain example embodiments, determining a genotype for each cell comprises lysing the cell in each bead such that genomic DNA is retained in the polymerized bead, releasing the beads from the outer capsule and re-encapsulating the beads in a second outer capsule, the second outer capsule comprising genomic DNA amplification reagents. The beads are maintained under conditions sufficient for genomic DNA amplification. The beads are then released from the second capsule and re-encapsulated in a third capsule comprising tagmentation reagents to generate genomic fragments, the tagmentation reagents comprising transposomes loaded with sequencing adapters. The sequencing adapters may further comprise a unique origin specific barcode or unique combination of origin specific barcodes. After maintaining the encapsulated beads under conditions sufficient for tagmentation, the tagmented DNA is then isolated to prepare a DNA sequencing library comprising the genomic DNA fragments. The genomic DNA library is then sequenced to determine a genotype for each microscale biological system. In certain example embodiments, the DNA amplification reagents are multiple displacement amplification reagents (MDA). In certain example embodiments, the method may further comprise a DNA sequencing library amplification step prior to the sequencing step. In certain example embodiments, the DNA sequencing library amplification step comprises releasing each encapsulated bead into a separate individual discrete volume comprising DNA amplification reagent, breaking the bead to release the genomic DNA fragments labeled with sequencing adapters, and optionally origin-specific barcodes, and maintaining the separate individual discrete volumes under conditions sufficient to allow for DNA amplification. In certain example embodiments, the amplification step may further comprise addition of a second barcode to each genomic DNA fragment.
In certain example embodiments, the contents of the bead, the outer capsule, or both may be altered over the time-course of a given assay. In certain example embodiments, the contents are altered by contacting the double encapsulated microscale biological system with one or more reagents that are diffusible into the outer shell and/or bead. The one or more reagents may be used to sustain replication or growth of the microscale biological system, or determine an additional biological function of the microscale biological system. In certain other example embodiments, altering the contents may comprise releasing the beads from the first outer capsule and re-encapsulating the beads in an additional outer capsule. The process of releasing and re-encapsulating the beads to introduce additional agents may be repeated over multiple iterations as needed per assay design. In addition, the beads may be sorted, for example, based on the readout of a reporter element, between each iteration of release and re-encapsulation. In some embodiments, a reporter element described herein may produce an optically detectable signal. In one embodiment, the reporter element comprises magnetic-based separation, and may comprise labeling a biological molecule of interest with a magnetic particle and isolating the biological molecule of interest using the magnetic particles. In another embodiment, the biological molecule of interest is selected from the group consisting of a protein, a cell surface marker, and a nucleic acid, or combinations thereof.
In certain example embodiments the reporter element is a reporter cell and the readout comprises cell growth, cell death, changes in reporter cell gene expression, changes in reporter cell epigenetic modifications or chromatin structure, or changes in cell phenotype. In certain example embodiments, the readout may be expression of a detectable gene expression product, such as an mRNA or protein. In certain example embodiments, the detectable gene expression product is operatively coupled to a promoter that is inhibited or induced by one or more biological functions of the microscale biological system. In certain embodiments, the promoter may be a stress promoter. In certain example embodiments, the detectable gene expression product is a fluorescent protein.
These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of illustrated example embodiments.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, +1-5% or less, +/−1% or less, and +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
As used herein, a “biological sample” may contain whole cells and/or live cells and/or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.
The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
Embodiments disclosed herein provide methods for multiplex screening of microscale biological systems for one or more biological functions. As used herein a microscale biological system refers to a cellular or acellular system capable of expressing one or more biomolecules exhibiting a biological function. In certain example embodiments, a microscale biological system may be a cell or population of cells. In certain example embodiments, a microscale biological system refers to an acellular system. In certain example embodiments, an acellular system may comprise one or more nucleic acid constructs encoding various nucleic acids and/or proteins. In general, each microscale biological system to be screened is placed within a series of nested individual discrete volumes, a first volume comprising the microscale biological system, and a second volume comprising one or more reporter elements for measuring a biological function of the microscale biological system.
