Patent Application
20230250139
The invention, in some aspects, includes compositions and methods for molecular recording protein expression and identifying protein expression records in cells.
The contents of the electronic sequence listing (SequenceListingUS.xml; Size: 40,942 bytes; and Date of Creation: Oct. 11, 2022) is herein incorporated by reference in its entirety.
Reading out biological signals and processes that take place over time, in living cells, intact organs, and organisms, is essential to advancing biological research, both basic science and translationally oriented. The imaging of genetically encoded fluorescent signal reporters, for example, enables specific biological activities to be monitored in real time in living cells [Greenwald, E. C., etal. Chem. Rev. 118, 11707-11794 (2018)]. However, long-term live imaging is laborious and equipment intensive, because a single microscope often has to be monopolized for the duration of the experiment, and furthermore the number of cells that can be observed is limited by the performance of live imaging methods, which are not as scalable as fixed-tissue imaging methods, which can benefit from sectioning, clearing, expansion, and other techniques that improve the number of cells that can be surveyed [Murray, E. et al. Cell 163, 1500-14 (2015); Ragan, T. et al. Nat. Methods 2012 93 9, 255-258 (2012); Gao, R. et al. Science (80-.). 363, (2019). Snapshot methods, that perform RNA FISH [Lin, D. et al. Nature 470, 221 (2011)], or protein immunostaining [Ceccatelli, S. et al. Proc. Natl. Acad. Sci. U.S.A 86, 9569-9573 (1989)], can enable one (and sometimes two) time points of a physiological signal to be inferred in fixed cells, but cannot support continuous recording of physiological signals for later fixed-cell readout.
According to an aspect of the invention, a composition including a sequence encoding an expression-recording island (XRI) is provided, when the composition is expressed, each encoded XRI includes one or more independently selected self-assembling filament-forming monomer, zero, one, or more independently selected detectable tag, and zero, one, or more independently selected protein spacer. In some embodiments, the self-assembling filament-forming monomer is an engineered protein. In certain embodiments, the self-assembling filament-forming monomer includes a 1POK or DHF40 protein. In some embodiments, the detectable tag includes an epitope tag. In some embodiments, the epitope tag is a human influenza hemagglutinin (HA) tag. In certain embodiments, the protein spacer includes a monomeric protein. In some embodiments, the monomeric protein comprise a mEGFP or maltose binding protein (MBP). In some embodiments, the encoded XRI, when expressed is capable of forming a linear protein assembly. In some embodiments, the linear protein assembly includes the protein spacer fused to a lateral edge of the filament forming monomer. In some embodiments, the encoded XRI comprises a self-assembling filament-forming monomer, a detectable tag, and optionally a protein spacer. In some embodiments, the encoded XRI includes 1, 2, 3, or 4 independently selected self-assembling filament forming monomers. In some embodiments, the encoded XRI includes 0, 1, 2, 3, or 4 independently selected detectable labels. In some embodiments, the encoded XRI includes 0, 1, 2, 3, or 4 independently selected protein spacers.
According to another aspect of the invention, a vector is provided, the vector including a nucleotide sequence encoding the encoded expression-recording island (XRI) composition of any embodiment of the aforementioned aspect of the invention.
According to another aspect of the invention, a cell is provided, the cell including any embodiment of an aforementioned vector of the invention. In certain embodiments, the cell is a vertebrate cell, a mammalian cell, and/or a human cell. In some embodiments, the cell is an excitable cell. In some embodiments, the cell is one or more of a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In certain embodiments, the cell is an in vitro cell. In certain embodiments, the cell is in a subject. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is a brain cell in a subject.
According to another aspect of the invention, an adeno-associated virus (AAV) including the encoded expression-recording island (XRI) composition of any embodiment of a composition of an aforementioned aspect of the invention is provided.
According to another aspect of the invention, a cell is provided, the cell including any embodiment of an aforementioned AVV of the invention. In some embodiments, the cell is a vertebrate cell, a mammalian cell, and/or a human cell. In certain embodiments, the cell is an excitable cell. In certain embodiments, the cell is one or more of a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In some embodiments, the cell is an in vitro cell. In some embodiments, the cell is in a subject. In certain embodiments, the cell is an ex vivo cell. In some embodiments, the cell is a brain cell in a subject.
According to another aspect of the invention, a method of identifying an expression history record in a cell is provided, the method including: expressing in a cell or in a plurality of cells the expression-recording island (XRI) encoded by one, two, or more independently selected XRI-encoding compositions of any embodiment of any aforementioned composition of the invention, and detecting the expressed XRI(s) in the one cell or the plurality of cells at a time point, wherein the detected expressed XRI(s) identify an expression record of the XRI(s) in the cell or the plurality of cells at the time point. In some embodiments, detecting the expressed XRI(s) in the plurality of cells includes detecting the expressed XRI(s) in one or more cells obtained from the plurality of cells. In some embodiments, the independently selected compositions each includes a different encoded XRI. In certain embodiments, the method also includes detecting the expressed XRI(s) in the plurality of cells at one or more additional independently selected time points providing a plurality of detections of detected expressed XRI(s) and identifying an expression record of the XRI(s) in the plurality of cells across the plurality of time points. In some embodiments, the method also includes comparing the identified expression record of the XRI(s) in the plurality of cells at two or more of the plurality of time points, wherein a difference between in the expressed XRI(s) detected at two or more of the plurality of time points identifies a change in the expression record of the XRI(s) in the plurality of cells. In some embodiments, the method also includes fixing the cells(s) prior to the detecting. In some embodiments, the detecting includes determining an amount of the expressed XRI. In certain embodiments, the detecting includes determining a pattern of epitope tags in the expressed XRI. In some embodiments, the detecting includes determining the identity of one or more epitope tags in the expressed XRI. In some embodiments, determining the amount of the expressed XRI(s) includes determining an amount of the detectable tag in the XRI(s). In certain embodiments, the time interval between any two of the plurality of the independently selected time points is at least 1 sec, 5 sec, 10 sec, 15, sec., 30 sec, 45 sec, 1 min, 30 min, 60 min, 240 min, 480 min, 1 day, 2, days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 60 days or 90 days. In some embodiments, the detected expressed of XRI(s) corresponds to a temporal history of expression of the XRI(s) in the cell or the plurality of cells. In some embodiments, the cell is a vertebrate cell, a mammalian cell, and/or a human cell. In some embodiments, the cell is an excitable cell. In certain embodiments, the cell is one or more of a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In some embodiments, the cell is an in vitro cell. In some embodiments, the cell is in a subject. In certain embodiments, the cell is an ex vivo cell. In some embodiments, each of the encoded XRIs include 0, 1, 2, 3, 4, or more independently selected detectable labels. In some embodiments, the number of detectable labels in each of the encoded XRIs is independently selected. In some embodiments, the encoded XRIs include 0, 1, 2, 3, 4, or more independently selected protein spacers. In some embodiments the number of protein spacers in each of the encoded XRIs is independently selected
According to another aspect of the invention, a method of identifying an effect of a candidate stimulus on expression in a cell, is provided, the method including: preparing a plurality of cells expressing the expression-recording island (XRI) encoded by any embodiment of an aforementioned composition of the invention; exposing the plurality of cells expressing the XRI to a candidate stimulus; detecting the expressed XRI in one or more cells in the exposed plurality of cells, and comparing the detected expressed XRI in the one or more cells to a control expressed XRI, wherein the control XRI is the expressed XRI in a cell including the expressed XRI but not exposed to the candidate stimulus; and wherein a difference in the detected expressed XRI in the exposed cells compared to the control XRI identifies an effect of the candidate stimulus on the XRI expression. In some embodiments, the detecting includes determining an amount of XRI expression. In some embodiments, the exposing to the candidate stimulus includes one or both of indirectly and directly contacting the plurality of cells with one or more of: electrical stimulus, a chemical stimulus, a biological stimulus, an inhibitory stimulus, an excitatory stimulus, a signaling molecule, a signaling chemical, a pharmaceutical stimulus, a cellular stimulus, a temperature stimulus, or a light stimulus. In some embodiments, the cell is a vertebrate cell, a mammalian cell, and/or a human cell. In certain embodiments, the cell is an excitable cell. In some embodiments, the cell is in a subject. In certain embodiments, the cell is in culture. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is one or more of a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In certain embodiments, the cell is an in vitro cell. In some embodiments, the XRI includes 0, 1, 2, 3, or 4 independently selected detectable labels. In some embodiments, the XRI includes 0, 1, 2, 3, or 4 independently selected protein spacers. In some embodiments, the XRI includes 1, 2, 3, or 4 independently selected self-assembling filament-forming monomers.
