HYBRID BIOELECTRONIC/ENGINEERED CELL WEARABLE SYSTEM FOR THERAPEUTIC AGENTS DELIVERY AND APPLICATIONS THEREOF

STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH [0001] This invention was made with government support under FA8650-21-2-7119 awarded by the Air Force Research Laboratory. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0002] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63/177,806, filed April 21, 2021, which is incorporated herein in its entirety by reference.

[0003] This application is also related to co-pending PCT patent applications, entitled “Hybrid Bioelectronic/Engineered Cell Implantable System for Therapeutic Agents Delivery and Applications Thereof’, with Attorney Docket No. 0116936.266W02, and “Engineered Cells for Producing Therapeutic Agents to be Delivered by a Hybrid Bioelectronic Device”, with Attorney Docket No. 0116936.266W023, respectively, which are filed on the same day that this application is filed, and with the same applicant as that of this application, which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0004] The present disclosure relates generally to the field of biomedical engineering, and more particularly to hybrid bioelectronics/engineered cell system providing delivery of therapeutic agents produced by genetically engineered cells into an individual’s body.

BACKGROUND OF THE INVENTION

[0005] The background description provided herein is for the purpose of generally presenting the context of the present invention. The subject matter discussed in the background of the invention section should not be assumed to be prior art merely as a result of its mention in the background of the invention section. Similarly, a problem mentioned in the background of the invention section or associated with the subject matter of the background of the invention section should not be assumed to have been previously recognized in the prior art. The subject matter in the background of the invention section merely represents different approaches, which in and of themselves may also be inventions.

[0006] For numerous medical conditions, the delivery of therapeutic agents having bioactivity requires precise control in terms of time and dosage, while the production of the therapeutic agents has to be in-situ and under an ideal condition. Currently technologies either provide a subpar cultivation environment for cells which significantly reduces the viability of the cells, or are incapable of providing precise delivery of the therapeutic agents to a degree necessary for treatment of many medical conditions.

[0007] Therefore, there remains an imperative need for a system to enhance the cell viability and potency, facilitate improved delivery, and remote control of activation, so as to provide a precise delivery of therapeutic agents having the best quality.

SUMMARY OF THE INVENTION

[0008] In light of the foregoing, this invention discloses a wearable bioelectronic system, which provides precise delivery of biomolecules synthesized by genetically engineered cells directly into the blood stream. Sensors on-board the implantable, in the external hub, in external wearable device, or coupled to other commercial off the shelf wearables sensors, provide input for dose delivery timing, depending on the application. The wearable device includes engineered mammalian cells that are genetically modified to deliver the biomolecule of interest, and to do so upon optoelectronic trigger. These cells are controlled by a series of LEDs and photodiodes and are supported via optoelectronic oxygen generation within an encapsulated, immuno-isolating cell-housing compartment. The wearable device includes multiple of the same type of cell in different compartments and/or other engineered cells for multi-molecule delivery. The wearable device also includes sensors, power management, and communication on a small form factor footprint. This work establishes a generalizable engineering framework for hybrid bioelectronic/engineered cell wearable system for therapeutic agents precise delivery.

[0009] In one aspect of the invention, a hybrid bioelectronic wearable device containing engineered cells for delivery of therapeutic agents to a subject, the device comprising a cell cartridge containing at least one type of engineered cells, wherein each of the engineered cells contains an optogenetic system; an optical stimulating system disposed adjacent to the cell cartridge, wherein the optical stimulating system has at least one light source, wherein the optogenetic systems of the engineered cells are configured to receive a light generated from the at least one light source such that the generated light operably controls production of at least one type of therapeutic agents and a reporter agent by the engineered cells; a media cartridge in fluid communication with the cell cartridge providing cell media to the cell cartridge; a pump in fluid communication with the media cartridge and the cell cartridge, wherein the pump is configured to pump the cell media from the media cartridge into the cell cartridge; a cannula having a first cannula end in fluid communication with the cell cartridge and a second cannula end in fluid communication with the subject; and a controller in communication with the optical stimulating system and the sensing system, wherein the controller is configured to control the production of the at least one type of therapeutic agents according to a control algorithm.

[0010] In one embodiment, the device further comprising a filter disposed between the cell cartridge and the second end of the cannula, wherein the filter is configured to selectively permit the at least one type of therapeutic agent passing through the filter and entering into the subject’s body through the second end of the cannula.

[0011] In one embodiment, the device further comprising a battery configured to provide power supply to the device.

[0012] In one embodiment, the engineered cells start production of the at least one type of therapeutic agent when the optogenetic systems of the engineered cells receive a signal light having a first wavelength from the optical stimulating system.

[0013] In one embodiment, the device further comprising a sensing system disposed adjacent to the cell cartridge, wherein the sensing system is configured to sense a fluorescent light signal or bioluminescence signal generated by the reporter agent, wherein the engineer cells stop the production of at least one type of therapeutic agent when either the optogenetic systems of the engineered cells receive a signal light having a second wavelength, or the sensing system detects a predetermined level of the fluorescent light signal or bioluminance signal generated by the reporter agent.

[0014] In one embodiment, the amount of the produced reporter agent and the amount of the produced at least one type of therapeutic agents are at a fixed ratio.