As used herein, a “discrete volume” or “discrete space” may refer to a container, receptacle, or other defined volume or space that can be defined by properties that prevent and/or inhibit migration of molecules, particles and/or nucleic acid containing specimens. For example, a discrete volume or space may be defined by physical properties such as walls of a discrete well, tube, or surface of a droplet which may be impermeable or semipermeable. The discrete volume or space may also refer to a reaction unit or region within a larger volume, where that region is not defined by walls but rather is defined spatially by location within the larger volume. For example, the discrete volume or space may be chemically defined, diffusion rate limited defined, electro-magnetically defined, or optically defined, or any combination thereof. By “diffusion rate limited” is meant volumes or spaces that are only accessible to certain species or reactions because diffusion constraints that would effectively limit the migration of a particular molecule, particle, or nucleic acid containing specimen from one discrete volume to another. By “chemically defined” is meant a volume or space where only certain molecules, particles, or nucleic acid containing specimens can exist because of their chemical or molecular properties. For example, certain gel beads may exclude certain molecules, particles, or nucleic acid containing specimens from entering the beads but not others by surface charge, matrix size, or other physical property of the gel bead. By “electro-magnetically defined” is meant volumes or spaces where the electro-magnetic properties of certain molecules, particles, or cells may be used to define certain volumes or spaces. For example, by capturing magnetic particles within a magnetic field or directly by magnets. By “optically defined” is meant volumes or spaces that may be defined by illuminating the volume or space with visible, ultraviolet, infrared, or other wavelengths of light such that only target molecules within the defined space or volume are detected.
In certain example embodiments, each individual microscale biological system may be identified by assigning each individual microscale biological system a unique barcode. This may be achieved by assigning a unique barcode to each individual discrete volume used in a screen and comprising a different microscale biological system. The individual discrete volumes may be pre-labeled with the unique barcode prior to loading each individual discrete volume with a microscale biological system, or the barcode may be introduced concurrently when loading the microscale biological systems. For example, small hydrogel beads labeled with a barcode may be added to each individual discrete volume. In certain example embodiments, a second barcode may be similarly used to identify the one or more reporter elements in an individual discrete volume. The barcode may be a nucleic acid-based barcode. The barcode may be an optical barcode.
A nucleic-acid based barcode is a short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier for an associated molecule, such as a target molecule and/or target nucleic acid. A nucleic acid barcode can have a length of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides, and can be in single- or double-stranded form. One or more nucleic acid barcodes can be attached, or “tagged,” to a target molecule and/or target nucleic acid. This attachment can be direct (for example, covalent or non-covalent binding of the barcode to the target molecule) or indirect (for example, via an additional molecule, for example, a specific binding agent, such as an antibody (or other protein) or a barcode receiving adaptor (or other nucleic acid molecule). Target molecule and/or target nucleic acids can be labeled with multiple nucleic acid barcodes in combinatorial fashion, such as a nucleic acid barcode concatemer. Typically, a nucleic acid barcode is used to identify a target molecule and/or target nucleic acids as being from a particular compartment (for example a discrete volume), having a particular physical property (for example, affinity, length, sequence, etc.), or having been subject to certain treatment conditions. Target molecule and/or target nucleic acid can be associated with multiple nucleic acid barcodes to provide information about all of these features (and more). Methods of generating nucleic acid-barcodes are disclosed, for example, in International Patent Application Publication No. WO/2014/047561.
Optically detectable barcodes are barcodes that can be detected with light or fluorescence microscopy. In certain example embodiments, the optical barcodes may comprise a sub-set of fluorophores or quantum dots of distinguishable colors from a set of defined colors. In certain example embodiments, beads are labeled with different ratios of dyes to form the set of defined colors from which the optical barcodes may be derived. For example, the beads may be polystyrene beads labeled with biotin conjugated dyes. Alternatively, the optical barcodes may be derived using a combination of optically detectable objects. For example, an optical barcode may be defined from a set of objects that can vary in size, shape, color, or any combination thereof that is distinguishable by light or fluorescence microscopy.
In certain example embodiments, microscale biological systems are first encapsulated in a bead by causing droplets to form around each individual microscale biological system. The fluid used to form the droplet comprises one or more polymers or polymer precursors. After successful droplet formation, the one or more polymers are then polymerized to form solid or semi-solid beads encapsulating the microscale biological system. For example, the polymers may be maintained at an elevated temperature, or under other physical conditions, that will maintain the polymer composition in a liquid or molten state. After droplet formation, the physical condition may be removed and/or changed such that polymerization of the polymer may occur. In some example embodiments, polymerization may be induced by addition of a polymerizing agent that triggers polymerization of polymer precursors, such as a free-radical generating agent. The bead may be further encapsulated in a second polymer in the same way. The polymer used to form the outer capsule may be the same or different from the polymer used to form the inner bead. In certain example embodiments, the inner bead may range in size from (diameter) 20 μm to 60 μm, and the outer capsule may range in size from 60 μm to 100 μm diameter.