According to another aspect of the invention, a composition including an expression-recording island (XRI) protein is provided, the XRI protein includes one or more self-assembling filament-forming monomers; zero, one, or more independently selected detectable tags; and zero, one, or more independently selected protein spacers. In some embodiments, the composition also includes zero, one, or more additional independently selected self-assembling filament-forming monomer(s). In some embodiments, the XRI is encoded by an embodiment of any aforementioned composition of the invention.
According to another aspect of the invention, a cell is provided, the cell including an embodiment of any aforementioned XRI protein composition of the invention. In certain embodiments, the cell is a vertebrate cell. In certain embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an excitable cell. In certain embodiments, the cell is one or more of a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In certain embodiments, the cell is an in vitro cell. In some embodiments, the cell is in a subject. In certain embodiments, the cell is an ex vivo cell. In some embodiments, the cell is a brain cell in a subject.
Table 1 provides a list of protein motifs used in certain embodiments of expression-recording island (XRI) compositions and methods set forth herein. For the motifs in Table 1, the protein sequences were mouse codon optimized into DNA sequences and then synthesized before experimenst using routine methods of protein engineering. The amino acid sequences fully determine the proteins' structures and functions. Based on the amino acid sequence information disclosed herein, it will be understood that routine methods can be used to determine and prepare DNA sequences that encode proteins such as motifs, spacers, detectable tags, self-assembling filament-forming monomers, and other proteins of the invention.
Certain aspects of the invention can be used to record cellular physiological information onto intracellular, steadily growing, protein chains made out of fully genetically encoded self-assembling proteins, and then read out via routine immunofluorescence and imaging techniques. Compositions of the invention include certain existing, human-created self-assembling protein candidates, which have now been engineered to add a novel “insulator” component to the self-assembling protein candidate. Compositions of the invention comprise one or more sequences encoding self-assembling filament forming monomers, zero, one, or more of a detectable tag or tags, and zero, one or more protein spacers. A composition of the invention, when expressed in a cell produces an engineered protein capable of stable, time-ordered longitudinal growth in the cell. Embodiments of compositions of the invention include a sequence encoding an expression-recording island (XRI).
It has now been determined that an expression-recording island (XRI) strategy such as described herein, can be used for long-term recording of gene expression time course, with single-cell precision, across cell populations. Because the linear protein assembly grows continuously over time, it acts like a molecular tape recorder that preserves the temporal order of the protein monomers made available by the cell depending on the cell's current state or function. For example, if protein monomers with the epitope tag ‘A’ are steadily expressed by the cell, and the expression of protein monomers with the epitope tag ‘B’ is increased by, say, a neural activity dependent promoter, then the neural activity dependent event will result in permanent storage of the activity record in the order of the epitope tags along the growing protein chain, enabling later readout via immunostaining against tags ‘A’ and ‘B’, followed by standard imaging. Recordings using embodiments of compositions and methods of the invention have shown that pharmacological modulation of gene expression histories in living cells and organisms can be read out post hoc.
Information disclosed herein defines, provides rationale for, and validates, a calibratable measure of time, and use of the fractional cumulative expression of a detectable tag bearing monomers, to calibrate the time axis onto the information recorded on the XRI via ordered epitope tags. A non-limiting example of an embodiment of a method of the invention, comprises application of XRIs of the invention to record c-fos promoter-driven gene expression in cultured mouse hippocampal neurons after depolarization, and application of the fractional cumulative expression of HA-bearing monomers to recover the time axis and c-fos promoter-driven gene expression solely from information read out from XRI via immunostaining and imaging. Studies disclosed herein provide evidence that XRI can preserve the temporal order of protein monomers expressed in a living cell, including but not limited to a cell in culture, a cell in a live subject, for example a cell in the brain of a live subject. Thus, XRIs of the invention are capable of functioning in multiple biological systems, including the cells in culture and cells in a live subject, cells in a live mammalian brain, and methods of the invention can use XRIs of the invention to encode cellular physiological signals into a linear, optically readable protein chain.
Compared to nucleic acid-based systems, which require nucleic acid sequencing methods that are destructive to cells [Kording, K. P. PLOS Comput. Biol. 7, e1002291 (2011); Perli, S. D., et al., Science 2016 Sep 9;353(6304):aag0511, doi: 10.1126/science.aag0511, Epub 2016 Aug 18 (2016); Rodriques, S. G. et al. Nat. Biotechnol. 2020 393 39, 320-325 (2020); Farzadfard, F. & Lu, T. K. Science 2014 Nov 14; 346(6211): 1256272; Farzadfard, F. & Lu, T. K. Science. 361, 870-875 (2018); Farzadfard, F. et al. Mol. Cell 75, 769-780.e4 (2019); Sheth, R. U., et al., Science 358, 1457-1461 (2017), the content of each of which is incorporated by reference herein in its entirety], reading out recorded information using compositions and methods of the invention through imaging, only requires routine immunofluorescence techniques and conventional microscopes, available in the art, without the need for additional hardware investment. Such preservation of cellular physiological information within the native environment offered by the protein-based compositions and methods of the invention also enable correlation of the recorded biological information with other kinds of structural and molecular information associated with the cellular population, such as the spatial location, cell type, and presence of RNA/protein markers in the recorded cells [Lin, D. et al. Nature 470, 221 (2011); Ceccatelli, S, et al., Proc. Nat/. Acad. Sci. U.S.A 86, 9569-9573 (1989); Guenthner, C. J., et al., Neuron 78, 773-784 (2013), the content of each of which is incorporated by reference herein in its entirety], some of which may be causally involved with the creation of the physiological signals, or that result from the physiological signals. Such kinds of multimodal data may enable the analysis of how specific cellular machinery drive, or result from, complex time courses of physiological stimuli. For example, by offering the ability to record gene expression time course in single cells, as described herein, the protein-based XRI compositions and methods of the invention enable the study of gene expression time course as a result of specific cellular inputs and/or drug treatments [Strober, B. J. et al. Science 364, 1287-1290 (2019); Gallo, F. T., et al., Front. Behav. Neurosci. Vol, 12, Article 79 (2018) doi.org/10.3389/fnbeh.2018.00079, the content of each of which is incorporated by reference herein in its entirety]. Non-limiting examples of the use of compositions and methods of the invention, include their use to investigate circadian genes [Zhang, R., et al, Proc. Natl. Acad. Sci. 111, 16219-16224 (2014), the content of which is incorporated by reference herein in its entirety] and other genes that change in complex ways over time; to record transcription factor activities [Elf, J., et al, Science 316, 1191 (2007), the content of which is incorporated by reference herein in its entirety]; and as an information storage platform to externally introduce unique cellular barcodes into single cells for cell identification [Viswanathan, S. et al. Nat. Methods 12, 568-576 (2015), the content of which is incorporated by reference herein in its entirety].
Certain embodiments of a composition of the invention comprise a nucleic acid sequence that encodes an expression-recording island (XRI). Such a composition of the invention comprises a nucleic acid sequence that encodes at least one self-assembling filament forming monomer at least one detectable tag (which may also be referred to herein as an epitope tag); and at least one protein spacer. In some embodiments, a composition of the invention includes a sequence that encodes 1, 2, 3, or more independently selected self-assembling filament forming monomers; 0, 1, 2, 3, or more independently selected detectable tags; and 0, 1, 2, 3, or more independently selected protein spacers. The term “independently selected” used herein in reference to multiple like components, means selection of each like component for inclusion in the composition, independent of the others selected. As a non-limiting example, in a composition of the invention comprising three independently selected protein spacers, the three spacers are considered to be “like components” and each may be selected independent of the others, meaning that in different embodiments of the invention, the three encoded protein spacers may be: all the same, each different from the others, or two the same and one different from the other protein spacers in the composition.