[0015] In one embodiment, the media cartridge is replaceable and detachable.

[0016] In one embodiment, the media cartridge is refillable.

[0017] In one embodiment, the pump is configured to create a pressure necessary for the at least one type of therapeutic agent passing through the filter and entering into the subject’s body through the second end of the cannula.

[0018] In one embodiment, the device further comprising an intake cannula in fluid communication with the subject and is configured to receive interstitial fluid from the subject. [0019] In one embodiment, the intake cannula is in fluid communication with the cell cartridge, wherein in use, the received interstitial fluid enters into the cell cartridge.

[0020] In one embodiment, the cell cartridge is detachable and replaceable.

[0021] In another aspect of the invention, a hybrid bioelectronic wearable device containing engineered cells for delivery of therapeutic agents to a subject, the device comprising a cell cartridge containing at least one type of the engineered cells, wherein each of the engineered cells contains an optogenetic system; an optical stimulating system disposed adjacent to the cell cartridge, wherein the optical stimulating system comprises at least one light source configured to generate a light; and a media cartridge in fluid communication with the cell cartridge configured to provide cell media to the cell cartridge.

[0022] In one embodiment, the generated light comprises a light of first wavelength and a light of second wavelength different from the light of first wavelength.

[0023] In one embodiment, the engineered cells start producing the at least one type of therapeutic agent when the optogenetic systems of the engineered cells receive the light of first wavelength.

[0024] In one embodiment, the engineered cells stop producing the at least one type of therapeutic agent when the optogenetic systems of the engineered cells receive the light of second wavelength.

[0025] In one embodiment, the device further comprising a sensing system disposed adjacent to the cell cartridge, the sensing system is configured to detect a signal generated by a reporter agent produced by the engineered cells, wherein the signal comprises at least one of a fluorescent light signal, a bioluminescence signal, a impedance signal, pigment signal, and a free radical signal.

[0026] In one embodiment, the engineered cells stop producing the at least one type of therapeutic agent when the signal detected by the sensing system reaches a predetermined level. [0027] In one embodiment, the sensing system comprises a photodiode.

[0028] In one embodiment, the amount of the produced reporter agent and the amount of the produced at least one type of therapeutic agents are at a fixed ratio.

[0029] In one embodiment, the device further comprising a media cartridge in fluid communication with the cell cartridge, wherein the media cartridge is configured to provide cell media to the cell cartridge. [0030] In one embodiment, the device further comprising a pump in fluid communication with the media cartridge and the cell cartridge, wherein the pump is configured to pump the cell media from the media cartridge into the cell cartridge.

[0031] In one embodiment, the device further comprising a cannula having a first cannula end in fluid communication with the cell cartridge and a second cannula end in fluid communication with the subject.

[0032] In one embodiment, the device further comprising a filter disposed between the cell cartridge and the second end of the cannula.

[0033] In one embodiment, the pump is configured to create a pressure necessary for the at least one type of therapeutic agent passing through the filter and entering into the subject’s body through the second end of the cannula.

[0034] In one embodiment, the device further comprising a control unit in communication with the stimulating system and the sensing system; and a memory unit in communication with the control unit.

[0035] In one embodiment, the memory unit is configured to store a control algorithm to regulate production of the at least one type of therapeutic agents.

[0036] In one embodiment, the device further comprising a battery configured to provide power supply to the device.

[0037] In one embodiment, the media cartridge is detachable and replaceable.

[0038] In one embodiment, the media cartridge is refillable.

[0039] In one embodiment, the cell cartridge is detachable and replaceable.

[0040] In one embodiment, the device further comprising an intake cannula in fluid communication with the subject and the cell cartridge, wherein the intake cannula is configured to receive interstitial fluid from the subject and the received interstitial fluid is configured to enter into the cell cartridge.

[0041] These and other aspects of the present invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS [0042] The accompanying drawings illustrate one or more embodiments of the invention and together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

[0043] Fig. 1 depicts phases of peripheral and central clocks in response to an 8hr shift, for normal entrainment (left), providing therapy affecting only the central clock (middle), and the proposed NTRAIN approach (right) with therapy targeting both central and peripheral clocks in accordance with an illustrative embodiment.

[0044] Fig. 2 is a table that depicts the rationale for using optical induction to perform control and feedback in accordance with an illustrative embodiment.

[0045] Figs. 3A-3D provide different embodiments of hybrid bioelectronic device. Fig. 3 A illustrates an implantable embodiment having a single cell housing containing engineered cells. Fig. 3B illustrates an implantable embodiment having plurality of cell housings containing same or different engineered cells. Fig. 3C illustrates an implantable embodiment having one or more cell housings integrated with power transduction management system, optoelectronics and other accessary systems e.g., O2 generation system. Fig. 3D illustrates a wearable embodiment having media cartridge separate from the cell housing.

[0046] Fig. 4 depicts operations performed to implement the proposed NTRAIN system in accordance with an illustrative embodiment.

[0047] Fig. 5 is a graphical depiction of the proposed synthetic biology circuit for optogenetic control of the peptide therapeutic Orexin A in accordance with an illustrative embodiment.

[0048] Fig. 6 depicts preliminary data showing that ARPE-19 cells can be made to express luciferase with high on/off ratio in response to blue light using an EL222 optogenetic system in accordance with an illustrative embodiment.

