This Patent Convention Treaty (PCT) International Application claims the benefit of priority to U.S. Provisional Application No. 62/457,879 filed February 11, 2017. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant nos. ROl HL098472; K02 HL109142; and ROl HL 121365. The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to mechanogenetics, cell biology and ultrasound technologies. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for remotely-controlled and non-invasive manipulation of genetic processes in live cells, e.g., for monitoring physiologic processes, for the correction or treatment of pathological processes and for control of therapeutic outcomes. In alternative embodiments, provided are ultrasound-based mechanical stimulations and a mechano-sensitive protein, e.g., a transmembrane protein or a channel or channels, either synthetically engineered or natively (endogenously) occurring, integrated to control the production of nucleic acids, genes and/or biological-active proteins, which can be used, in alternative embodiments, for diagnostic or therapeutic purposes. In alternative embodiments, exemplary mechanogenetic systems provided herein, being based on ultrasound, allow a deep penetration of stimulation and manipulation in vivo at centimeter-level depth with high spatiotemporal precision.

BACKGROUND

Fluorescent proteins (FPs) and their derived biosensors based on fluorescence resonance energy transfer (FRET) have revolutionized the biology/medicine by allowing the visualization of dynamic molecular activities in live cells. In parallel, it has long been a dream in biology to be able to dynamically manipulate molecular activities in live cells. Together with chemical inducers (e.g. dimerizers),

optogenetics integrating optical and genetic methods has emerged to enable the control of specific molecular events in the superficial parts of living systems.

However, there is a critical need to non-invasively manipulate cells deep in the body, particularly for clinical and translational applications.

Ultrasound can be focused to deliver mechanical energy safely and

noninvasively into small volumes of tissue deep inside the body. Microbubbles are highly responsive to ultrasound due to a large difference in acoustic impedance between the surrounding media and the gas inside the bubble. Therefore, oscillatory pressure of ultrasound waves can exert strong mechanical force on cells to which the microbubbles are physically coupled.

While disparate trial-and-error approaches utilizing ultrasound have been applied to mechanically perturb cells and C. elegans, there is no established method to convert the mechanical signals into genetic controls of cells.

SUMMARY

In alternative embodiments, provided are: products of manufacture, multiplexed systems and kits for practicing methods for remotely-controlling and noninvasively manipulating a physiologic and/or a genetic process in a cell, or for expressing an exogenous protein in a cell; and, methods for remotely-controlling and non-invasively manipulating a physiologic and/or a genetic process in a cell, or for expressing an exogenous protein in a cell, comprising:

(a) providing a cell or plurality of cells expressing on its extracellular surface a mechanoresponsive protein, e.g., a mechanoresponsive transmembrane protein or channel, wherein optionally the mechanoresponsive protein or channel comprises a MechanoSensitive channel (MS channels) such as a Piezol,

and optionally the mechanoresponsive protein or channel is an exogenous protein or an endogenous protein, or a recombinantly engineered protein, or for example, a exogenous protein encoded by a nucleic acid implanted in the genome of a transgenic (non -human) animal, or an endogenous cell implanted, infected or transfected with nucleic acid encoding as exogenous protein the mechanoresponsive protein or channel, or an exogenous cell capable of expressing the mechanoresponsive protein or channel implanted into an animal or a human; (b) providing a microbubble, or a plurality of microbubbles, capable of responding to ultrasound or equivalent, wherein the microbubble or plurality of microbubbles are linked or attached to at least one, or two or more, proteins, small molecules or moieties capable of specifically binding to the mechanoresponsive protein on the extracellular surface of the cell, such that energy generated by

(remotely initiated) ultrasound stimulation of the ultrasound-responsive microbubble, or a plurality of microbubbles is transmitted to the mechanoresponsive protein to activate the mechanoresponsive protein, wherein activation of the mechanoresponsive protein causes the mechanoresponsive protein to transmit or generate an intracellular response or signal, wherein optionally the intracellular response or signal comprises an ion (optionally, a calcium) influx into the cell, or the intracellular response or signal comprises any change in the cell that results in the activation of an inducible promoter, or activation or deactivation (or inhibition of activity of) of a protein or enzyme,

and optionally the microbubble, or the plurality of microbubbles, have a size ranging between a micrometer (μπι) to a millimeter (mm) in size, for example, having a size of anywhere between 1 μπι to 500 mm in size, and optionally comprising or having stretchable shells, optionally the shells comprising a lipid or mixture of lipids, biocompatible polymers, or other biomaterials and mixtures thereof;

(c) stimulating the cell with an ultrasound (optionally remotely exposing the cell to ultrasound) in an amount sufficient to stimulate the plurality of ultrasound- responsive microbubbles, thereby causing the mechanoresponsive protein on the cell surface to transmit or generate the intracellular response or signal into the cell (wherein optionally the intracellular response or signal comprises an ion, e.g., a calcium or sodium, influx to the cell or efflux from the cell), thereby remotely- controlling and non-invasively manipulating a physiologic and/or a genetic process in the cell.