Some embodiments of a composition of the invention comprise an expressed XRI protein. Components of an expressed XRI comprise one or more independently selected self-assembling filament-forming monomers, zero, one, or more independently selected detectable tag (which may also be referred to herein as epitope tags), and zero, one, or more independently selected protein spacers. In some embodiments, a composition of the invention includes 1, 2, 3, 4, 5, 6, or more independently selected self-assembling filament forming monomers; 0, 1, 2, 3, 4, 5, 6, or more independently selected detectable tags; and 0, 1, 2, 3, 4, 5, 6, or more independently selected protein spacers. Some embodiments of a composition of the invention comprise an expressed XRI protein. Components of an expressed XRI in some embodiments, comprise a self-assembling filament-forming monomer, a detectable tag (which may also be referred to herein as epitope tags), and a protein spacer.
Non-limiting examples of self-assembling proteins that may be present in, or encoded in certain embodiments of a composition of the invention, are 1POK and DHF40 proteins. Self-assembling protein encoding sequences and the expressed self-assembling protein may, in some embodiments of the invention be engineered sequences and proteins, respectively. Non-limiting examples of a detectable tag that may be present in, or encoded in certain embodiments of a composition of the invention, is a human influenza hemagglutinin (HA) tag. Non-limiting examples of monomer protein spacers that may be present in, or encoded in certain embodiments of a composition of the invention, are a monomeric mEGFP and a maltose binding protein (MBP).
Certain embodiments of the invention include compositions as described and methods of using XRI components of the invention for recording cellular physiological histories in living cells. As used herein the term XRI system, XRI recorder, XRI recorder system, and recorder system, refer to an embodiment in which an XRI of the invention is expressed in a living cell, and used to determine an expression history of the cell. In certain aspects of the invention, an XRI reporter system is used in a cell to determine characteristics such as, but not limited to the timing of protein expression and the effect of stimuli on expression in the cell. In some embodiments, a baseline determination of one or more characteristics of expression in a “control” cell can be performed using a method and/or system of the invention. Such baseline determinations may be made for the same characteristics that are also determined in similar cells but under different circumstances. For example, a baseline determination may indicate a “control” characteristic, which can be compared to the characteristic in a “test” cell that is exposed to one or more different stimuli, environmental changes, etc. to which the control cell was not exposed. For example, though not intended to be limiting, a test cell that includes an XRI system of the invention can be contacted with a candidate stimulus and a difference in one or more characteristics in the test cell compared to a control cell not contacted with the candidate stimulus in order to ascertain whether there is an effect of the candidate stimulus on expression in the cell. Non-limiting examples of candidate stimuli are: test agents are: electrical stimulus, a chemical stimulus, a biological stimulus, an inhibitory stimulus, an excitatory stimulus, a signaling molecule, a signaling chemical, a pharmaceutical stimulus, a cellular stimulus, a temperature stimulus, a light stimulus, a candidate compound, a pharmaceutical compound, an electrical stimulus, a chemical stimulus, a biological stimulus. Additional stimuli that are suitable for use in embodiments of the invention are known and routinely used in the art.
It will be understood that in some aspects of the invention, a candidate stimulus may be delivered directly to a cell that includes an XRI recorder system of the invention, or may be delivered to another cell that is in communication with a cell that includes an XRI recorder of the invention. As used herein, the term “in communication with” used in reference to a cell that includes an expressed or encoded XRI recorder of the invention, includes cells, for example, that influence the cell comprising the expressed or encoded XRI recorder, for example, though not intended to be limiting, via a neurotransmitter means, an electrical means, etc. Communication can be direct communication from a cell immediately (directly) upstream from the cell that includes an expressed or encoded XRI recorder of the invention, or can be indirect communication, such as the result of activity of a cell further (indirectly) upstream that impacts the cell in which an expressed or encoded XRI recorder of the invention is included. Stimulation of one or more of a cell directly upstream and a cell indirectly upstream may result in a change in expression in a cell that includes an expressed and/or encoded XRI recorder of the invention, and the presence of the expressed and/or encoded XRI recorder permits determination of changes in characteristics of expression in that cell using methods of the invention. As used herein a change in expression means an alteration in the expression characteristic, for example an increase in a rate or timing of expression, a decrease in a rate or timing of expression, the start of expression, a delay in the start of expression, and the like.
Methods and XRI recorder systems of the invention can be used to assess one or more changes in: (1) an internal environment of a cell, (2) an external environment of a cell, (3) an internal environment of an upstream cell, and (4) an external environment of an upstream cell. Non-limiting examples of events and situations that may change in a cell's internal or external environment and that can directly or indirectly effect expression in a cell comprising an expressed and/or encoded XRI recorder of the invention include, an action potential, a disease or injury condition in the cell or subject comprising the cell, contact of the cell with a candidate stimuli agent or compound, contact of the cell with a pharmaceutical agent or compound, a surgical procedure in the subject, contact of the cell with radiation, light, electric stimulation, etc. Other types of events and actions that alter the internal or external environment of a cell are known in the art, and can also be assessed using methods and XRI recorders ofthe invention.
Components of XRI-based recorder systems of the invention are well suited for targeting cells, expression in cells, and for use to detect and assess expression levels and changes associated with stimuli and/or cell activities. In some embodiments, an expressed and/or encoded XRI recorder system of the invention can be utilized to detect one or more of conductance changes across cell membranes, the impact of endogenous signaling pathways (such as calcium dependent signaling, etc.), and the effect of applied candidate stimuli on a cell that includes the expressed and/or encoded XRI recorder of the invention. Thus, certain aspects of the invention include methods of using XRI-based recorders to screen putative therapeutic agents, known therapeutic agents, combinations of two or more independently selected known and putative therapeutic agents.
One or more XRI-based recorders of the invention can also be used in some embodiments of methods of the invention to assess the effect of internal cellular conditions, environmental conditions external to the cell, and to assess the result diseases, injuries, treatments, etc. on expression in the cell comprising the expressed and/or encoded XRI recorder. Methods and systems of the invention can also be used to examine normal cells in vitro and in vivo. For example, in some embodiments, an XRI recorder system can be used to determine expression events in normal cells and subjects and the resulting information on expression characteristics can be applied in the study of normal cell development, non-limiting examples of which are cell development in regeneration, embryonic cell development, establishment of cell connectivity, and the like.
The present invention, in part, includes novel XRI-based recorder systems and components thereof, their expression in cells, and their use to determine alterations in characteristics of expression in the cell, which may also be referred to herein as a “host cell.” As used herein, the term “host cell” means a cell that includes one or more components of an expressed or encoded XRI-based recorder system of the invention. Non-limiting examples of components of XRI recorder systems of the invention are described herein, see for example, Tables 1-2 and the Examples section. Aspects of the invention also include additional functional variants of components of XRI-based recorder systems described herein, including polynucleotides, polypeptides, compositions comprising the components and functional variants thereof, and methods of using the components and functional variants thereof to perform XRI-based recording in a cell, or in a plurality of cells. As used herein the term “plurality of cells” means more than one cell, which in some embodiments of the invention is more than 1, more than 10, more than 100, more than 1000, more than 10,000, or more than 100,000, and more than 1,000,000, including all integers within the range from 1 to at least 1,000,000.
It is understood that the terms: XRI-based recorder system components encompass molecules, polypeptides, and polynucleotides described herein, as well as functional variants thereof. The invention also includes compounds and compositions that comprise one or more components of an expressed and/or encoded XRI-based recorder system of the invention. A compound or composition that comprises a component of an expressed and/or encoded XRT recorder of the invention may in some embodiments, include one, two, three, four, five, six, or more additional components. Non-limiting examples of additional components are a vector, a promoter, a trafficking sequence, a delivery molecule sequence, an additional sequence, etc.
Certain embodiments of the invention include polynucleotides comprising nucleic acid sequences that encode a component of a XRI recorder system of the invention, and some aspects of the invention comprise methods of delivering and/or using such polynucleotides in cells, tissues, and/or organisms. XRI-based recorder-component polynucleotide sequences and amino acid sequences used in aspects and methods of the invention may be “isolated” sequences. As used herein, the term “isolated” used in reference to a polynucleotide, nucleic acid sequence, polypeptide, or amino acid sequence means a polynucleotide, nucleic acid sequence, polypeptide, or amino acid sequence, respectively, that is separate from its native environment and present in sufficient quantity to permit its identification or use. Thus, a nucleic acid or amino acid sequence that makes up a component of an XRI-based molecular recorder molecule that is present in one or more of a vector, a cell, a tissue, an organism, etc., may be considered to be an isolated sequence if it is not naturally present in that cell, tissue, or organism, and/or did not originate in that cell, tissue, or organism.