[0049] Fig. 7 shows a biohybrid precision control scheme based on co-production of therapeutic peptide and proxy reporter fluorophore (GFP*) in accordance with an illustrative embodiment.

[0050] Fig. 8 shows a comparison of traditional optogenetic control strategies that use constant illumination to activate the ion channels to the proposed step-function opsin control strategy that utilizes a blue LED to open the light-gated channels and an orange LED to close the channels in accordance with an illustrative embodiment.

[0051] Figs. 9A-B show wired and wireless prototype devices. Fig. 9A shows design of the top side of the circuit board with the LEDs and photodiodes. Fig. 9B shows wired prototype device with the wires extending out the right side of the device.

[0052] Fig. 10 shows charts regarding utilization of blue LEDs and photodiodes to measure green fluorescence of cells.

[0053] Fig. 11 shows top view of blue and orange LEDs on an encapsulated circuit board at the end of the soak test.

[0054] Figs. 12A-C show a filter and its application to isolate the photodiode from the blue LED. Fig. 12A shows a chart of optical density of the Wratten 12 filter. Fig. 12B shows the laser cut optical filter is bent into a box shape before being glued to the photodiode. Fig. 12C shows a chart of photovoltages obtained with and without the optical filter.

[0055] Figs. 13A-B show device molds. Fig. 13 A shows an aluminum device mold. Fig. 13B shows a PDMS device molded by the aluminum mold.

[0056] Figs. 14A-B show synthesis reaction for RZA15. Fig. 14A shows a process for RZA15 synthesis. Fig. 14B shows NMR and ES MS characterization of the resulting RZA15 product.

[0057] Figs. 15A-B show synthesis reaction for RZA15 UPVLVG. Fig. 15A shows systhesis process; Fig. 15B shows NMR and elemental characterization of the product.

[0058] Fig. 16 shows an aid device for loading the bioelectric device with cells.

[0059] Fig. 17 is a block diagram of a computing system to perform operations described herein in accordance with an illustrative embodiment.

DETAILED DESCRIPTION OF THE INVENTION [0060] The invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.

[0061] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0062] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0063] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the invention. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0064] It will be understood that, as used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

[0065] It will be understood that when an element is referred to as being “on”, “attached” to, “connected” to, “coupled” with, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0066] It will be understood that, although the terms first, second, third etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the invention.

[0067] Furthermore, relative terms, such as “lower” or “bottom” and “upper” or “top,” may be used herein to describe one element’s relationship to another element as illustrated in the figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation depicted in the figures. For example, if the device in one of the figures is turned over, elements described as being on the “lower” side of other elements would then be oriented on “upper” sides of the other elements. The exemplary term “lower”, can therefore, encompasses both an orientation of “lower” and “upper,” depending of the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as “below” or “beneath” other elements would then be oriented “above” the other elements. The exemplary terms “below” or “beneath” can, therefore, encompass both an orientation of above and below.

[0068] It will be further understood that the terms “comprises” and/or “comprising”, or “includes” and/or “including”, or “has” and/or “having”, or “carry” and/or “carrying”, or “contain” and/or “containing”, or “involve” and/or “involving”, “characterized by”, and the like are to be open-ended, i.e., to mean including but not limited to. When used in this disclosure, they specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[0069] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the invention, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0070] As used in the disclosure, “around”, “about”, “approximately” or “substantially” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about”, “approximately” or “substantially” can be inferred if not expressly stated.

[0071] As used in the disclosure, the phrase “at least one of A, B, and C” should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. [0072] As used in the disclosure, the term “implantable” refers to an ability of a device to be positioned at a location within a body of a user, such as subcutaneously, within a body cavity, or etc. Furthermore, the terms “implantation” and “implanted” refer to the positioning of a device at a location within a body of a user, such as subcutaneously, within a body cavity, or etc.

[0073] As used in the disclosure, the term “wearable” refers to articles, adornments or items designed to be worn by a user, incorporated into another item worn by a user, act as an orthosis for the user, or interfacing with the contours of a user's body.

[0074] As used in the disclosure, “biocompatible” material is a material that is compatible with living tissue or a living system by not being toxic or injurious and not causing immunological rejection.

[0075] As used in the disclosure, “therapeutic agent” refers to any substance that provides therapeutic effects to a disease or symptom related thereto. In certain embodiments, a therapeutic agent refers to a substance that provides therapeutic effects to any diseases or biological or physiological responses to the diseases. [0076] As used in the disclosure, the term “therapy” refers to any protocol, method, and/or agent that can be used in the management, treatment, and/or amelioration of a given disease, or a symptom related thereto. In certain embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies known to one of skill in the art, such as medical personnel, useful in the management or treatment of a given disease, or symptom related thereto.

[0077] As used in the disclosure, “treat”, “treatment”, and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a given disease resulting from the administration of one or more therapies (including, but not limited to, the administration of microspheres disclosed herein). In certain embodiments, the terms refer to the reduction of pain associated with one or more diseases or conditions.