In alternative embodiments, methods as provided herein further comprise engineering into the cell or cells a Gene Transducing Module (GTM), or any equivalent thereof, such as an expression cassette or vector, such that upon stimulating the cell with ultrasound and causing the mechanoresponsive protein, e.g., mechanoresponsive transmembrane protein or channel, to transmit or generate an intracellular response or signal, a gene or nucleic acid sequence in the GTM is expressed or is optimally expressed, wherein optionally the gene or nucleic acid sequence encodes a protein, and optionally the protein affects cell physiology, or is expressed on the cell's surface, or the protein is secreted from the cell or causes a molecule to be secreted from the cell (optionally a peptide, another protein, a steroid or a hormone), and optionally the protein comprises a chimeric antigen receptor (CAR).

In alternative embodiments of the methods, or the multiplexed systems or kits, the cell is a human cell or a mammalian cell, or is a cell transplanted into an organism or an individual, or is a non-human transgenic animal genetically engineered to express a Gene Transducing Module (GTM) and an exogenous mechanoresponsive protein, e.g., mechanoresponsive transmembrane protein or channel.

In alternative embodiments of the methods, or the multiplexed systems or kits, the microbubble, or a plurality of microbubbles are connected to or caused to be operably connected to the mechanoresponsive protein by linkage or attachment directly or indirectly to at least one, or two or more, proteins, small molecules, polysaccharides, or a moiety capable of specifically binding to the

mechanoresponsive protein,

and optionally the at least one, or two or more, proteins, small molecules, polysaccharides, or moiety comprises a streptavidin (optionally bound to the microbubble, or a plurality of microbubbles) bound to an antibody or peptide

(optionally an RGD peptide) linked to a biotin, wherein the antibody specifically binds to the mechanoresponsive protein, or the RGD peptide specifically binds to an integrin, which by binding the RGD peptide transmits the ultrasound signal to the mechanoresponsive protein,

or optionally the microbubble, or a plurality of microbubbles are linked to a protein, small molecule, polysaccharide or moiety capable of specifically binding to the mechanoresponsive protein.

In alternative embodiments, provided are multiplexed systems or kits for, or used for, remotely-controlling and non-invasively manipulating, activating or inhibiting a physiologic and/or a genetic process in a cell, comprising:

(a) a cell or plurality of cells expressing on its extracellular surface a mechanoresponsive protein,

wherein optionally the mechanoresponsive protein is a mechanoresponsive transmembrane protein or channel, and optionally the mechanoresponsive protein comprises a MechanoSensitive protein or channels (MS channels), wherein optionally the MechanoSensitive protein or channel is a Piezol or equivalent, and optionally the mechanoresponsive protein is an exogenous protein or an endogenous protein, or is a recombinantly engineered protein,

and optionally the cell is an animal (a non-human) or a human cell, and optionally the non-human or human cell is implanted into an animal; and,

(b) a microbubble, or a plurality of microbubbles, capable of responding to ultrasound or equivalent, wherein the microbubble or plurality of microbubbles are linked or attached to at least one, or two or more, proteins, small molecules, polysaccharides, or moieties capable of specifically binding to the mechanoresponsive protein on the extracellular surface of the cell, such that energy generated by ultrasound stimulation of the ultrasound-responsive microbubble, or a plurality of microbubbles is transmitted to the mechanoresponsive protein to activate the mechanoresponsive protein, wherein activation of the mechanoresponsive protein causes the mechanoresponsive protein to transmit or generate an intracellular response or signal,

wherein optionally the intracellular response or signal comprises a calcium influx into the cell.

In alternative embodiments, provided are Uses of multiplexed systems or kits as described herein, for remotely-controlling and non-invasively:

- manipulating, activating or inhibiting a physiologic and/or a genetic process in a cell;

- activating a mechanico-responsive cell surface polypeptide;

- generating an intracellular signal initiated at the cell surface, wherein optionally the intracellular signal is an ion influx, optionally a calcium influx into the cell;

- treating a microbial, optionally a protozoal, a bacterial or a viral infection, or treating an intracellular microbial infection, or

- activating expression of an exogenous nucleic acid, optionally a Gene Transducing Module (GTM), an RNA-expressing cassette, or a vector, or activating expression of an endogenous nucleic acid (optionally a gene), in a cell, wherein optionally the cell is in a tissue in vivo.