A host cell means a cell that comprises one or more components of an expressed and/or encoded XRI-based recorder. In certain aspects of the invention, one or more components of an expressed and/or encoded XRI-based recorder system of the invention are delivered into and/or expressed in a cell. Examples of cells that may be used in embodiments of the invention include, but are not limited to vertebrate cells, mammalian cells (including but not limited to non-human primate, human, dog, cat, horse, mouse, rat, etc.), insect cells (including but not limited to Drosophila, etc.), fish, worm, nematode, and avian cells. In some embodiments of the invention, a cell is a plant cell.
One or more components of an XRI-based reporter system of the invention may be derived from (also referred to herein as “being a variant of”) one or more components disclosed herein, and they may exhibit the same qualitative function and/or characteristics of the molecular reporter system component from which they have been derived, and/or may show one or more increased or decreased level of a function or characteristic of the parent component. In some embodiments of the invention an effectiveness of a variant or derived component of an XRI reporter system set forth herein may differ from the parent component. For example, in some instances a variant or derived component is capable of faster determination of a characteristic of expression in a host cell than is possible for its parent component.
It is understood in the art that the codon systems in different organisms can be slightly different, and that therefore where the expression of a given protein from a given organism is desired, the nucleic acid sequence can be modified for expression within that organism. Thus, in some embodiments, a polynucleotide that encodes a component of an XRI-based recorder system of the invention comprises a mammalian-codon-optimized nucleic acid sequence, which may, in some embodiments, be a human-codon optimized nucleic acid sequence. Codon-optimized sequences can be prepared using routine methods.
Delivery of one or more components of an XRI-based recorder of the invention to a cell and/or expression of the component in a cell can be done using art-known delivery means. [see for example, Chow et al. Nature 2010 Jan 7;463(7277):98-102; and for Adeno-associated virus injection: Betley, J. N. & Sternson, S. M. (2011) Hum. Gene Ther. 22, 669-677; for In utero electroporation: Saito, T. & Nakatsuji, N. (2001) Dev. Biol. 240, 237-46; for microinjection into zebrafish embryos: Rosen J. N. et al., (2009) J. Vis. Exp. (25), e 1115, doi:10.3791/1115; and for DNA transfection for neuronal culture: Zeitelhofer, M. et al., (2007) Nature Protocols 2, 1692-1704, the content of each of which is incorporated by reference herein in its entirety].
In some embodiments of the invention a component of an XRI-based recorder of the invention is included as part of a fusion protein. It is well known in the art how to encode, prepare, and utilize fusion proteins that comprise a polypeptide sequence. In certain embodiments of the invention, a vector that encodes a fusion protein can be prepared and used to deliver a component of an XRI-based recorder system of the invention to a cell and can also in some embodiments be used to target delivery of a component of an XRI-based recorder system of the invention to a specific cell, cell type, tissue, or region in a subject. Suitable targeting sequences useful to deliver a component of an XRI-based recorder of the invention to a cell, tissue, region of interest are known in the art. Delivery of a component of an XRI-based recorder system of the invention to a cell, tissue, or region in a subject can be performed using art-known procedures. A fusion protein of the invention can be delivered to a cell by delivery of a vector encoding the XRI-containing fusion protein. The delivered fusion protein is then expressed in a specific cell type, tissue type, organ type, and/or region in a subject, or in vitro, for example in culture, in a slice preparation, etc.
In certain aspects of the invention, a component of an XRI-based recorder system of the invention is non-toxic or substantially non-toxic to the cell into which it is delivered and/or expressed. In some embodiments of the invention, a component of an XRI-based recorder of the invention is genetically introduced into a cell, and reagents and methods are provided for genetically targeted expression of components of an XRI-based recorder system of the invention. Genetic targeting can be used to deliver one or more components of an XRI-based recorder system of the invention to specific cell types, to specific cell subtypes, to specific spatial regions within an organism. In some embodiments of the invention, targeting can be used to control of the amount of a component of an XRI-based recorder system of the invention that is expressed and the timing of the expression. Preparation, delivery, and use of a fusion protein and its encoding nucleic acid sequences are well known in the art. Routine methods can be used in conjunction with teaching herein to express one or more XRI-based recorder system components and optionally additional polypeptides, in a desired cell, tissue, or region in vitro or in a subject.
Some embodiments of the invention include a reagent for genetically targeted expression of a component of an XRI-based recorder of the invention, wherein the reagent comprises a vector that contains the gene for the component. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. The term “vector” may also refer to a virus or organism that is capable of transporting the nucleic acid molecule. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Other useful vectors, include, but are not limited to viruses such as lentiviruses, retroviruses, adenoviruses, and phages. Vectors useful in some methods of the invention can genetically insert an XRI-based recorder system of the invention into dividing and non-dividing cells and can insert an XRI-based recorder system of the invention into an in vivo, in vitro, or ex vivo cell.
Vectors useful in methods of the invention may include additional sequences including, but not limited to one or more signal sequences and/or promoter sequences, or a combination thereof. Expression vectors and methods of their use are well known in the art. Non-limiting examples of suitable expression vectors and methods for their use are provided herein. In certain embodiments of the invention, a vector may be a lentivirus comprising the gene for an XRI-based recorder system of the invention. A lentivirus is a non-limiting example of a vector that may be used to create stable cell line. The term “cell line” as used herein is an established cell culture that will continue to proliferate given the appropriate medium.
Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Methods for selecting and using cell-specific promoters and general promoters are well known in the art. A non-limiting example of a general purpose promoter that allows expression of an XRI-based recorder system of the invention in a wide variety of cell types —thus a promoter for a gene that is widely expressed in a variety of cell types, for example a “housekeeping gene” can be used to express an XRI-based recorder system component(s) of the invention in a variety of cell types. Non-limiting examples of general promoters are provided elsewhere herein and suitable alternative promoters are well known in the art. In certain embodiments of the invention, a promoter may be an inducible promoter, examples of which include, but are not limited to tetracycline-on or tetracycline-off, or tamoxifen-inducible Cre-ER.
In some embodiments of the invention a reagent for expression of a component of an XRI-based recorder system of the invention is a vector that comprises a gene encoding the component, and optionally a gene encoding one or more additional polypeptides. Vectors useful in methods of the invention may include additional sequences including, but not limited to, one or more signal sequences and/or promoter sequences, or a combination thereof. In certain embodiments of the invention, a vector may be a lentivirus, adenovirus, adeno-associated virus, or other vector that comprises a gene encoding XRI-based recorder system component(s) of the invention. An adeno-associated virus (AAV) such as AAV8, AAV1, AAV2, AAV4, AAV5, AAV9, are non-limiting examples of vectors that may be used to express a fusion protein of the invention in a cell and/or subject. Expression vectors and methods of their preparation and use are well known in the art. Non-limiting examples of suitable expression vectors and methods for their use are provided herein. Other vectors that may be used in certain embodiments of the invention are provided in the Examples section herein.
Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Non-limiting examples promoters that can be used in vectors of the invention are: ubiquitous promoters, such as, but not limited to: CMV, CAG, CBA, and EFla promoters; and tissue-specific promoters, such as but not limited to: Synapsin, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, and aMHC promoters. In some embodiments, a promoter included in a method and/or vector of the invnetnio is a UBC promoter. Methods to select and use ubiquitous promoters and tissue-specific promoters are well known in the art. A non-limiting example of a tissue-specific promoter that can be used to express a component of an XRI-based recorder system of the invention in a cell such as a neuron is a synapsin promoter, which can be used to express the component in certain embodiments of methods of the invention. Additional tissue-specific promoters and general promoters are well known in the art and, in addition to those provided herein, may be suitable for use in compositions and methods of the invention. Other non-limiting examples of promoters that may be used in certain embodiments of methods of the invention are provided in the Examples section.
Non-limiting examples of detectable label polypeptides that may be included in a composition comprising a component of an XRI-based recorder system of the invention are: green fluorescent protein (GFP); enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP); yellow fluorescent protein (YFP), dtTomato, mCardinal, mCherry, DsRed, cyan fluorescent protein (CFP); far red fluorescent proteins, etc. Numerous fluorescent proteins and their encoding nucleic acid sequences are known in the art and routine methods can be used to include such sequences in fusion proteins and vectors, respectively, of the invention.