These encapsulated beads may then be screened for one or more biological functions, at least in part, by detecting the readout of the one or more reporter elements. In certain example embodiments, the biological function may be determined by observation of the encapsulated beads using light or fluorescent microscopy, or similar imaging technique, in place of or in addition to other readouts of the one or more reporter elements.
In certain example embodiments, the methods may further comprise sorting the encapsulated beads based, at least in part, on the detected readout of the one or more reporter elements and analyzing the sorted encapsulated beads identified as having the one or more desired biological functions. The method may further comprise isolating and/or purifying said biological agents and optionally further characterizing the isolated biological agents. In certain example embodiments, the method may comprise sequencing of the microscale biological system and/or expression products of the microscale biological system.
In certain example embodiments, the microscale biological systems encapsulation may be carried out, in whole or in part, on a microfluidic device.
Microfluidic devices disclosed herein may be silicone-based chips and may be fabricated using a variety of techniques, including, but not limited to, hot embossing, molding of elastomers, injection molding, LIGA, soft lithography, silicon fabrication and related thin film processing techniques. Suitable materials for fabricating the microfluidic devices include, but are not limited to, cyclic olefin copolymer (COC), polycarbonate, poly(dimethylsiloxane) (PDMS), and poly(methylacrylate) (PMMA). In one embodiment, soft lithography in PDMS may be used to prepare the microfluidic devices. For example, a mold may be made using photolithography which defines the location of the one or more flow channels and the array of microwells. The substrate material is poured into a mold and allowed to set to create a stamp. The stamp is then sealed to a solid support such as, but not limited to, glass.
Due to the hydrophobic nature of some polymers, such as PDMS, which absorbs some proteins and may inhibit certain biological processes, a passivating agent may be necessary (Schoffner et al. Nucleic Acids Research, 1996, 24:375-379). Suitable passivating agents are known in the art and include, but are not limited to, silanes, parylene, n-Dodecyl-b-D-maltoside (DDM), pluronic, Tween-20, other similar surfactants, polyethylene glycol (PEG), albumin, collagen, and other similar proteins and peptides.
The microfluidic devices may further comprise inlet and outlet ports, or openings, which in turn may be connected to valves, tubes, channels, chambers, and syringes and/or pumps for the introduction and extraction of fluids into and from the microfluidic device. The microfluidic devices may be connected to fluid flow actuators that allow directional movement of fluids within the microfluidic device. Example actuators include, but are not limited to, e.g., syringe pumps, mechanically actuated recirculating pumps, electroosmotic pumps, bulbs, bellows, diaphragms, or bubbles intended to force movement of fluids.
Polymers
The polymers used in the present invention to form the bead and the outer capsule may be the same or different polymers. In certain example embodiments, the polymers are different. In certain example embodiments, the polymer may be a hydrogel polymer. Hydrogel polymers may comprise polysaccharides, synthetic polymers, natural polymer, block copolymers or a combination thereof. Example polysaccharides include hydroxyethyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, agarose, agar, hydroxyethyl agarose, galactomannan, dextran, or a combination thereof. Example synthetic hydrogels include poly(hydroxyalkyl methacrylates), poly(acrylamide, poly(methacrylamide), poly(N-vinyl-2-pyrrolidone), polyelectrolyte complexes, poly(vinyl alcohol), polyethylene glycol, acylamide matrigel, alginate, functionalized alginate, PAMAM, and derivatives thereof.
In certain example embodiments the first polymer is agarose. In certain other example embodiments, the first and second polymer is agarose. In certain other example embodiments, the first polymer is agarose and the second polymer is polyethylene glycol (PEG).
Microscale Biological Systems
Microscale biological systems that may be screened in the present invention include cell and acellular systems. The cells may be eukaryotic cells, prokaryotic cells, or plant cells. In certain example embodiments, the cells may be naturally occurring. In certain example embodiments, the cells may be isolated from clinical isolates. Any suitably clinical isolate may be used in the present invention including, but are not limited to, a biopsy or other tissue sample, a blood sample, a saliva sample, and a urine sample. In certain example environments the cells may be isolated from an environmental sample. Environmental samples include, but are not limited to household/commercial/industrial surfaces (metal, wood, plastic), soil samples, and water samples (fresh and saline).