In alternative embodiments, provided are multiplexed systems or kits as described herein, for remotely-controlling and non-invasively: - manipulating, activating or inhibiting a physiologic and/or a genetic process in a cell;

- activating a mechanico-responsive cell surface polypeptide;

- generating an intracellular signal initiated at the cell surface, wherein optionally the intracellular signal is an ion influx, optionally a calcium influx into the cell;

- treating a microbial, optionally a protozoal, a bacterial or a viral infection, or treating an intracellular microbial infection, or

- activating expression of an exogenous nucleic acid, optionally a Gene Transducing Module (GTM), an RNA-expressing cassette, or a vector, or activating expression of an endogenous nucleic acid (optionally a gene), in a cell, wherein optionally the cell is in a tissue in vivo.

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 is a diagram of an exemplary system and method as provided herein comprising a mechano-controlled cell activation to produce biologically active molecules and activate cellular functions by ultrasound, as described in further detail, below.

FIG. 2 schematically illustrates an exemplary ultrasound-based mechanical stimulation and detection system provided herein, including exemplary use parameters, as described in further detail, below.

FIG. 3 schematically illustrates an exemplary ultrasound-based mechanical stimulation and detection system provided herein, wherein microbubbles can be stimulated at a depth of at least about 5 centimeters; Left image: the detected microbubble responses in a time domain upon ultrasound stimulation, with amplitude (V) as a function of time in μ8εϋ; Right image: the detected microbubble responses in a frequency domain upon ultrasound stimulation, with amplitude (db) as a function of frequency in mHz. In this exemplary the excitation frequency is 2.5 mHz, and the right image illustrates the fundamental, the second harmonic and the third harmonic.

FIG. 4 schematically illustrates an exemplary ultrasound-based mechanical stimulation and detection system provided herein, wherein HEK cells expressing an exemplary mechanosensitive Piezol and coupled to microbubbles can be stimulated by ultrasound. Ultrasound stimulation caused calcium response in HEK cells expressing Piezol and coupled to microbubbles:

FIG. 4A schematically illustrates exemplary microbubbles coated with streptavidin, which are coupled to biotinylated RGD peptides; the RGD peptides and their ligation to or specific binding to integrins allows the (indirect) physical connection between the microbubbles and the mechanosensitive channel Piezol via cytoskeleton; the calcium FRET (Forster resonance energy transfer, fluorescence resonance energy transfe ) biosensor expressed in HEK cells can detect the calcium influx when Piezol channels are activated remotely (in long distance) by the ultrasound-mediated mechanical perturbation;

FIG. 4B graphically illustrates the time course of calcium signaling detected by the exemplary FRET biosensor provided herein, as illustrated in FIG. 4A, upon ultrasound stimulation, with the emission ratio of (yellow fluorescent protein for energy transfer) Ypet/ECFP (enhanced cyan fluorescent protein) as a function of time in seconds (sec); and

FIG. 4C schematically illustrates: Left image shows the cells expressing the Piezol channel with the cell in (red) frame stimulated by an ultrasound pulse (1 MHz ultrasound) as demonstrated by the heightened fluorescence; and the two images on the right show the FRET signals of the cells before and after ultrasound stimulation (1 MHz ultrasound), where the scale on the right indicates the color or shading corresponding to the level of fluorescence, with 5 being the highest level.

FIG. 5 schematically and graphically illustrates an exemplary ultrasound- based mechanical stimulation and detection system provided herein, wherein HEK cells expressing the mechanosensitive Piezol but without microbubbles showed no response to ultrasound stimulation: the image on the left shows the cells expressing the Piezol channel, with the cell in boxed (red) frame stimulated by a ultrasound pulse; the curves on the right graphically illustrate the time courses of calcium signaling of the FRET biosensor before and after ultrasound stimulation, with the emission ratio of (yellow fluorescent protein for energy transfer) Ypet/ECFP

(enhanced cyan fluorescent protein) as a function of time in seconds (sec); no calcium response can be detected in F£EK cells expressing Piezol in the absence of microbubbles.

FIG. 6 schematically and graphically illustrates exemplary systems and methods as provided herein for ultrasound stimulation to cause calcium and gene activations in engineered F£EK cells:

FIG. 6 A schematically illustrates exemplary microbubbles coated with streptavidin and coupled to biotinylated RGD peptides, which are attached on integrins and hence connected to Piezol; the ultrasound-induced calcium influx and FAT activation can drive the reporter production via a 1 -stage (left) or 2-stage (right) Gene Transducing Module (GTM); the initial product of 2-stage GTM is LexA-VPR, which can drive the final reporter production via a second gene cassette;

FIG. 6B illustrates that the exemplary calcium FRET biosensor expressed in engineered HEK cells can detect the calcium influx when targeted by ultrasound, note the cells within the marked broken (pink) circles; no calcium change was observed in cells without Piezol (data not shown); the images taken at 0 seconds, and at plus 10 and 40 seconds, noting the fluorescence at plus 10 seconds, where the scale on the left indicates the color or shading corresponding to the level of fluorescence, with 8 being the highest level;

FIG. 6C graphically illustrates the ratios of the inducible firefly luciferase and the constitutively expressed renilla luciferase increased upon chemical (iono:

ionomyosin) or ultrasound (Ultras) stimulation: Left: 1 -stage GTM; Right: 2-stage GTM;

FIG. 6D illustrates the reporter FP expression (lower panels) can be induced upon ionomyosin or ultrasound stimulation; the upper panels show the expression of Piezol fused to tdTomato.