[0078] As used in the disclosure, “engineered cell(s)” refers herein to cells having been engineered, e.g., by the introduction of an exogenous nucleic acid sequence or specific alteration of an endogenous gene sequence. An exogenous nucleic acid sequence that is introduced may comprise a wild type sequence of any species that may be modified. An engineered cell may comprise genetic modifications such as one or more mutations, insertions and/or deletions in an endogenous gene and/or insertion of an exogenous nucleic acid (e.g., a genetic construct) in the genome. An engineered cell may refer to a cell in isolation or in culture. Engineered cells may be “transduced cells” wherein the cells have been infected with e.g., an engineered virus. For example, a retroviral vector may be used, such as described in the examples, but other suitable viral vectors may also be contemplated such as lentiviruses. Non-viral methods may also be used, such as transfections or electroporation of DNA vectors. DNA vectors that may be used are transposon vectors. Engineered cells may thus also be “stably transfected cells” or “transiently transfected cells”. Transfection refers to non-viral methods to transfer DNA (or RNA) to cells such that a gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposomal or lipoplex transfection of nucleic acids. Such a transfection may be transient, but may also be a stable transfection wherein cells can be selected that have the gene construct integrated in their genome.

[0079] Present system described herein features a wearable device housing engineered cells, e.g., engineered ARPE-19 cells, that produce or are capable of producing one or more therapeutic agents. The therapeutic agent may be a biological substance, such as a nucleic acid (e.g., a nucleotide, DNA, or RNA), a polypeptide, a lipid, a sugar (e.g., a monosaccharide, disaccharide, oligosaccharide, or polysaccharide), a small molecule, etc. In some embodiments, the therapeutic agent is a polypeptide. Each engineered cell comprises a promoter operably linked to a nucleotide sequence encoding the polypeptide. In such an implementation, the promoter can essentially be a nucleotide sequence. In some embodiments, the therapeutic agent is a replacement therapy or a replacement protein, e.g., useful for the treatment of a blood clotting disorder or a lysosomal storage disease in a subject.

[0080] In some embodiments, the wearable device includes one or more engineered cells, which can be provided as a cluster or disposed in a microcarrier. In some embodiments, the engineered cells produce or release a therapeutic agent (e.g., a polypeptide) for at least 0.5 day, 1 day, 10 days, or more, when the wearable device being worn by the subject. In one embodiment, more than one therapeutic agent are produced by the engineered cells. In one embodiment, the wearable device may include one or more types of engineered cells, one type of the engineered cells may produce a therapeutic agent which is different from the therapeutic agent produced by other types of the engineered cells.

[0081] In another aspect, the present disclosure features a method of treating a subject comprising connecting administering to the subject a wearable device housing the engineered cells producing at least one therapeutic agents.

[0082] In one illustrative embodiment, the subject is a human and the engineered active cell is a human cell. Alternatively, the subject may be a dog, cat, or other animal. In some embodiments, the therapeutic agent produced by the engineered cell(s) is a replacement therapy or a replacement protein, e.g., useful for the treatment of metabolic diseases.

[0083] The produced therapeutic agents can be evaluated by an art-recognized reference method, e.g., polymerase chain reaction or in situ hybridization for nucleic acids; mass spectroscopy for lipid, sugar and small molecules; microscopy and other imaging techniques for agents modified with a fluorescent or luminescent tag, and ELISA or Western blotting for polypeptides. In some embodiments, the implantable device or the wearable device comprises an encapsulating component (e.g., formed in situ on or surrounding the engineered cells, or preformed prior to combination with the engineered cells). In other embodiments, the implantable device or the wearable device is chemically modified, as described herein.

[0084] Thus, described herein is a hybrid bioelectronics/engineered cells pharmacy that enables the production of therapeutic agents within the subject. The therapeutic agents can be used to control pain, treat metabolic disorders, treat immune system disorders, treat psychiatric disorders, improve fertility, and any other medical or health conditions requiring a frequent and/or precise administration of therapeutic agents.

[0085] The present invention provides a therapy having a timing and dosing control which far exceeds the existing therapies and/or bioelectronics. The proposed system is able to achieve 1) specific biological action on select target receptors or molecules that cannot be accomplished with current bioelectronics, and 2) precise control of timing and dosage that cannot be accomplished with current synthetic biology.

[0086] In this embodiment, the living pharmacy includes engineered cells that produce therapeutic peptides with a timing and dose profile that is tightly controlled by optical triggers from a wearable bioelectronic carrier device. The system overcomes the major challenges facing hybrid bioelectronic devices, including: 1) selective activity on biological targets, 2) precise control of biomolecule production, 3) high dose to load volume ratio. In alternative implementations, the system may have fewer, additional, and/or different features.