In certain example embodiments, the cells may be engineered to comprise one or more genetic perturbations. For example, the sample set could comprise a set of cells, each cell carrying a different genetic perturbation or combination of genetic perturbations to be screened. Genetic perturbations may include gene knock-outs, gene knock-ins, transpositions, inversions, and/or one or more nucleotide insertions, deletions, or substitutions. The set could then be screened using the embodiments disclosed herein to assess an impact the one or more genetic perturbations has on one or more biological functions, to determine a mechanism action, or to identify components of a cell signaling pathway.
In certain example embodiments, the cells are microbial cells. In certain example embodiments, the microbial cells are bacterial cells. In certain other example embodiments, the microbial cells are fungal cells. Any type of microbial cell may be screened using the present assay. For example, the bacterial cells may be screened for production of an antibiotic. In certain example embodiments, the cells are bacterial cells isolated from soil samples. In certain other example embodiments, the cells are bacterial cells isolated from soil samples and screened for antibiotic production. Bacterial genera or species that may be useful in accordance with the invention include, but are not limited to, Streptomyces, Paenibacillus, Clostridium. Eukaryotes that may be useful with the invention include, but are not limited to, Aspergillus, Penicillium, Cephalosporium, and Tolypocladium.
In certain example embodiments, the microscale biological system is an acellular system. An acellular sample may include, but is not limited to, a cellular extract, a cellular fraction or sub-fraction (including nuclear or cytoplasmic fractions or isolated organelles), a cell-free system, and/or an in vitro solution, including but not limited to, an aliquot from an assay screen, probe or experimental solution or reaction. Cell-free systems may comprise a nucleic acid construct or set of nucleic acid constructs encoding a set of gene expression products. Each nucleic acid construct may comprise a different combination of regulatory elements and genes and/or ordering of regulatory elements and genes. Hence, the present invention may be used to screen a set of synthetic biology constructs for one or more target biological functions or to select an optimal construct architecture.
Biological Functions
The embodiments disclosed herein may be used to screen a wide array of biological functions. In certain example embodiments, the microscale biological systems may be screened for a particular type or particular class of biological agent. Example biological agents include expression of certain RNA molecules, production of certain proteins, lipids, polysaccharides, metabolites, small molecules or a combination thereof. In certain other example embodiments, the microscale biological systems may be screened for an ability to impact a biological function of a target cell type. For example, the microscale biological systems may be screened for the ability to induce or inhibit expression of certain expression products in a target cell, to induce phenotypic changes (changes in cell membrane or cell wall morphologies, axonal growth, changes in nuclear or organelle morphologies, changes in cytoskeletal arrangement and/or vesicle trafficking), the ability to induce or inhibit target cell growth, to induce target cell death, the ability to induce neoplastic transformation of a target cell, and/or the ability to induce migration of a target cell.
It should be further understood, the screens for particular biological functions may be done in the further context variable physical parameters, such as different temperatures, atmospheric pressures, pH, growth media conditions, atmospheric CO2 concentrations, atmospheric O2 concentrations, and/or sheer stresses. The microscale biological systems may be exposed to such parameters either before or after encapsulation. Exposure to the above physical parameters may be done on the same microfluidic device used to generate and encapsulate the microscale biological systems, or a separate microfluidic device configured to vary such parameters, or in other discrete volumes outside of a microfluidic device.
In addition, it is possible to alter contents of the capsule or bead to which the microscale biological systems are exposed via diffusion. For example, the contents of the capsule or the bead may be altered by contacting the encapsulated beads with one or more reagents to be diffused into the outer capsule and/or bead. As understood by one of ordinary skill in the art, the type of reagents to be diffused into the outer capsule or bead will further determine the type of polymer that is used in the formation of the outer capsule and bead to ensure the necessary degree of porosity and/or permeability. Example reagents for diffusion into the capsule or bead may include, but are not limited to, reagents necessary to sustain replication or growth of the microscale biological system in the bead, or to sustain replication or growth of a reporter cell (described further below) residing in the outer capsule, reagents to inhibit or induce expression by the microscale biological system, or reagents to detect the presence of target biological agents such as capture agents. Accordingly, the encapsulated beads may be exposed to a carrier comprising the reagent to be diffused into the beads. For example, the encapsulated beads may be maintained in culture medium necessary to sustain growth and expression of the microscale biological system, the make-up of the culture medium being changed or replenished over time. Diffusion of reagents into the encapsulated beads may be done on a microfluidic device or in another suitable discrete volume.