FIG. 7 schematically and graphically illustrates that GTMs can be integrated into the endogenous molecular network of Jurkat cells to sense the stimulation of calcium signaling and guide gene expressions for the control of cellular functions in T cells, as discussed in detail in Example 2, below: FIG. 7 A schematically illustrates exemplary schemes of the ionomycin- induced reporter production via a 1 -stage (left) or 2-stage (right) GTM;

FIG. 7B graphically illustrates data showing the 1 -stage (left image) or 2-stage (right image) level of GTM activation (normalized bioluminescence measured with our without Ionomycin at 30 minutes (min)), where Ionomycin-activated the inducible firefly luciferase production in Jurkat T cells.

FIG. 8 graphically illustrates data showing that ultrasound can induce different reporters and Gene Transducing Module (GTM) expression, in particular, in this study, ultrasound can induce CD19-CAR gene expression:

FIG. 8A illustrates Jurkat cells transfected with inducible luciferase with minimal promoter (with relative protein expression measured with or without induction); the ultrasound stimulation can induce the luciferase expression in these Jurkat cells, note the 2.4x induction of protein expression;

FIG. 8B illustrates Jurkat cells were transfected with inducible luciferase with CMV minimal promoter (with relative protein expression measured with or without induction); the ultrasound stimulation can induce the luciferase expression in these Jurkat cells, note the 2.4x induction of protein expression;

FIG. 8C illustrates the ultrasound stimulation can induce the CD19CAR expression in these Jurkat cells at an mRNA level (with relative mRNA expression measured with or without induction), note the 1.3x induction of mRNA expression.

FIG. 9 graphically illustrates data showing that ultrasound can induce CD 19- chimeric antigen receptor (CAR) gene expression and activation of Jurkat cells against target cancer cells:

FIG. 9A schematically illustrates how Jurkat cells were transfected with inducible recombinant chimeric antigen receptor (CAR) (ReCoM-CAR): after ultrasound stimulation, the transfected Jurkat cells were mixed with antigen CD 19- expressing target tumor cells and evaluated for their activation level (CD69 expression);

FIG. 9B-C illustrate that ultrasound stimulation can induce calcium elevation in the ReCoM-CAR transfected Jurkat cells without additional exogenous Piezol or mechano-sensors, possibly because Jurkats express high levels of endogenous Piezol and other mechanosensitive channels: in FIG. 9B left image is an unstimulated cell, and FIG. 9B right image illustrates the fluorescent emission of the stimulated cell, where the scale on the right indicates the color or shading corresponding to the level of fluorescence, with 5 being the highest level; and FIG. 9C graphically illustrates the normalized ratio of FRET/ECFP is measured with and without ultrasound stimulation;

FIG. 9D graphically illustrates that ultrasound stimulation can induce transfected CD19CAR expression in these Jurkat cells, with relative protein expression measured with and without ultrasound stimulation.

FIG. 10 graphically illustrates data showing that ultrasound can induce transfected CD19-CAR activation in Jurkat cells against target cancer cells:

FIG. 10A illustrates the engineered Jurkat cells can be stimulated by ultrasound to allow the engagement of Toledo cells, which leads to the expression of CD69 as an activation marker of Jurkat cells; representative histograms of T cell activation in

Jurkat cells were shown by quantifying the expression of cell surface protein marker CD69;

FIG. 10B graphically illustrates the bar graphs representing CD69 up- regulation (normalized percentage of CD69 positive cells) in ultrasound-induced Jurkat cells upon Toledo cell engagement; these results demonstrate that GTMs can be engineered into Jurkats for the ultrasound-induced production of CARs which can mediate the immune engagement with cancer cells to activate Jurkats.