Additional sequences that may be included in a fusion protein comprising a component of an XRI-based molecular recorder system of the invention are trafficking sequences, including, but not limited to: Kir2.1 sequences and functional variants thereof, KGC sequences, ER2 sequences, etc. Trafficking polypeptides and their encoding nucleic acid sequences are known in the art and routine methods can be used to include and use such sequences in fusion proteins and vectors, respectively, of the invention.
Table 2 provides a list of constructs that have been prepared and used in XRI-based recorder systems and components of the invention.
UBC-1POK(E239Y)-Linker25-HA-Linker3-MBP_tag (also known as
UBC-1POK(E239Y)-Linker12-gg-HA
UBC-1POK(E239Y)-Linker13-HA-mEGFP
UBC-1M3U(D157L,E158L,D161L)-Linker14-HA
UBC-HA-Linker14-2CG4(K126Y,D131Y)
UBC-2VYC(K491L,D494L,D497L)-Linker14-HA
CMV-1POK(E239Y)-Linker8-HA
UBC-1POK(E239Y)-Linker7-HA-Linker3-MBP_tag
UBC-HA-Linker3-MBP_tag-Linker18-1POK(E239Y)
UBC-1POK(E239Y)-Linker5-HA-mEGFP
UBC-mEGFP-HA-Linker12-1POK(E239Y)
UBC-1POK(E239Y)-Linker25-HA-g-mEGFP
UBC-1POK(E239Y)-Linker7-HA-Linker3-Top7
UBC-1POK(E239Y)-Linker25-HA-gsg-Top7
UBC-1POK(E239Y)-Linker5-mEGFP-Linker2-HA-Linker3-MBP_tag
UBC-1POK(E239Y)-Linker24-mEGFP-HA-Linker6-MBP
UBC-1POK(E239Y)-Linker5-mEGFP-HA-Linker3-Top7
UBC-Top7-Linker12-1POK(E239Y)-Linker13-HA-mEGFP
UBC-1POK(E239Y)-Linker24-mEGFP-HA-Linker6-Top7
UBC-HA-dTor_12x31L-Linker24-1POK(E239Y)
UBC-NLS-Linker4-1POK(E239Y)-Linker13-HA-mEGFP
UBC-NLS-Linker4-1POK(E239Y)-Linker14-HA
UBC-DHF40-Linker14-HA
UBC-DHF40-Linker13-HA-mEGFP
UBC-DHF58Four-Linker14-HA
UBC-DHF58Six-Linker14-HA
UBC-DHF58Six-Linker14-mRuby2_smFP(HA)
UBC-DHF79-Linker14-HA
UBC-DHF 119-Linker14-HA
CMV-DHF40-Linker8-HA
CMV-DHF46-Linker8-HA
CMV-DHF47-Linker8-HA
CMV-DHF50-Linker8-HA
CMV-DHF77-Linker8-HA
UBC-γPFD-Linker8-HA
Sequences of three non-limiting examples of XRIs, each with a unique epitope tag, are set forth herein as (1) Synthetic construct XRI-HA gene, Genbank® OK539810 (amino acid sequence is SEQ ID NO: 26; DNA sequence is SEQ ID NO: 27); (2) synthetic construct XRI-FLAG gene, Genbank© OK539811 (amino acid sequence is SEQ ID NO: 28; DNA sequence is SEQ ID NO: 29); and (3) synthetic construct XRI-V5 gene, Genbank® OK539812 (amino acid sequence is SEQ ID NO: 30; DNA sequence is SEQ ID NO: 31).
Some aspects of the invention include cells used in conjunction with an XRI-based recorder system of the invention. Cells in which an XRI-based recorder system component may be expressed, and that can be used in methods of the invention, include prokaryotic and eukaryotic cells. Certain embodiments of the invention include use of mammalian cells; including but not limited to cells of humans, non-human primates, dogs, cats, horses, rodents, etc. In some embodiments of the invention, cells that are used are non-mammalian cells; including but not limited to insect cells, avian cells, fish cells, plant cells, etc. An XRI-based recorder system of the invention may be included in non-excitable cells and in excitable cells, the latter of which include cells able to produce and respond to electrical signals. Examples of excitable cell types include, but are not limited, to neurons, muscle cells, visual system cells, sensory cells, auditory cells, cardiac cells, and secretory cells (such as pancreatic cells, adrenal medulla cells, pituitary cells, etc.), cardiac cells, immune system cells, etc.
Cells in which an XRI-based recorder system of the invention can be used include embryonic cells, stem cells, pluripotent cells, mature cells, geriatric cells, as well as cells in other developmental stages. Non-limiting examples of cells that may be used in methods of the invention include neuronal cells, nervous system cells, cardiac cells, circulatory system cells, kidney cells, liver cells, epidermal cells, visual system cells, auditory system cells, secretory cells, endocrine cells, and muscle cells.
In some embodiments, a cell used in conjunction with methods and an XRI-based recorder system of the invention is a healthy normal cell that is not known or suspected of having a disease, disorder, or abnormal condition. In some embodiments of the invention, a cell used in conjunction with methods and an XRI-based recorder system of the invention may in some embodiments be a normal cell or in some embodiments is an abnormal cell. Non limiting examples of elements of an abnormal cell are: (1) a cell that has a disorder, disease, or condition; (2) a cell obtained from a subject that has, had, or is suspected of having disorder, disease, or condition; (3) a cell known to be or suspected of being involved in a disorder, disease, or condition; and (4) a cell that is a model for a disorder, disease, or condition, etc. Non-limiting examples of such cells are a degenerative cell, a neurological disease-bearing cell, a cell model of a disease or condition, an injured cell, a cell downstream from a disease-bearing or injured cell, etc. In some embodiments of the invention, a cell may be a control cell. A cell that is directly or indirectly upstream from a cell in which an XRI-based recorder system may be included may be a normal cell or may be an abnormal cell.
An embodiment of an XRI-based recorder system of the invention may be included in a cell from or in culture, a cell in solution, a cell obtained from a subject, and/or a cell in a subject (in vivo cell). In some embodiments of the invention, an XRI-based recorder system is present in and monitored in cultured cells, cultured tissues (e.g., brain slice preparations, etc.), and in living subjects, etc. As used herein, a the term “subject” may refer to a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, bird, rodent, fish, insect, or other vertebrate or invertebrate organism. In certain embodiments, a subject is a mammal and in certain embodiments, a subject is a human. Additional non-limiting examples of cell types that may be used in certain methods of the invention are provided in the Examples section, as are non-limiting examples of organisms that may subjected to certain methods of the invention.
A cell that includes an XRI-based recorder system and/or component of the invention may be a single cell, an isolated cell, a cell in culture, an in vitro cell, an in vivo cell, an ex vivo cell, a cell in a tissue, a cell in a subject, a cell in an organ, a cell in a cultured tissue, a cell in a neural network, a cell in a brain slice, a neuron, a cell that is one of a plurality of cells, a cell that is one in a network of two or more interconnected cells, a cell in communication with another cell, a cell that is one of two or more cells that are in physical contact with each other, etc. It will be understood that methods of the invention can be carried out in a plurality of cells such that one or more cells comprises the XRI-based recorder system of the invention. Inclusion of a system of the invention in a plurality of cells permits monitoring and determining one or more alterations in expression across the plurality of cells. It will be understood that when assessing expression and history in a plurality of cells, a plurality of cells may be prepared to contain an expressed and/or encoded XRI recorder of the invention and the status of expression in the plurality of cells can be determined at one or more different time points by obtaining one or more cells from the plurality at the one or more different time points and determining the expression and/or history in the obtained cell or cells. At a different time point, another cell or other cells may be obtained from the plurality of cells and also assessed. Results of two or more assessments done in cells obtained from the plurality at different times can be compared to determine a change in expression in the plurality of cells. Results using cells obtained at two or more times can be used to assess changes in expression in the plurality of cells over time and under different conditions. For example, one or more cells may be obtained from a plurality of cells comprising expressed and/or encoded XRI of the invention and assessed for expression, then the plurality of cells may be contacted with a candidate stimuli and another cell or cells obtained following the contact can be assessed and compared to the initial assessment or a control assessment as a determination of the expression history of the plurality of cells.