[0087] The biohybrid system of the present invention provides a general platform for precise drug delivery and regulation that can be worn long-term to treat short term or long term diseases, physical and/or mental health conditions, as well as improve user health and performance, without the need to carry pharmaceuticals. In one exemplary embodiment discussed below, the proposed system minimizes the adverse health consequences of circadian misalignment by achieving at least a 50% reduction in entrainment time using a living biohybrid pharmacy that remains functional for an extended period of time (e.g., 30 days, 60 days, 90 days, etc.). In other embodiments, the disclosed invention provides treatments to diseases and/or physiological conditions including metabolic diseases, e.g., obesity and diabetes (e.g., Type 1, Type 2) by producing metabolically active molecules, e.g., leptin, ACTH, insulin, and GLP-1; cancers by producing therapeutic cytokines e.g., IL-2, IL-12, IL-15, GCSF; autoimmune diseases by producing regulated molecules e.g., IL-10, IL-35, treatment resistant depression and pains by producing neuropeptides e.g., GLYX-13, rapastinel, and ziconotide; osteoporosis by producing PTH; infertility by producing gonadotropin releasing hormone GnRH; and etc. To achieve these metrics, the system focuses on five main innovations to overcome barriers of current bioelectronic and synthetic biology technologies, as well as an innovative approach to accelerating entrainment. These innovations, which are described in detail below, include performing selective activity on biological targets using natural peptides, precisely controlling biomolecule production, obtaining a high dose to load volume ratio. [0088] With respect to selective activity on biological targets using natural peptides, the inventors have proposed using engineered allogeneic cells to produce select peptides that are otherwise naturally produced by the body to control pain, fight disease, regulate sleep cycles, treat metabolic related conditions, and etc. It is to be understood that in other applications the system can be used to produce other types of therapeutic agents. The body naturally produces these native peptides which are structurally similar to their recombinant counterparts. However, the native peptides diverge in potency and bioactivity. Significantly, it is noted that native peptides have not been commercialized due to their instability. However, the inventors have determined that a cell delivery platform which supports on demand in situ production use of native peptides as therapeutics is feasible. These naturally produced molecules are excellent candidates to regulate the specific activity of central and peripheral circadian clocks because they act selectively on these biological targets and do not elicit the immune responses that are shortcomings of recombinant peptides or exogenous drugs (i.e., anti drug antibodies). Furthermore, these peptides feature short metabolic half-lives, and are useful for relatively fast cessation of drug production. These traits make the proposed cell platform uniquely suited to deliver such biologies on demand. The novel system is also able to perform precision dosing with closed-loop bioelectronic control. A key challenge for biological production of therapeutic agents is controlling the production levels, which can vary due to cell health, temperature, and metabolism. To overcome this challenge, the proposed system includes a state-of-the art bioelectronic feedback control system based on optogenetically controlled therapy production and fluorescent tracking of therapy production levels. In alternative embodiments, the feedback control system may not be used. Cells engineered with optogenetic systems start protein production in response to exposure to specific activating light signals. By controlling light exposure, production of therapeutic agents can be controlled.

[0089] Another innovation of the proposed system is a bioelectronic feedback loop based on fluorescent tracking of the production levels. To create this feedback control loop, the cells are engineered to produce a fluorescent protein at a fixed ratio relative to the therapeutic agents. Using this fluorescence measurement as the feedback signal, the system is able to regulate the on time of the engineered cells to maintain a stable fixed point of therapeutic agents production with precision that exceeds synthetic biological feedback loops.

[0090] In some embodiments, in addition to the fluorescent signal provided by the fluorescent proteins, the bioelectronic feedback loop is based on biochemical signals which can be electronically detected. In addition to the fluorescent signal, the biochemical signals may include bioluminescence signal, impedance signal, pigment signal, and free radical signal.

[0091] In some embodiments, the proposed system also provides high-dose to load volume with on-chip life support and engineered cells. To support a higher concentration of therapeutic agents produced by the wearable device, one could increase the density of the engineered cells inside the chassis. However, the maximum cell density is currently limited by the amount of diffuse oxygen available in the subcutaneous space. To reach higher cell densities, the carrier is engineered to produce local O2 with the bioelectronic carrier. Furthermore, using synthetic biology tools, the system amplifies transcription of the therapeutic peptides and programs cells to be resilient to senescence and cell death.

[0092] The proposed system uses battery power or energy harvesting from the body.

[0093] The proposed system can be used for delivery of single or multiple therapeutics. In one embodiment, the engineered cells produce one or more therapeutic agents. In another embodiment, there are more than one type of engineered cells housed either in one cell housing, or each type of the engineered cells is housed in separated housings. In one exemplary embodiment of multiple therapeutic delivery, multi-clock targeting with precision timing for circadian rhythm regulation can be performed by the proposed system, as discussed below.

[0094] Unlike bioelectronic or gated biofluidic systems that feature pre-filled (or even refillable) reservoirs of drugs, the proposed system delivers naturally-occurring peptides throughout its functional lifetime without the need to stock, carry, or refill therapies that are vulnerable to loss, degradation, or that add to the already burdensome load carried by the user. The developed technology will serve as a platform whereby the optical control and feedback to achieve precision therapies can be applied to delivery of a broad swath of naturally occurring peptides/proteins by following the procedures and protocols described herein.

[0095] Thus, the disclosed system provides a hybrid bioelectronics platform and forms the basis and components for a number of bioelectronic and biohybrid tools to address or alleviate dysfunction and injury, to enhance readiness and performance, to treat pain, to treat disease, improves metabolism, and etc. The rationale behind the proposed system, along with details of its implementation, use, and testing are described in more detail below.