FIG. 11 graphically illustrates data showing that ultrasound can induce the CD 19 CAR expression and activation of PBMC cells against target cancer cells (Nalm6):

FIG. 11 A schematically illustrates how PBMC cells were transfected with inducible ReCoM-CAR; and after ultrasound stimulation they were mixed with antigen CD19-expressing target tumor cells and evaluated for killing efficiency;

Fig 1 IB graphically illustrates data showing that the ultrasound stimulation can induce calcium elevation in these PBMCs without additional exogenous Piezol or mechano-sensors, possibly because Jurkats express high levels of endogenous Piezol and other mechanosensitive channels, where normalized fluoro-4 intensity was measured with or without ultrasound stimulation;

FIG. l lC graphically illustrates data showing that the ultrasound stimulation can induce the CD19CAR expression in these PBMC cells, where relative protein expression was measured with or without ultrasound stimulation;

FIG. 1 ID graphically illustrates data showing that the cytotoxicity of PBMC cells transfected with ReCoM-CAR for target Nalm6 tumor cells upon ultrasound stimulation measured by luciferase based killing assay (the "relative killing effect"); ultrasound induced ReCoM PBMCs can cause significantly more toxicity for Nalm6 cells than the ReCoM PMBCs not exposed to ultrasound or the plain PBMCs exposed to ultrasound but without ReCoM GTMs.

Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION

In alternative embodiments, provided are compositions, including products of manufacture, kits and multiplexed systems, and methods, for remotely-controlled and non-invasive manipulation of genetic processes in live cells, e.g., for monitoring physiologic processes, which in alternative embodiments are used in vivo for the correction or control of pathological processes or genetic conditions, for the treatment of diseases or infections, and/or for the control of therapeutic outcomes.

We developed novel ReCoM (remote-controlled mechanogenetics) systems and methods based on ultrasound that can allow the remotely-controlled and noninvasive manipulation of physiologic and genetic processes in live cells for, e.g., monitoring physiologic processes, and optionally used in vivo for the correction or control of pathological processes or genetic conditions, for the treatment of diseases or infections, and/or for the control of therapeutic outcomes. Our results provide clear evidence that mechano-sensors and genetic transducing modules (GTMs) can be engineered and integrated into the endogenous molecular network of live cells for the sensing of remote ultrasound stimulation to guide a cell surface event, e.g., the activity of a mechanoresponsive protein, which optionally can activate an ion influx into the cell, which can result in a gene activation or expression of a protein or other physiologic process.

In alternative embodiments, the ReCoM approach provided herein can remotely control cell physiology or a nucleic acid (e.g., a gene) activation, and thus remotely control or change a cell behavior, at a distance in centimeters in vivo with high spatiotemporal precision in a non-invasive and biomedically compatible manner. ReCoM brings the full power of remote control of cell physiology and gene or cell activation to the general scientific and clinical community similar to how fluorescent proteins and optogenetics have revolutionized live biological sensing and actuating.

In alternative embodiments, several exemplary components of the ReCoM approach provided herein include: Cells: A biologically-active cell population, wherein the cell optionally can comprise human or non-human cells, or the cells can be transduced or transfected with a nucleic acid, e.g., a GTM.

Gene Transducing Modules (GTMs): desirable protein-encoding, antisense, miRNA and/or gene sequences are engineered into the cells, such that upon activation by the remotely controlled systems as provided herein, the nucleic acids or gene sequences are inducibly expressed, e.g., optimally expressed. In alternative embodiments, a desirable nucleic acid or gene sequence product may be biologically active, for example, it can be an antibody or a receptor such as a chimeric antigen receptor (CAR). In alternative embodiments, the nucleic acid or gene comprises (is operatively linked to) an element responsive to a signal, such as calcium, produced by certain sensors/proteins upon ultrasound stimulation; for example, the nucleic acid or gene are operatively linked to a promoter that is activated directly or indirectly when the cell surface mechanoresponsive protein is stimulated by its remotely controlled mechanic-stimulation.

MechanoSensors: mechanoresponsive proteins, either membrane bound, cytosolic, or compartmentalized in subcellular organelles, including

MechanoSensitive channels (MS channels) such as Piezol (Piezol is a mechanosensitive ion channel protein that in humans is encoded by the gene PIEZOl; for example, in one embodiment Homo sapiens mRNA for PIEZOl, or KIAA0233 gene GenBank ACCESSION D87071, or recombinant proteins encoded by genes or mRNA encoding PIEZOl, or active fragments thereof, are used), are engineered into cells for protein and cell surface expression and ultrasound sensing. MS channels which can be used are ion channels found in a number of tissues and organisms and can be sensors for systems including the senses of touch, hearing and balance, cardiovascular regulation and osmotic homeostasis such as thirst.

Microbubbles: responsive to ultrasound, which is an optimal method for amplifying mechanical stimulation of ultrasound and allowing the remote delivery of an active signal (e.g., a medically active signal) to the cells, which can be physically coupled to the microbubbles. Optionally the microbubble, or the plurality of microbubbles, have a size ranging between micrometer to millimeter in size, for example, having a size of anywhere between 1 μιη to 500 mm in size, and optionally comprising or having stretchable shells, optionally the shells comprising lipids, biocompatible polymers, or other biomaterials; and optionally the microbubble, or the plurality of microbubbles, have a composition or can be prepared as described by e.g., Heureaux Cell Mol. Bioeng. 2014 Sep;7(3):307-319, e.g., comprise TARGESPHERE™-SA microbubbles

(Targeson, San Diego, CA).