An XRI-based recorder system of the invention and methods of using such recorder systems can be utilized to assess changes in cells, tissues, and subjects in which the system is included. Some embodiments of the invention include use of an XRI-based recorder system of the invention to identify effects of candidate stimuli on cells, tissues, and subjects. Results of testing cell expression activity using an XRI-based recorder of the invention can be advantageously compared to a control. In some embodiments of the invention, an XRI-based recorder system may be in a cell or cell population and used to test the effect of candidate stimuli on the cell or population, respectively. A “test” cell, tissue, or organism may be a cell, tissue, or organism in which activity of an XRI-based recorder system of the invention can be determined or assayed. Results obtained using assays and tests of a test cell, tissue, or organism may be compared with results obtained from the assays and tests performed in other test cells, tissues, or organisms or assays and tests performed in control cells, tissues, or organisms.
As used herein a control value may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as cells or tissues that include an XRI-based recorder system of the invention that is under essentially the same conditions of test cells but are not contacted with a candidate compound. Another non-limiting example of a comparative group includes cells or tissues that have a disorder or condition and groups without the disorder or condition. Another non-limiting example of comparative group includes cells from a subject or subjects with a family history of a disease or condition and cells from a subject or subjects without such a family history. A predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups based on results of testing. Those skilled in the art are able to select appropriate control groups and values for use in comparative methods of the invention.
Administration of a component of an XRI-based recorder system of the invention may include, but is not limited to: administering to a cell or subject a composition that includes a vector comprising a polynucleotide sequence that encodes the XRI, administering to a cell or subject a composition comprising the vector, and administering to a subject a cell in which the vector and/or encoded XRI is present. A composition of the invention optionally includes a carrier, which may be a pharmaceutically acceptable carrier.
A component of an XRI-based recorder system of the invention may be administered to a cell and/or subject in a formulation, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally additional ingredients. In some aspects, a pharmaceutical composition comprises one or more XRI-based recorder system component(s) of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically acceptable carriers are well known to the skilled artisan and may be selected and utilized using routine methods. As used herein, a pharmaceutically acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and others are known by those in the art.
The terms “delivery into” and “include” when used herein to describe an action that results in a component of an XRI-based recorder system of the invention being present in a cell, are intended to encompass delivery of the component(s) into the cell (for example, though not intended to be limiting, in the form of a fusion protein), and delivery of a polynucleotide sequence that encodes the component and that is subsequently expressed in the cell. A component of an XRI-based recorder system of the invention may be administered using art-known methods. The absolute amount to be delivered can be determined using routine methods. The delivery may be done in a single administration, a single or multiple deliveries, and if delivered into a subject may be based on individual subject parameters including age, physical condition, size, weight, and the stage of a disease or condition, test parameters to be followed, etc. These factors can be addressed with no more than routine experimentation.
Various modes of administration will be known to one of ordinary skill in the art that can be used to effectively deliver one or more components of an XRI-based recorder system of the invention in a desired cell, tissue, cell of a subject, organ of a subject, or region of a subject. Methods for administering a composition comprising a component of an XRI-based recorder system of the invention may include, but are not limited to: injection, microinjection, perfusion, electroporation, or other suitable means. The invention is not limited by the particular modes of administration disclosed herein and additional art-known delivery means may be suitable for administration of components of an XRI-based recorder system of the invention.
Other protocols suitable for administration of one or more components that are part of an XRI-based recorder system of the invention are known to those in the art. Embodiments of methods of the invention to administer a cell or vector to increase a level of a component of an XRI-based recorder system of the invention in an animal other than a human; and administration and use of an XRI-based recorder system of the invention for testing purposes or veterinary purposes, are substantially the same as described above. It will be understood by a skilled artisan that this invention is applicable to both human and animals.
Disorders, conditions, and events that may be assessed using methods of the invention to include an XRI-based recorder of the invention in a cell, tissue, and/or subject and to use the system to determine characteristics of expression in the cell. Methods and systems of the invention may be used to assess early stage development, cell and tissue regeneration, cell communication, disease, etc. Diseases that may be examined using methods and systems of the invention include, but are not limited to injury, brain damage, spinal cord injury, epilepsy, metabolic disorders, cardiac dysfunction, vision loss, blindness, deafness, hearing loss, and neurological conditions (e.g., Parkinson's disease, Alzheimer's disease, and seizure), degenerative neurological conditions, drug contact, toxins, etc. In some embodiments of the invention, a disorder or condition may be monitored by including an XRI-based recorder system of the invention in at least one cell and monitoring characteristics of expression in the cells using the recorder system. In some embodiments of the invention, such methods can be used in methods such as, but not limited to, assessing therapeutic agents and treatments, assessing putative therapeutic agents and treatments, expanding understanding of connectivity between cells, and exploring expression activity patterns in a cell or cells. An XRI-based recorder system of the invention may be targeted to cells and used to monitor expression changes in such cells.
The present invention in some aspects, includes one or more of preparing nucleic acid sequences that encode one or more components of an XRI-based recorder system of the invention, expressing in cells one or more components of an XRI-based recorder system encoded by the prepared nucleic acid sequences; and monitoring changes expression in the cell by assessing changes in a level of expressed protein, determine an amino acid sequence of proteins expressed in the cell, and/or determining the presence, location, and/or amount of one or more XRI detectable tags expressed in the cell. The ability to specifically, consistently, reproducibly, and sensitively monitor changes in the XRI composition using methods such as imaging and amino acid sequence determination and single cell assessment of such characteristics has been demonstrated. The present invention enables monitoring of expression changes in in vivo, ex vivo, and in vitro, and the XRI-based recorder system and its use have broad-ranging applications for drug screening, disease assessment, treatment assessment, and research applications, some of which are describe herein.
Animals and neuron cultures. All procedures involving animals at Massachusetts Institute of Technology were conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Massachusetts Institute of Technology Institutional Animal Care and Use and Biosafety Committees.
Molecular Cloning. The DNAs encoding the protein motifs used in this study were mammalian-codon optimized and synthesized by Epoch Life Science and then cloned into mammalian expression backbones, pAAV-UBC (for constitutive expression), pAAV—UBC-FLEx (for Cre-dependent expression), or pAAV-cFos (for expression driven by the c-fos promoter) for DNA transfection in cultured neurons and AAV production by Janelia Viral Tools. See Table 1 for sequences of the motifs; see Table 2 for all tested constructs.
DNA Transfection and AAV Transduction. For experiments with results shown in
For experiments with results shown in
Chemical Treatments and Stimulations of Cultured Cells. In 4-OHT induction experiments, (results in
For potassium chloride (KCl) treatments (results in
DNA Transfection in Cultured U2OS cells. Human bone osteosarcoma epithelial cells (U20S cells; ATCC) were maintained between 10% and 90% confluence at 37° C. with 5% C02 in DMEM (Gibco) with the addition of 10% heat inactivated fetal bovine serum (HI-FBS) (Corning), 1% penicillin/streptomycin (Gibco), and 1% sodium pyruvate (Gibco), in glass-bottom 24-well plates pre-treated with 75 μL diluted Matrigel (250 μL Matrigel (Corning) diluted in 12 mL DMEM) per well at 37° C. for 30-60 minutes. The DNA plasmid was transiently transfected into U20S cells using the TransIT-X2 Dynamic Delivery System kit (Mirus Bio) according to the manufacturer's protocol.
Electrophysiology. For experiments with results shown in
Animals and Mouse Surgery. All procedures involving animals at Boston University were conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Boston University Institutional Animal Care and Use and Biosafety Committees.
For experiments with results shown in
For experiments with results shown in
4-Hydroxytamoxifen Injection. For experiments with results shown in
Histology. For experiments with results shown in
Immunofluorescence ofcultured cells. In studies with results shown in
Immunofluorescence of brain slices. In studies with results shown in
Expansion microscopy of cultured cells. In studies with results shown in
Acryloyl-X (6-((acryloyl)amino)hexanoic acid, succinimidyl ester (AcX) (Invitrogen) was resuspended in anhydrous DMSO (Invitrogen) at a concentration of 10 mg/ml, and stored in a desiccated environment at −20° C. For anchoring, cells were incubated in 200 μL of AcX at a concentration of 0.1 mg/ml in a 2-(N-morpholino)ethanesulfonic acid (MES)-based saline (100 mM MES, 150 mM NaCl) overnight at 4° C. Then, cells were washed with 1×PBS three times at RT for 5 minutes each.