Reporter Elements
As can be appreciated by one of ordinary skill in the art, the biological function to be screened will in part determine the reporter system used. The reporter system may comprise one or more reporter elements that generate a detectable readout. The reporter system may be a reagent or set of reagents that generate a detectable readout upon interaction with a target protein, nucleic acid, metabolite, lipid, polysaccharide, a small molecule, or other indicator of a desired biological function produced by a microscale biological system.
In certain example embodiments, the reporter system produces a detectable signal upon detection of a particular biological function. The detectable signal may be a colorimetric signal, such as a change in color by a pH indicator.
In certain example embodiments, the reporter element may comprise one or more detectable labels. Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. A wide variety of appropriate detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as digoxigenin, ß-galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.
The detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
Examples of the labeling substance which may be employed include labeling substances known to those skilled in the art, such as fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, and radioactive substances. Specific examples include radioisotopes (e.g., 32P, 14C, 125I, 3H, and 131I), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin, and ruthenium. In the case where biotin is employed as a labeling substance, preferably, after addition of a biotin-labeled antibody, streptavidin bound to an enzyme (e.g., peroxidase) is further added.
In certain example embodiments, the detectable label is a fluorescent label. Examples of fluorescent labels include, but are not limited to, Atto dyes, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes; cyanosine; 4′,6-diamidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives; eosin, eosin isothiocyanate, erythrosin and derivatives; erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
The fluorescent label may be a fluorescent protein, such as blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, red fluorescent protein, yellow fluorescent protein or any photoconvertible protein. Colorimetric labeling, bioluminescent labeling and/or chemiluminescent labeling may further accomplish labeling. Labeling further may include energy transfer between molecules in the hybridization complex by perturbation analysis, quenching, or electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes. The fluorescent label may be a perylene or a terrylene. In the alternative, the fluorescent label may be a fluorescent bar code.
In an advantageous embodiment, the label may be light sensitive, wherein the label is light-activated and/or light cleaves the one or more linkers to release the molecular cargo. The light-activated molecular cargo may be a major light-harvesting complex (LHCII). In another embodiment, the fluorescent label may induce free radical formation.
In certain other example embodiments, the reporter system may comprise a reporter microscale biological system. A reporter microscale biological system may be a cell or acellular system designed to detect one or more biological functions of the test microscale biological system. For reporter microscale biological systems, the readout may be a phenotypic assessment of the reporter microscale biological system, or detection of one or more reporter elements produced by the reporter microscale biological system. The phenotypic readout may be done using standard cell imaging techniques known in the art. Given the high throughput nature of the method disclosed herein, the cell imaging techniques may comprise high throughput imaging techniques known in the art. Conversely, detection of one or more reporter elements may be done in the same fashion described above for detecting the presence of an indicator of biological function directly from the test microscale biological system itself.
In certain example embodiments, the reporter system detects one or more nucleic acids produced by the test microscale biological systems. Accordingly, the reporter system may comprise one or more reagents that can bind to and produce a detectable signal upon binding to the one or more nucleic acids produced by the test microscale biological system. In certain example embodiments, the reporter system may comprise nucleic acids that hybridize the one or more nucleic acids produced by the microscale biological system. The one or more nucleic acids may be DNA or RNA. In certain example embodiments, the reporter elements bind to one or more mRNAs produced by the microscale biological systems. In certain example embodiments, the nucleic acid reporter elements may undergo a conformational shift upon binding the one or more target nucleic acids that results in emission of a detectable signal. For example, the conformational shift may result in removal of a quencher molecule that otherwise masks the detectable signal until binding to the one or more target nucleic acids. Alternatively, the reporter element may comprise two nucleic acids each labeled with a detectable label that bind to adjacent regions on the one or more target nucleic acids, and wherein binding to the adjacent regions produces emission of a shifted signal from the detectable label, such as that produced through fluorescence resonance energy transfer (FRET).
In certain example embodiments, the reporter system detects a biological molecule produced by the test microscale biological system, including, but not limited to, a protein or peptide, a lipid, a polysaccharide, a metabolite, a small molecule, or any combinations thereof. In some embodiments, a reporter system as described herein may detect more than one such biological molecule as described herein, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more biological molecules. The limits of the reporter system of the present invention may be determined by one of skill in the art as appropriate based on the particular application.