- In alternative embodiments, microbubbles used to practice methods and multiplexed systems as provided herein are those that have been well established as ultrasound imaging contrast agents and approved by the FDA for clinical use; microbubbles serve to amplify the ultrasound-induced mechanical signals for the cell activation compatible to treating medical conditions in vivo.

- In alternative embodiments, microbubbles used to practice methods and multiplexed systems as provided herein are injected or otherwise placed into a tissue, e.g., a tissue in vivo, for binding to a desired cell; in alternative embodiments, for these embodiments the microbubbles are formulated as sterile formulations, or formulations appropriate for injection in a tissue in vivo, and can be formulated or stored as a carpule, ampule or cartridge; and optionally are formulated at between about 10 ² to 10 ¹⁰ microbubbles/ml in e.g., a sterile saline. Combination: In alternative embodiments, microbubbles that are coupled to the cell body or cell surface do not inhibit the mechanosensor's proper (natural) functions. In alternative embodiments, the microbubbles are coupled to the cells by their linkage or attachment to at least one, or two or more, proteins, small molecules or moieties capable of specifically binding to the mechanoresponsive protein on the extracellular surface of the cell.

Combination: In alternative embodiments, the microbubbles are coupled to a Surface Protein mechanosensor, and do that do not inhibit the Surface Protein's proper (natural, wild type) function.

In alternative embodiments, provided are methods comprising:

(1) Genes or other nucleic acids encoding MechanoSensors for expression, and Gene Transducing Modules (GTMs) (optionally, as vectors), are delivered or transduced into a target cell (or they can be delivered into a tissue in vivo), and

(2) Microbubbles are delivered to the target cell or a desired tissue, and the microbubbles then become coupled to the cell surface or the cell's body; where optionally such gene or nucleic acid and/or microbubble delivery is done in the presence of target cells, and in such quantities to achieve a biological effect, e.g., mechanic-stimulation of a mechanic-responsive cell surface protein such as e.g., an ion channel protein. Ultrasound stimulation is remotely provided to stimulate the microbubbles such that the

mechanoresponsive domain of the Surface Protein Mechanosensors is activated, causing a signal to pass into or within the cells (e.g., such as an increase in an ion, e.g., resulting in an increase in intracellular calcium), e.g., that stimulates the nucleic acid or gene expression in a GTM, or stimulates an endogenous gene, leading to the production of a biologically-effective protein encoded in the GTM or gene. The new or increased presence of a

biologically-effective protein in the cell achieves a desirable effect, such as expression of a receptor, e.g., a chimeric antigen receptor (CAR) on a T cell to result in the killing of a target cell such as a cancer cell.

It is described here for the first time methods and systems that integrate an ultrasound-based mechanical stimulation (or an ultrasound-mediated mechanical perturbation) and a cell surface mechano-sensitive channel control or initiate a physiologic process, e.g., the synthesis of a nucleic acid (e.g., production of a message or a gene, exogenous or endogenous) and/or a biological-active protein, which can be used, in alternative embodiments, for diagnostic or therapeutic purposes. In alternative embodiments, exemplary mechanogenetic systems provided herein, being based on ultrasound, allow a deep tissue penetration of ultrasound stimulation and manipulation in vivo at a centimeter-level depth with high spatiotemporal precision.

While previously ultrasound was applied to disrupt microbubbles and release anti-cancer drugs, this approach has a relatively transient effect and is limited by the microbubble targeting deficiency; and in alternative embodiments exemplary methods and systems provided herein overcome these difficulties to allow a deep penetration in vivo to control cells and their biological functions, thus enabling exemplary applications for e.g., therapeutics and diagnostics.

FIG. 1 schematically illustrates an exemplary activation mechanism for acoustic mechanogenetics as provided herein for the remote control of cell activation, or to elicit any desired response from a cell. FIG. 1 illustrates a diagram of an exemplary system and method as provided herein comprising a mechano-controlled cell activation to produce biologically active molecules and activate cellular functions by ultrasound. Cells are engineered to express a mechanosensitive channel such as Piezol, or any equivalent, and optionally also a calcium responsive construct encoding genes of interest capable of producing biologically active molecules (which will have a desired effect on the cell). An ultrasound stimulation is applied to deliver mechanical perturbation on a microbubble or plurality of microbubbles, e.g., 1 to 2 or more microbubbles (as a mechanical amplifier), which in this exemplary embodiment are coated with streptavidin to couple with a biotinylated antibody against (which can specifically bind to) the mechanosensitive channel (e.g., Piezol). This exemplary system allows the deep penetration into a tissue, e.g., penetration in centimeters, e.g., 1 to 10 cm, to mechanically perturb the engineered cells and ion channels, e.g., calcium channels, to result in an ion influx, e.g., a calcium influx. In one