Gelation solution which contains 1.1 M sodium acrylate (Sigma), 2 M acrylamide (Sigma), 90 ppm N,N′-methylenebisacrylamide (Sigma), 1.5 ppt ammonium persulfate (APS) (Sigma), and 1.5 ppt tetramethylethylenediamine (TEMED) (Sigma) in 1×PBS was prepared fresh. Cells were first incubated on ice for 10 min with shaking to prevent premature gelation and enable diffusion of solution into samples. A gelation chamber was prepared by placing two No. 1.5 coverslips on a glass slide spaced by about 8 mm to function as insulators on either end of the neuronal coverslip to avoid compression and each coverslip containing a neuronal cell culture sample was placed on a gelation chamber with the cells facing down. The gelation chamber was filled with gelation solution and a coverslip placed over the sample and across the two insulators to ensure the sample was covered with gelling solution and no air bubbles were formed on the sample. Samples incubated at 37° C. for 1 hours in a humidified atmosphere to complete gelation. Following gelation, the top coverslip was removed from the samples, and only the sample gel was transferred into a 1.5 mL tube containing 1 mL of denaturation buffer, consisting of 5% (w/v) sodium dodecyl sulfate (SDS), 200 mM NaCl, and 50 mM Tris at pH 8. Gels were incubated in denaturation buffer overnight at RT and 3 hour at 80° C., followed by washing in water overnight at RT to remove residual SDS. Gels were then stored in 1×PBS at 4° C. before immunostaining.
For immunostaining and imaging, gels were first incubated in bovine serum albumin (BSA) blocking solution that contains 1% BSA, 0.5% Triton-X in 1×PBS for 1 hour at RT then with primary antibodies overnight at 4° C. Gels were washed three times in BSA blocking solution for 30 minutes each at RT and incubated with fluorescently-labeled secondary antibodies overnight at 4° C. Gels were then washed three times in BSA blocking solution for 30 minutes each at RT and expanded in water overnight at 4° C. before imaging.
Fluorescence Microscopy of Immunostained Samples. Fluorescence microscopy was performed on a spinning disk confocal microscope (a Yokogawa CSU-W1 Confocal Scanner Unit on a Nikon Eclipse Ti microscope) equipped with a 40X 1.15 NA water immersion objective (Nikon MRD77410), a Zyla PLUS 4.2 Megapixel camera controlled by NIS-Elements AR software, and laser/filter sets for 405 nm, 488 nm, 561 nm, and 640 nm optical channels. For each field of view, multi-channel volumetric imaging was performed at 0.4 μm per Z step. Imaging parameters were kept the same for all samples within a set of experiments (e.g., a set of 4-OHT induction experiments in which samples were treated with 4-OHT at different time points).
Antibodies and Nissl Stain. The following antibodies and Nissl stain were used in certain studies described herein: primary antibodies, anti-HA (Santa Cruz, cat #sc-7392), anti-FLAG (Invitrogen, cat #740001), anti-V5 (Abcam, cat #ab9113), anti-NeuN (Synaptic Systems, cat #266004), anti-GFAP (Cell Signaling Technology, cat #12389), anti-Ibal (Wako Chemicals, cat #019-19741), anti-Synaptophysin (Sigma, cat #S5768), anti-Cleaved Caspase-3 (Cell Signaling Technology, cat #9664), anti-gH2AX (Millipore, cat #05-636), anti-Hsp70 (Cell Signaling Technology, cat #4872), anti-Hsp27 (Cell Signaling Technology, cat #2402); fluorescent secondary antibodies from Invitrogen, cat #A-21241, cat #A-21133, cat #A-32933, cat #A-32733, cat #A-11035, and cat #A-11073; fluorescent secondary antibodies from Biotium, cat #20308; Nissl stain, NeuroTrace Blue Fluorescent Nissl Stain (Invitrogen, cat #N21479).
Fluorescence Microscopy of Live Cells and Immunostained Samples. Fluorescence microscopy was performed on a spinning disk confocal microscope (a Yokogawa CSU-W1 Confocal Scanner Unit on a Nikon Eclipse Ti microscope) equipped with a 40X 1.15 NA water immersion objective (Nikon MRD77410), a 10X objective, a Zyla PLUS 4.2 Megapixel camera controlled by NIS-Elements AR software, and laser/filter sets for 405 nm, 488 nm, 561 nm, and 640 nm optical channels. For each field of view under 40X objective, multi-channel volumetric imaging was performed at 0.4 μm per Z step. Imaging parameters were kept the same for all samples within a set of experiments (e.g., a set of 4-OHT induction experiments in which samples were treated with 4-OHT at different time points).
RNA-Seq. For studies with results shown in
Image Analysis. Image analysis was performed in ImageJ (ImageJ National Institutes of Health) and MATLAB (MathWorks).
Intensity profile measurements. First, the somata of neurons in the images were identified by the Nissl staining (in samples without ExM) or anti-NeuN staining (in samples with ExM) channel, and XRI(s) in the soma of each neuron were identified by the anti-HA channel. If multiple XRIs were present in a soma, the XRI with the longest length as well as any XRI with length above half of that longest length was selected for downstream analysis. For each XRI, a curved centerline was drawn along the longitudinal direction of XRI in the anti-HA channel. The centerline width was set to half of the width of the XRI. The intensity profiles along this centerline with width were measured in the anti-HA channel (as HA line profile) and in other XRI epitope staining channels, such as in the anti-FLAG channel (as FLAG line profile) or anti-V5 channel (as V5 line profile).
Readout information from intensity profiles. For studies with results shown in
Then these line integrals of HA were normalized to the maximum integral value (integral from the split point (d=0) to the end of XRI (d=End)) so that each line integral of HA started at the value 0 at the geometric center point of the XRI, and gradually increased to the value 1 at the end of the XRI. We define this quantity as the ‘fraction of HA intensity line integral (H fraction integral)’:
For the corresponding half FLAG (or V5) line profiles (F), line integrals (F integral) were also calculated but not normalized:
At this point, the line integrals of HA and FLAG (or V5) had been identified, which corresponded to the cumulative HA and FLAG (or V5) intensities along each half of the XRI. The line integrals of FLAG (or V5) line profiles were then converted from the position axis (p) into the axis of the fraction of HA intensity line integral (H fraction integral) via variable substitution from p to H fraction integral (p):
The FLAG (or V5) intensity change per unit change in the cumulative HA intensity, defined as the FLAG (or V5) signal (F signal), was calculated by taking the derivative of the line integral of FLAG (or V5) with respect to the fraction of HA intensity line integral:
At this stage, the line integral of HA and the FLAG (or V5) signal was obtained from each of the halves of the XRI, and the final extracted FLAG (or V5) signal from this XRI was defined as the point-by-point average of the two FLAG (or V5) signals from the two halves of the XRI. Step 4: the two obtained FLAG (or V5) signals from the same XRI were found to have small but noticeable differences. It was determined that such small but noticeable discrepancies between the two halves of the same XRI were due to the asymmetry of the XRI, and the choice of the exact geometric center as the split point may not be optimal. To minimize the discrepancy between the two FLAG (or V5) signals from the two halves of the same XRI, a search was performed to identify an optimal split point near the geometric center of the XRI (searching range was the geometric center +/−10% of the total XRI length), so that using this optimal split point, instead of the geometric center, as the split point results in the least difference (in sum of squared differences) between the two FLAG (or V5) signals from the two halves of the splitted XRI. Step 5: Same as Step 3, except that the optimal split point, instead of the geometric center, was used to split the intensity profiles into two halves. The resulting final FLAG (or V5) signal (after averaging those from the two halves) when using the geometric center as the split point was found to be similar to that when using the optimal split point as the split point. Nevertheless, the optimal split point was used as the split point to analyze XRIs throughout studies described herein.
Calculation of the fraction of HA line integral when FLAG signal begins to rise. The FLAG signal minus the FLAG signal at the center of XRI (i.e., the optimal split point as defined above) was plotted against the fraction of HA line integral. The initial rising phase of the FLAG signal (defined as the portion of the FLAG signal between 10% to 50% of the peak FLAG signal) was fitted as a linear function, which was then extrapolated onto the axis of the fraction of HA line integral. The intersection point at the axis of the fraction of the HA line integral was defined as the fraction of HA line integral when the FLAG signal began to rise.