In certain example embodiments, the reporter system may comprise a reporter cell. The readout may comprise detecting a change in gene expression, epigenetic modification or chromatin structure of a reporter cell. In certain example embodiments, the readout may comprise detecting phenotypic changes in the reporter cell. Phenotypic changes may include changes in reporter cell morphology, including changes in cell membrane, nuclear, organelle, and/or cytoskeletal morphological changes. Phenotypic changes may also include detecting cell death, neoplastic transformation, or migration of the reporter cell. Phenotypic changes may be assessed using cell imaging techniques known in the art, including high throughput imaging techniques such as those disclosed in Blasi et al. (Nature Communications 2016, DOI:10.1038/ncomms10256). Changes in chromatin structure may be detected using nucleic acid proximity detection assays such as Hi-C described in International Patent Application Publication No. WO 2016/089920.
In certain example embodiments the reporter system comprises a reporter cell that is modified so that the readout is expression of a detectable gene expression product. The modification may comprise insertion of a gene expression module into the genome of the reporter cell or insertion of a nucleic acid construct, such as a plasmid, into the reporter cell. In certain example embodiments the gene expression product is operatively connected to a promoter that is inhibited or induced by one or more biological functions of the microscale biological system. In some embodiments, a promoter useful with the invention may be a response promoter. For example, a response promoter may be identified through an RNAseq pathway that is activated upon detection of an extracellular signal or condition that elicits a response in the cell. The signal may convert one or more promoters of genes in a particular pathway. In certain other example embodiments, the promoter may be a stress promoter, such as including, but not limited to, DNA damage response, desiccation response, starvation response, replication checkpoints, cell cycle checkpoints, unfolded protein response, heat/cold, temperature shock response, salt stress, etc. In certain example embodiments, the gene expression product may be a protein. In certain example embodiments, the protein may be a fluorescent protein such as GFP, YFP, RFP, or similar protein. An example reporter cell system is shown in
In certain example embodiments, the gene expression product may be an mRNA transcript. The mRNA transcript may comprise a defined target sequence recognized by a corresponding probe, wherein the probe is labeled with a detectable label. The reporter cell may comprise different promoters controlling the expression of different mRNA transcript, each separate mRNA transcript comprising a unique sequence recognized by a different probe comprising a different label and thereby able to screen for multiple biological functions at one time.
Sorting and Detecting Biological Agents
In certain example embodiments, the method disclosed herein may further comprise sorting encapsulated beads based at least in part on the detected readout of the one or more reporter elements. As understood by one of ordinary skill in the art, the type of readout will dictate the type of sorting technology that is required. For example, the encapsulated beads may be sorted using flow cytometry, flow imaging cytometry, and flow label-free imaging cytometry. In flow cytometry and flow imaging cytometry, sorting may be based on detection of a detectable label, such as a fluorescent label, of the reporter elements described above. In label-free imaging cytometry, sorting may be done on the basis of differences in size, morphology, or other cellular characteristics and without requiring a detectable label.
In some embodiments, detection may be performed using an antibody pull-down approach. Encapsulated cells can produce proteins that can be captured on the surface of, for example, an agarose bead. These beads can be targeted by magnetic nanoparticle-antibody conjugates (the pore size of an agarose gel is ˜200-500 nm). The beads with the corresponding product can then be pulled down by a magnet and further analyzed downstream or in later steps. Such an approach would be similar to an ELISA, but instead of a fluorescent readout with a counter antibody, it may be enriched with a magnet.
In further embodiments, the encapsulated cells can be engineered to produce a compound or enzyme that would degrade or denature the gel. A common choice for this can be matrix metalloproteins, which can break down peptide crosslinks in gels. Any cells that remain embedded within an agarose bead can be discarded through filtration. Cells that manage to break out or proliferate can be separated and followed for further analysis.
Altering Contents of Capsules Via Re-Encapsulation of Beads
In certain example embodiments, the beads may be cycled through a series of re-encapsulations to alter the contents of the outer capsule, to control the types of materials that diffuse into the beads and/or to screen for multiple biological functions within a single assay run. In general, re-encapsulation comprises de-polymerizing the existing outer capsule to release the beads and then re-encapsulating the released beads in a second polymer. The process of re-encapsulation may be carried out as described above. The polymer used in the re-encapsulation process may be the same or different as the capsule used in the initial encapsulation. In certain example embodiments, each re-encapsulation results in the introduction of a new reporter element configured to detect an additional biological function of the microscale biological system. The number of re-encapsulations per assay may be dictated by the number of biological functions to be screened in a single assay. In certain example embodiments, the beads may be exposed to an additional sorting step as described above after each re-encapsulation such that a sub-set of microscale biological systems having the desired combination of biological functions is obtained. In certain other example embodiments, re-encapsulation may be used to introduce additional reagents to sustain replication and/or growth of the biological system or induce or silence expression by the microscale biological system.