Biohybrid System For On Demand Therapy

[0096] Engineering a cell-based hybrid bioelectronic system for on demand therapy is a challenge involving a careful balance between cellular and bioelectronic device engineering. Several factors have to be taken into consideration to balance the strengths of each pillar and to minimize their drawbacks and deficiencies. Cells possess their own natural machinery to synthesize and release specific biomolecules. Furthermore, their machinery can be hacked to externally induce such production, but without the level of timing, control, and user interfacing that is possible with bioelectronics. On the other hand, optoelectronic components have inherent chemical and mechanical mismatches with cells and tissue that can limit their lifetime (degradation, rejection), or make them otherwise incompatible with reliable use in vivo. Striking the proper balance between the two is important.

[0097] Key design decisions that permeate the proposed system focus on (i) promoting long term viability/efficiency and (ii) controlling and creating feedback-loop of therapeutic agents production. To promote viability of engineered cells, the proposed system both genetically engineers cells to be more resilient, and in some embodiments can also produce O2 to support them. For control and feedback-loop, optical induction can be used. Compared to other cellular control mechanisms, optical induction methods enable fast response, tunable, localized induction properties (wavelength control), and are readily integrated into the platform with minimal power and size demands. Furthermore, optoelectronic cell interfacing enables innovative precision low- power dosing control. Fig. 2 is a table that depicts the rationale for using optical induction to perform control and feedback in accordance with an illustrative embodiment.

Technological Components of the System

[0098] In an illustrative embodiment, the proposed system includes a wearable device, featuring individually-controlled cell housing/cartridge It should be noted that, in the application, the term “cell house”, “cell housing” “cell well”, “cell cartridge” are used interchangeably.

[0099] Fig. 3 A provides an illustrative diagram showing the general structure of a unit of the implantable bioelectronics device. As shown in the lower panel, the bioelectronics may comprising a unit of the implantable device 10 inside the body of a user, and an external hub 20 located outside the body of the user. The external hub 20 is in communication with the implantable device 10 for power charging and data exchanging/transmission. As shown in the upper panel, the implantable device 10 comprising a cell housing 11 for containing engineered cells 1000 which produces the therapeutic agents 1010 and a reporter agent/molecule 1020. A stimulator 17 for triggering the production of the therapeutic agents 1010 and the reporter agent/molecule 1020 by the engineered cells 1000 locates in the cell housing 11. A sensor 21 for sensing the reporter agent/molecule 1020 also locates in the cell housing 11. Both the stimulator and the sensor locate in vicinity to the engineered cells 1000 such that they effectively stimulate the production of the therapeutic agents 1010 and the reporter agent/molecule 1020 and sensing the production of the reporter agent/molecule 1020. At least one side of the implantable device 10 are coated or encapsulated with a permeable material/membrane 23 which shield the implantable device 10 from the immune system/cells of the user.

[00100] Fig. 3B provides an illustration diagram of another embodiment of the implantable device 30 which has multiple cell wells/housings 31. The implantable device has more than one cell well/housing 31 attached to an electronic layers 40. Each of the cell well/housing 31 has an individual stimulator 37 and an individual sensor 41. In one embodiment, the stimulators 37 and sensor 41 locate in vicinity to the engineered cells 1000 such that they effectively stimulate the production of the therapeutic agents 1010 and the reporter agent/molecule 1020 and sensing the production of the reporter agent/molecule 1020. In one embodiment, cell wells/housings 31 contain engineered cells producing same therapeutic agent 1010. In another embodiment, different types of engineered cells 1000 producing different therapeutic agents 1010 may be each contained in a separate cell well/housing 31, such that the each type of engineered cells may be individually controlled by the stimulator and the sensor in each cell well/housing 31, so as to produce a particular therapeutic agents 1010 for a specific amount and/or at a specific time different from that of in the other cells/housings.

[00101] Fig. 3C depicts an alternative embodiment showing a subcutaneous NTRAIN device 110, including a cross-section that depicts method of operation and associated tasks for engineered components in accordance with an illustrative embodiment. The implanted subcutaneous device includes (i) genetically engineered allogeneic mammalian cells programmed to deliver peptide therapeutics in accordance with an optical trigger 117, (ii) hybrid synthetic biology/bioelectronic feedback control to provide precision dosing 121, (iii) O2 generation capabilities/device 115, (iv) a custom application-specific integrated circuit (ASIC) 125 for low-power feedback control, temperature sensing, and power management, , and(v) a near field communication (NFC) coil 127 for wireless data uplink. In alternative implementations, the device can have fewer, additional, and/or different features.

[00102] In one embodiment, each cell well/housing houses about 240k cells, 2x2xlmm in size. Alternatively, a different number of cells and/or a different compartment size may be used. At the base of the compartment is a bioelectronic carrier, on which control LEDs (stimulator) 117 initiate and stop peptide production. Specifically, an LED/photodiode pair (sensor) 121 is used to probe production of destabilized fluorescent proteins which are produced as a proxy for the delivered peptide, providing optical feedback of production levels, for closed loop dosage control. The compartment also contains O2 generating particles or an O2 generating electrochemical device 115 in one embodiment, which allows the system to have increased density of engineered cells within the chassis. In an illustrative embodiment, the housings that form the cell compartments can be made opaque by using opaque PDMS walls 113 between the compartments 111 to minimize crosstalk of the optical control signals between cell compartments.