embodiment, a calcium signal can activate a phosphatase such as a calcineurin to induce in the cell to dephosphorylate a transcription factor such as, e.g., a Nuclear Factor of Activated T-cells (NFAT) (e.g., NFATcl, NFATc2, NFATc3, NFATc4 or NFAT5), and subsequently its nucleus translocation. In alternative embodiments, the nuclear localization of the transcription factor, e.g., NFAT, together with other calcium-sensitive transcription factors, can activate upstream promoters and turn on the gene expression of biologically active proteins.

FIG. 2 schematically illustrates an exemplary ultrasound-based mechanical stimulation and detection system provided herein, where microbubbles are placed in a hollow tube which is submerged under water in a cuvette. An excitation ultrasound transducer can deliver a mechanical stimulation at 2.5 MHz while a receiver transducer is positioned to detect the microbubble deformation responses. The parameters of the experiment are shown on the left of the figure, which comprise 100 cycles, an applied voltage of 500 mV after power amp (a 40 dB gain) of 40 V; and a pulse repetition frequency of 1 kHz. In this exemplary embodiment, the receiving transducer receives at 10 mHz with a 170% bandwidth; and the excitation transducer emits at 2.5 mHz with a 13% bandwidth. In alternative embodiment, equivalent amounts and frequencies of ultrasound are applied to a tissue comprising an engineered cell or a cell that will be responsive to the applied ultrasound, e.g., equivalent amounts and frequencies of ultrasound are applied to and directed to a tissue of interest in an animal.

In alternative embodiments, exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are engineered to remotely control cells by ultrasound at a distance to produce biologically functional molecules and cellular outcomes. In alternative embodiments, remote control of cells by exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are used in the treatment or amelioration of diseases or abnormal cells or tissues, e.g., cells with specific cell surface markers, such as cancer cells.

In alternative embodiments, biosensors based on fluorescence resonance energy transfer (FRET) are provided and used to monitor and quantify molecular events in these cells to serve as "digital multimeters" to allow the characterization of each molecular module for the functional optimization of the engineered cells.

In alternative embodiments, exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are engineered to treat bacterial or viral infections. For example, T cells can be engineered to express a microbial, e.g., a viral or bacterial, antigen. In alternative embodiments, a cell can be engineered to express a protein that targets an intracellular pathogen.

In alternative embodiments, exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are used to manipulate the physiology of a cell, e.g., cells are engineered to express an inducible protein that causes or induces apoptosis, or inhibit mitosis, or any biochemical pathway in the cell. For example, in one embodiment the ultrasound stimulation activates secretion from the cell of a hormone or a protein, e.g., a steroid, or insulin, and the like.

In alternative embodiments, exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are engineered to have the capability of controlling production of RNAs (including but not limited to e.g., microRNA, long non-coding RNAs), epigenetic and genetic modulation molecules for the treatment and amelioration of a disease, infection or condition, e.g., a genetic condition.

In alternative embodiments, exemplary systems and methods comprising a mechano-controlled cell activation as provided herein are engineered to comprise and integrate wireless devices, e.g., wearable wireless devices, to couple ultrasound transducers such that remote-controlled cell activations can be conducted via wireless and remote controls. The remote-control can initiate ultrasound stimulation at varying and periodic time points for continuous, pulsatory or episodic expression of nucleic acids/ proteins linked to (expression is dependent on) a promoter whose activity is activated by (or alternatively, inhibited by) an ultrasound-mediated mechanical perturbation.

The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in

McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

Example 1 : Engineering and characterization of ultrasound-controllable cells

This example demonstrates that methods and compositions as provided herein using the exemplary embodiment comprising microbubbles coated with streptavidin and coupled to biotinylated RGD peptides, which themselves become attached to integrins and hence connected to Piezol, are effective and can be used for ultrasound- induced calcium influx and NFAT activation to drive reporter production, or more broadly, this example demonstrates the effectiveness of methods and compositions as provided herein to stimulate an ultrasound-sensitive response in a cell, e.g., a cell in vivo.

We synthetically introduced mechano-sensors and gene transducing modules

(GTMs) to engineer cells to acquire the capability to remotely sense ultrasound mechanical perturbation and transduce it into synthetic protein production. In this exemplary embodiment, a Piezol is used to serve as a membrane mechano-sensor to conduct calcium influx into mammalian cells.

We established and integrated an ultrasound stimulation system with our FRET imaging microscope. We showed that the expression of exogenous Piezol, but not control vector, in Piezol -deficient Hek293 cells allowed a microbubble-mediated calcium response upon 2 MHz ultrasound stimulation at a long working distance of about 5 cm (Fig. 6A-B).