Statistical analysis. All statistical analysis was performed using the built-in statistical analysis tools in Prism (GraphPad) or MATLAB, except for the statistical analysis of the RNA-Seq data, which was performed using DESeq2. The statistical details of each statistical analysis can be found in the figure descriptions provided elsewhere herein, except for the statistical details of the RNA-Seq data.
Initial studies were performed to test if human-designed proteins known to self-assemble into filaments, could be coaxed to reliably form continuously growing linear chains in cultured mammalian cells. In the studies 14 human-designed filament-forming proteins (previously characterized in buffers, bacteria, and yeast) were fused to a short epitope tag (HA, for immunofluorescence imaging after protein expression and cell fixation) and expressed in primary cultures of mouse hippocampal neurons (see Table 1 for sequences of the motifs; see Table 2 for all tested constructs). Upon immunofluorescence staining, followed by imaging under confocal microscopy, two filament-forming proteins produced clear and stable fiber-like structures in the cytosol: 1POK(E239Y), a human-engineered filament-forming protein based on an E. coli isoaspartyl dipeptidase (Garcia-Seisdedos, et al. Nature 548, 244 (2017);
Because linear protein assembly would enable useful information encoding that could then be easily read out, next protein engineering was performed on 1POK to reduce the unstructured aggregates in cells. It was reasoned that unstructured aggregates could be present due to unwanted lateral growth (
Next, studies were performed that tested if the mEGFP or MBP tag-bearing variants could encode information along their linear extent while preserving temporal order of the information along their corresponding protein assemblies. If protein monomers with, say, the epitope tag HA are constantly expressing, and the expression of protein monomers with, say, the epitope tag FLAG are induced at a specific time point, then at that time point, monomers with the FLAG tag will be more common, and thus preferentially added over those containing HA, along the growing protein chain; then, the period of time at which FLAG is expressed could be easily read out via immunostaining against both HA and FLAG tags (FIG. 1F). In certain studies the ERT2-iCre-ERT2 based chemically inducible Cre system [Matsuda, T., & C. L. Cepko Proc. Natl. Acad. Sci. U.S.A 104, 1027-1032 (2007)] was used to activate the expression of protein monomers with the FLAG tag, in a Cre-dependent FLEX vector, by 4-hydroxytamoxifen (4-OHT) treatment at defined times (
In order to characterize the electrophysiological integrity of neurons expressing XRIs, a bicistronic adeno-associated virus (AAV) construct was prepared, that contained mEGFP-P2A-XRI-HA, where P2A is a well-known self-cleaving peptide [Kim, J. H. et al. PLoS One 6, e18556 (2011)] (
This bicistronic AAV construct was then used to track XRI formation over time in live neurons, by imaging the GFP fluorescence in the same neurons daily for 7 days post AAV transduction (
Next, electrophysiology and RNA-Seq analysis of cultured neurons expressing XRI was performed and it was observed that XRI expression did not alter the electrophysiology and endogenous gene expression in these neurons (
To study how accurate this XRI protein assembly could preserve time information, the chemically-inducible Cre system was again used and different neuron cultures expressing the XRI were treated with 4-OHT at different times after beginning of expression. To increase the efficiency of gene delivery, adeno-associated viruses (AAVs) were also used to deliver the chemically-inducible Cre system and the XRI genes into cultured mouse neurons. Because the expression of AAV is slower compared to DNA transfection, the expression time window was increased from 3 days to 7 days before fixation, immunofluorescent labeling, and imaging. The neuron cultures were divided into 7 groups, and 4-OHT treatment was added at 1, 2, 3, 4, 5, or 6 days after AAV transduction, or not at all (
Next, studies were performed to quantify the relationship between the times of 4-OHT treatment and the resulting FLAG immunofluorescence patterns on XRI assemblies in neurons. Because the XRI growth is bidirectional over the 7-day experiment, the fractional cumulative HA expression (i.e., the normalized, unidirectional line integral of HA immunofluorescence starting from the center of the XRI) at the center of the XRI was defined as ‘0’ and at the end of the XRI was defined as ‘1’ (see
Results were then examined to determine whether HA-bearing and FLAG-bearing monomers were adding independently, each at a rate independent of the presence of the other monomer. If the binding and retention of HA-bearing monomers and FLAG-bearing monomers onto the XRI were both rare enough in time that the chance of both types of monomers competing for the same slot on the XRI was insignificant, then this would be plausible. And, in this case, the fractional cumulative HA expression would still be a proper, calibratable measure of time. That is, if units with a new tag are supplementing the units being constitutively synthesized bearing an old tag, the latter units would not be added at a slower rate (i.e., there is no competition between the new units and the old units for being added to the growing chain), but instead would be added at the same rate, but simply be spaced out further from each other, separated by the units bearing the new tag. This would make the line integral the appropriate measure for extracting absolute time measurements.
Experiments were performed to empirically test the hypothesis that absolute time measurements could be extracted from this specific measure. The FLAG signals across the two halves of the XRI (because XRIs are symmetric) were averaged, to obtain the final FLAG signal (
To quantify the fraction of the line integral of HA intensity at which the FLAG signal began to rise, the net waveform of the FLAG signal was generated with respect to the fraction of the line integral of HA intensity, by subtracting the baseline (i.e., the FLAG signal when the fraction of the line integral of HA intensity is zero) from the FLAG signal (
Experiments were then performed to explore whether XRIs could be used to record gene expression timecourse under mammalian immediate early gene (IEG) promoter activation. IEG promoters, such as the c-fos promoter [Roy, D. S. et al. Nat. 2016 5317595 531, 508-512 (2016)], are widely used to couple the expression of reporter proteins to specific cellular stimuli [Kawashima, T., et al., Frontiers in Neural Circuits 8, 37 (2014)]. By using the c-fos promoter to drive the expression of XRI subunits tagged by a unique epitope tag, here the V5 tag, the time course of c-fos promoter driven expression could be recorded along the XRI filament, and read out by measuring the intensity profiles of V5 immunostaining signals along the filament. The V5 tag was chosen to use here, instead of the previously used FLAG tag, so that each new XRI construct would be tagged by a unique epitope tag: in future usage of XRIs, one may want to co-express multiple XRI constructs in the same cell to achieve multiplexed recording of several different kinds of biological signals, readable via multiplexed immunostaining against distinct epitope tags. HA-bearing XRI, driven by the UBC promoter, was expressed in neurons using AAV as in the experiments in
To validate the XRI-recorded time course of c-fos promoter driven expression, studies were carried out that included performing time-lapse imaging, one image per day, of cultured neurons transduced with an AAV construct encoding c-fos promoter-driven expression of GFP, under the same KCl stimulation (
To assess the sensitivity of the XRI fos recorder, experiments were carried out that included XRI recording of c-fos promoter driven XRI expression with different doses and durations of KCl stimulation (
Next, experiments were performed to test whether XRI could preserve temporal information in the living mammalian brain. The same XRI AAVs used in
The XRIs in 835 CA1 neurons were analyzed in confocal imaged volumes and plotted the absolute, baseline subtracted (baseline defined as the signal at the center of XRI) FLAG signals with respect to the fraction of the line integral of HA intensity, and the same analysis was performed on XRIs in 475 CA1 neurons in another mouse that underwent the same experimental pipeline but without 4-OHT injection (
Statistical analysis. All statistical analysis was performed using the built-in statistical analysis tools in Prism (GraphPad) or MATLAB. The statistical details of each statistical analysis can be found in the figure descriptions.
It is to be understood that the methods, compositions, and apparatus that have been described above are merely illustrative applications of the principles of the invention. Numerous modifications may be made by those skilled in the art without departing from the scope of the invention. Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the following claims. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.
This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional application Ser. No. 63/254,829 filed Oct. 12, 2021, the disclosure of which is incorporated by reference herein in its entirety.
This invention was made with government support under grants 1R24MH106075, R44EB021054, 1R01DA045549, 1R01MH114031, 2R01DA029639, 1R01EB024261, 1DP1NS087724, and 1R01GM104948 awarded by the National Institutes of Health; grant W911NF1510548 awarded by the U.S. Army Research Laboratory and the U.S. Army Research Office; and grant CBET 1344219 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63254829 | Oct 2021 | US |