[00103] Fig. 3D illustrates another embodiment of the invention. Instead of an implantable device, this embodiment is directed to a wearable external device housing and controlling the engineered cells for production of therapeutic agents. The external wearable device 50 may include (i) a cell housing/cartridge 51 containing engineered cells, (ii) a replaceable cell media cartridge 52, (iii) a pump 53 pumping the media from the media cartridge 52 to the cell housing/cartridge 51, (iv) a cannula 58 extending from the cell housing/cartridge 51 and providing the therapeutic agents to a user, (v) a filter between the cell housing/cartridge 51 and the outlet of the cannula 58 for removing unwanted agents and compositions, (vi) a stimulating system 57 providing light source to the engineered cells housed in the cell housing/cartridge 51, (vii) a sensing system 61 detecting the fluorescent signals produced by the engineered cells for feedback control, (viii) a control unit 63 controlling the stimulation system 57 and sensing system 61, and (ix) a battery unit 65 providing power supply.

[00104] In this embodiment, the engineered cells are housed and supported in the wearable external device 50, which delivers the in situ synthesized and excreted therapeutic agents in a regulated manner via the cannula 58 into the body, e.g., subcutaneously, intraperitoneally, and etc.

[00105] In one embodiment, the cell housing/cartridge 51 can be a separate, replaceable modular chamber, so as to flexibly customize the production of the therapeutic agents. In one embodiment, the cell housing/cartridge 51 includes microcarriers.

[00106] In one embodiment, the stimulating system 57 providing light source(s) of one or more wavelength is disposed in vicinity to the cell housing/cartridge 51. The stimulating system 57 and the cell housing/cartridge 51 are aligned in a manner maximizing the engineered cells’ exposure to the light source, e.g., substantially parallel with each other.

[00107] In one embodiment, fresh media can be exchanged in the cell housing/cartridge 51, and an on board pump 54 circulated fresh media through the cell housing/cartridge 51 and carries the excreted therapeutic agents through the filter 56 and into the body through the cannula 58. In one embodiment, the replaceable cell media cartridge 52 is replaceable and detachable modular, and the media inside the cell media cartridge 52 can be refilled or replaced.

[00108] In another embodiment, a user’s interstitial fluid can be collected into the device through a different cannula, circulated through the cell housing/cartridge 51 and then through the filter, before being transferred back into the user’s body through the cannula 58 or a separate delivery cannula attached to the cell housing/cartridge 51.

[00109] The pump system 54 and the control unit 63 are housed in the wearable external device 50. The battery 65 provides power supply to the wearable external device 50. The battery is replaceable and/or rechargeable.

[00110] The wearable external device can be worn by a user or can be attached to the user’s skin with adhesive.

[00111] The engineered cells housed in the wearable external device 50 have optogenetic system which controls the production of one or more therapeutic agents upon receiving the light signal from the stimulating system 57. The coordination between the optogenetic system in the engineered cells and the stimulating system 57 and sensing system 61 is the same as described for the implantable device in Figs. 3A-3C.

[00112] In this embodiment, the wearable external device relieves the demand for the immune-isolating barrier and the external hub, which may be necessary for certain embodiments of the implantable device. In addition, the wearable external device permits a more flexible size and design choice for the cell housing/cartridge 51.

Biohybrid Device Operation

[00113] In some embodiments of the device the current state of the patient would be evaluated before therapy in order to improve therapeutic timing and dosing. Generally, parameters for determining the dose, timing and etc. of a therapeutic agent delivery schedule are either detected by the wearable device via sensing the relevant parameters, e.g., heart rate, blood pressure, core temperature activity status, locations of the user, and etc., or entered manually by the user or another via a terminal either integrated with the wearable device or in wireless communication with the wearable device. Based on these parameters, a customized therapeutic agent delivery schedule is determined by the control unit of the wearable device or the terminal. Therefore, the delivery schedule can be precisely customized to the user’s situation. Once the delivery schedule is determined, the user would be asked to initiate the schedule in the wearable device or the terminal, and confirm its execution by engaging a button on the wearable device or the terminal. The therapeutic schedule is then stored on board the wearable device or the terminal, which triggers the therapy at the appropriate times. Cancellation can be done by the user at any time through the wearable device or the terminal.

[00114] In one illustrative example regarding circadian rhythm control, before first therapeutic activation of a system designed to control circadian rhythm, the user undergoes a baseline period of approximately 3 days (typically at least 1 day) to establish circadian phase with respect to light/dark cycle. When requesting therapy, the user enters a value for a magnitude of an upcoming or recently experienced clock shift in terms of number of time zones, numbers of hours, etc. The magnitude value can be entered in an application in communication with the wearable device. . The ideal therapeutic schedule (dose, duration, and timing of both peptide therapeutics) is determined by the device based on the magnitude of the clock shift. The user is asked to initiate the schedule in the wearable device or the terminal, and confirm its execution by engaging a button on the wearable device. The therapeutic schedule is then stored on board the wearable device, which triggers the therapy at the appropriate times. This procedure allows the therapy to be scheduled for delivery at times that may be inconvenient for the user to initiate. In an illustrative embodiment, the user is able to cancel a set schedule at any time using either the wearable device controls or the application. Measurements from sensors confirm the progress of entrainment. Additional instructions related to suggested behavior can be implemented at the wearable device or the terminal level as appropriate (e.g., suggested use of sunglasses). At any time, the user can input a new shift, for example, return home, and initiate a new therapeutic schedule. Fig. 4 depicts operations performed to implement the proposed hybrid bioelectronics system in accordance with an illustrative embodiment. It is to be understood that other procedures, schedules, and user interaction can be used to treat other conditions.