Piezol was then introduced as the mechano-sensor together with a GTM, in which a regulatory region composed of three calcium response elements in cis: serum response element (SRE), cyclic adenosine monophosphate response element (CRE), and the nuclear factor of activated T cell response element (NFAT RE), is placed upstream to a minimal promoter and a reporter gene (firefly luciferase, fLuc) (Fig. 6A-1). The ultrasound-induced mechanical oscillation (20 min) of microbubbles coupled to integrins, and hence Piezol, clearly led to an activation in the reporter gene (Fig. 6C-1). To reduce the potentially leaky protein production in cells at the basal level and enhance the induction specificity upon stimulation, a two-stage GTM was designed (Fig. 6A-2), in which the first induced protein product upon ultrasound stimulation is a DNA binding domain (DBD) LexA connected to a highly efficient transcription activator VPR (LexA-VPR). This LexA-VPR upon induction can activate a second gene cassette for the production of reporters or target proteins (Fig. 6A-2). Indeed, ultrasound caused a clear induction of reporter genes, either luciferase or a new GFP mNeonGreen, similar to the chemical stimulation by ionomysin (Fig. 6C-2 and 6D).

These results provide the proof-of-concept that exemplary mechano-sensors and GTMs provided herein can be integrated into an endogenous cellular molecular network for the sensing of ultrasound stimulation to guide gene activations, or for the activation or stimulation of an ultrasound-sensitive response in a cell, e.g., a cell in vivo.

Example 2: Genetic transducing modules (GTMs) are functional in T cell lines

This example demonstrates that genetic transducing modules (GTMs) are functional in T cell lines.

We cloned two GTMs into lentiviral vectors and tested them in Jurkat T cells (Fig. 7A). Ionomycin treatment for 30 min to induce the calcium influx clearly triggered the activation of the reporter gene with the one-stage GTM (Fig. 7B-1, left). A two-stage GTM to reduce leaky protein productions at the basal level also allowed the induction of reporter production upon ionomycin treatment for 30 min (Fig. 7B-2, right). Ultrasound stimulation for 10 min also clearly triggered the calcium influx (Fig. 9B-C) and the activation of the reporter gene with two one-stage GTMs (Fig. 8A-B).

These results provide the proof-of-concept that GTMs can be integrated into the endogenous molecular network of Jurkat cells to sense the stimulation of calcium signaling and guide gene expression, or GTM expression, for the control of a cellular function in a cell, e.g., a T cell, or expression of an exogenous protein in a cell, e.g., ultrasound-inducible and functional expression of a CAR on the surface of a T cell.

Example 3 : Remoted-controlled activation of T cells with ReCoM

This example demonstrates that remote controlled, ultrasound-inducible recombinant chimeric antigen receptors (ReCoM-CAR) as provided herein are functional in T cell line and primary T cells:

We applied ReCoM to control anti-CD 19 CAR production by ultrasound in Jurkat cells (Fig. 9A). The encoding mRNA and expression percentage of anti-CD19 CAR in Jurkat cells was significantly increased after ultrasound stimulation at protein (Fig. 9D) and mRNA expression level (Fig. 8C).

The Jurkat cells with the ultrasound-induced CAR expression were then incubated with CD 19 antigen-expressing target tumor cells (Toledo lymphoma tumor cells which express high levels of CD 19). Upon the engagement of ultrasound- induced Jurkat and target Toledo cells for 24 hr, the surface marker CD69 reflecting T cell activation was clearly upregulated in the Jurkat cells (Fig.10A-B). These results indicate that the ultrasound-induced CAR production in Jurkat T cells can

functionally mediate the engagement with antigens on the target tumor cells and activate Jurkat cells.

We then applied ReCoM to remotely control the CAR production in PBMCs and examine their efficacy in tumor cell killing (Fig. 11 A). Calcium influx in PBMCs can be clearly observed upon ultrasound stimulation (Fig. 1 IB). We further measured the ReCoM-mediated expression of anti-CD19 CAR in PBMCs. The average expression level of anti-CD 19 CAR was significantly increased after ultrasound stimulation (Fig. 11C). These ultrasound-induced PBMCs were incubated with target B cell leukemia cell line (Nalm6) expressing CD19-antigen and luciferase (27, 28), which allowed a convenient tracking of the target tumor cell growth by measuring the luciferase activities. The results of luciferase activity in reflecting Nalm6 cell numbers revealed that the ultrasound-induced ReCoM PBMCs can cause significantly more toxicity of the target Nalm6 cells than the ReCoM PBMCs not exposed to ultrasound or the plain PBMCs exposed to ultrasound but without ReCoM GTMs (Fig. 11D).

A number of embodiments of the invention have been described.

Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.