Sequencing of Microscale Biological Systems
In certain example embodiments, the methods disclosed herein may further comprise sequencing of the microscale biological systems. Sequencing may comprise DNA sequencing, RNA sequencing, or both. In certain example embodiments, a genotype of the microscale biological systems is determined. In the context of cells, this refers to determining a genotype of the cell in a classical sense. In the context of microscale biological systems comprising a set of nucleic acid constructs determining a “genotype” may refer to sequencing said nucleic acid constructs.
As noted above, the individual discrete volumes comprising the microscale biological systems may be identified using barcodes. In certain example embodiments, the microscale biological system may comprise a unique barcode sequence. In certain example embodiments, the barcode sequence may be a unique naturally occurring sequence that identifies the microscale biological systems, such as a 5S, 16S, or 23S rRNA sequence. Alternatively, in some example embodiments, the microscale biological system may be modified to include a barcode sequence. For example, a unique barcode sequence may be inserted into the genomic sequence of a cellular microscale biological system or into a nucleic acid construct of an acellular or synthetic system. Nucleic acid barcodes may be used to de-convolute sequencing data and correlate nucleic acid sequences with individual discrete volumes from which the sequence information was obtained.
In certain example embodiments, determining a genotype of the microscale biological system may comprise lysing the cell in each bead such that genomic DNA is retained in the gel beads. As one of the ordinary skill in the art will appreciate, the lysing step is not required in cell free systems. The beads are then released from the first outer capsule and re-encapsulated in a second outer capsule comprising genomic DNA amplification reagents. The beads are then maintained under conditions sufficient for genomic DNA amplification. After genomic DNA amplification, the beads are released from the second outer capsule and re-encapsulated in a third outer capsule comprising tagmentation reagents. In certain example embodiments, the tagmentation reagents are delivered such that each droplet receives a transposome complex comprising a unique origin-specific barcode or unique combination of origin specific barcodes. The beads are then maintained under conditions sufficient for tagmentation resulting in genomic DNA fragment labeled with sequencing adapters, and optionally, origin-specific barcodes. The genomic DNA fragments are then isolated and used to prepare a DNA sequencing library. The genotype of each microscale biological system may then be determined by sequencing the DNA sequencing library.
In certain example embodiments, sequencing may comprise determining the sequence of some or all of the RNA transcripts expressed by a microscale biological system. RNA sequencing may be done using known RNA sequencing methods in the art. In certain example embodiments, transcriptome sequencing is done using a method such as drop-seq as disclosed in Macosko et al. “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.” Cell, 2015, 161:1202-1214 and Klein et al. “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell, 2015, 161:1187-1201.
In one example embodiment, the embodiments disclosed herein are used to screen bacterial cells for biological functions. In certain example embodiments, the biological function is production of one or more bacterial cell products exhibiting antibiotic activity. In certain example embodiments, the bacteria may comprise different species of bacteria, different strains of the same species of bacteria, or a combination thereof. The bacteria may be isolated from a natural environment. For example, many existing antibiotics are produced by bacterial species commonly found in the soil. Therefore, one example application would be the screening of bacteria obtained from soil samples for novel antibiotic activity.
The bacteria to be screened are encapsulated in a first polymer. In certain example embodiments, the first polymer is agarose. In certain example embodiments, the cells to be screened are loaded into a microfluidic device in combination with molten agarose, wherein the microfluidic device induces droplet formation from the molten agarose by flowing a stream of carrier oil into a stream comprising the molten agarose and bacteria cells to be screened. The flow rate of the molten agarose and carrier oil streams may be controlled using known methods in the art to result in Poisson loading of individual cells into individual agarose droplets.
The agarose droplets are then cooled and allowed to polymerize. This may be achieved by flowing the newly formed droplet through an incubation channel or series of incubation channels that exposed the agarose droplets to reduced temperatures and thereby allow the agarose to cool and polymerize. The result is a library of beads encapsulating individual bacteria i.e. “producer cells” or “producer strains.”
The beads are then again encapsulated in an outer capsule (i.e. second individual discrete volume) by substantially repeating the process used to generate the beads. However, in addition to adding the beads to a solution comprising a second polymer, the solution further comprises a second bacterium, or reporter strain. This reporter stain may be a single species or combination of species. In certain example embodiments, the reporter strain is a representative species of a bacterium to which effective antibiotics are desired. The outer capsule is formed the same way as the initial bead. Where the second polymer is agarose it will be understood that the initial agarose bead will depolymerize and repolymerize with the outer capsule such that the initial discrete volume formed by the first bead may become continuous with the larger agarose droplet upon re-polymerization. However, where a second polymer is used that polymerizes under different conditions, the initial bead may remain distinct with the larger final droplet. The second polymer should allow diffusion of products generated by the produced strain in the bead to diffuse out into the outer capsule in order to contact the reporter strain.
Once formed, the system may be incubated to allow for growth of the reporter and producer strains. Antibiotic activity may be assessed by observation of growth characteristics of the reporter strain. For example, the reporter strain may be assigned for signs of cell death, inhibited growth relative to controls, or for zones of inhibition around the producer strain or inner bead. In certain example embodiments, the reporter strain may further comprise a reporter element. In certain example embodiments the reporter element is a nucleic acid construct introduced into the report strain, the nucleic acid construct comprising a fluorescent protein operably connected to a stress reporter. As shown in
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
In one example embodiment, determining a genotype of the microscale biological system comprises encapsulation of single cells into a first droplet. In certain example embodiments, the polymer used to form the first droplet is agarose. The agarose is maintained at a molten temperature during droplet formation. After encapsulation in droplets the droplets are cooled, allowing the agarose to polymerize. The cells are then lysed with the agarose bead, for example, by exposure of the beads to alkaline conditions or other cell lysis conditions known in the art and suitable for lysing a cell within the agarose bead. The beads containing lysed cells are then encapsulated in a second polymer. In certain example embodiments, the second polymer is PEG. The beads may be encapsulated in a polymer comprising PEG precursors and multiple displacement amplification (MDA) reagents. The PEG precursors are polymerized to form the outer capsule. The MDA reaction may be carried out under conditions and for a time known in the art to be sufficient for the MDA reaction. After the MDA reaction, the beads are released from the outer capsule. In certain example embodiments, the emulsions may be broken using perfluorooctanol. At this point, the amount of genomic DNA per gel bead can be quantified using qPCR. In order to prepare for sequencing, the individual beads are then re-encapsulated with a tagmentation mix. After the tagmentation reaction, the encapsulated beads are Poisson loaded into an additional individual discrete volume, such as the wells of a 96-well plate, containing library amplification reagents. The amplified, fragmented, and transposon-labeled DNA is then released from the bead and capsule, for example using electrocoalescence, then subjected to library amplification in the individual discrete volume.
First Encapsulation
Second encapsulation
First Encapsulation)
Approach 1 (Droplet Microfluidics))
Approach 2 (Vortex)
MDA (Same Protocol as Before)
MDA was performed according to manufacturer's protocol (Qiagen Repli G single cell kit).
Loading TN5 with Fluorescent Adapters
Protocol adapted from Amini, S. et al Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing. Nat. Gen. 46, 1343-1349 (2014).
Transposases were expressed, purified, and assembled following methods published previously. DNA containing unique fluorescent tag and the 19 bp mosaic end were annealed by incubating 100 uM of DNA from 95 C to 25 C at 0.1 C/sec and holding at 88, 74, 60, and 37 C for 2 min each. The annealed oligos and transposases were mixed at 12.5 uM and incubated at 37 C for 1 hour before storing at −20 C.
Loading TN5 with Barcode Adapters (Same as Above, but Loaded with Different DNA Adapters)
Transposases was expressed, purified, and assembled followed methods published previously. DNA containing barcodes, read 1 sequencing primer, read 2 sequencing primer, and the 19 bp mosaic end were annealed by incubating 100 uM of DNA from 95 C to 25 C at 0.1 C/sec and holding at 88, 74, 60, and 37 C for 2 min each. The annealed oligos and transposases were mixed at 12.5 uM and incubated at 37 C for 1 hour before storing at −20 C.
Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.
This application is a 35 U.S.C. § 371 national stage application of International Patent Application No. PCT/US2018/027631 filed Apr. 13, 2018, and entitled “High-Throughput Screens for Exploring Biological Functions of Microscale Biological Systems,” and claims the benefit of U.S. Provisional Application No. 62/485,529, filed Apr. 14, 2017. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.
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| PCT/US2018/027631 | 4/13/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2018/191701 | 10/18/2018 | WO | A |
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| 20210139941 A1 | May 2021 | US |
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| 62485529 | Apr 2017 | US |