[00115] In an ideal use case, the wearable device can be worn for a needed duration of time (e.g., length of a deployment, length of a project or job, etc.). Depending on the materials used and the implementation, the proposed system can have a 60 day lifetime, a 130 day lifetime, a lifetime measured in years, etc. For example, it is anticipated that, using the technology described herein, the system could last for years and that repeated administration would be possible. In one embodiment, the engineered cells can be developed to include a genetically inducible safety kill switch to ensure that the cell therapy can be terminated should there be an untoward event during patient use. In the event that the device needs to be rendered non functional, kill switch activation is initiated by an FDA-approved small molecule biologic. In an illustrative embodiment, viability of cells can be tracked optically to confirm efficacy of the kill switch.

Engineering Cell-Based Drug Factories

[00116] In an illustrative embodiment of a system used for circadian rhythm control, ARPE- 19 cells are engineered to produce high levels of the desired therapeutic proteins (e.g., GLP-1 and Orexin A) on an optical trigger. In one embodiment, a melanopsin based optogenetic system can be used. In other embodiments a step-function opsin or dimerizable transcription factor (e.g., EL222) or split transcription factor (e.g., PhyB-TAD, DBD-PIF6) can be used. Additionally, the cells can be engineered to co-express a fluorescent reporter protein, for example, a destabilized GFP (GFP*) in a fixed ratio with GLP-1 and Orexin A, such as 1:1, allowing the system to observe the expression of GLP-1 and Orexin A in real time by using the easily readable destabilized GFP* fluorescence as a proxy. Additionally, a small-molecule-inducible kill switch can be engineered into the cells to allow for easy termination of the cells, rendering the device inactive. Fig. 5 is a graphical depiction of the proposed synthetic biology circuit for optogenetic control of the peptide therapeutic Orexin A in accordance with an illustrative embodiment. Preliminary data demonstrates the utility and feasibility of this architecture.

Optical Induction of Production

[00117] Each of the engineered cells have an optogenetic system. Using engineered cells enables the use of an optogenetic control system to control and produce the desired therapies. Using optogenetic systems, dosing can be controlled by modulating the amount of time that the cells are in the on state. Cells are activated to the “ON” state by exposure to light from LEDs of the stimulating system housed within the bioelectric device. Cells in this “ON” state actively transcribe the therapeutic agents needed to produce the therapeutic. Achieving Enhanced Dosing for Therapy

[00118] In order to engineer the cells to reach therapeutic dosing, and produce higher quantities of GLP-1 and Orexin A, a catalytically dead version of a CRISPR/Cas9 system (termed dCas9) can be used. The dCas9 system binds to a DNA site-specifically, but does not make any cuts or double-strand breaks. In an illustrative embodiment, the dCas9 can be deployed to recruit transcription activation domains to inserted copies of the NFAT promoter. This will allow amplification of the therapeutic protein and GFP* in a stoichiometrically equal manner amenable to high throughput screening of activation levels and quantification of kinetics. Using this system enables target-agnostic gene activation in a highly specific manner and provides a toolbox of validated synthetic biology tools to tailor activation and kinetics to ideal levels, such as synthetic promoters (NFAT or others), protein degradation tags, and 3’UTR variants among others to facilitate gene amplification only when desired.

Fluorescent Reporter

[00119] Since some embodiments of system utilizes the destabilized GFP (GFP*), co expressed with the therapeutic, one can observe the production of the therapeutic in real time by observing the fluorescence from the GFP*. Since this protein has a half-life of approximately 7 minutes, it provides an accurate real-time indicator of production levels. This real-time observation of protein production enables feedback for precision dosing control, which can be quantified.

Resilience to Apoptosis

[00120] In an illustrative embodiment, the engineered cells are designed to be durable to apoptosis and senescence, which is important for prolonged and durable expression over the course of usage. To do this, parallel genetic screening is conducted to find genetic modifications that confer resistance to apoptosis and senescence, but that retain the ability for robust kill switch operation. By applying a selective pressure that elicits these phenotypes in the engineered cells and then sequencing them, the system will enrich for cells harboring genotypes robust to these conditions. These genotypes are then recapitulated in an engineered cell line to be encapsulated as a living drug factory.

Kill Switch [00121] An important consideration with cell-based therapeutics is that the body may reject the cells, leading to a harmful immune response. Additionally, the user may want to render the system inactive. To address this issue, a kill switch is engineered into the cells. Because it has been used in multiple clinical trials and has shown to be safe, the small molecule inducible kill switch iCaspase 9 (iCasp9) can be used in one embodiment. This will allow for the controlled apoptosis of the implanted cells by administering the small molecule AP1903. The molecule can be administered orally or intravenously in some embodiments. Alternatively, in in one embodiment, the system can feature a small on-board payload of the molecule to be released electronically. In other alternative embodiments, a different type of kill switch may be used. [00122] As shown in Fig. 5, in an illustrative embodiment, plasmids are designed for therapeutic protein expression. In one embodiment, 4 plasmids can be used as follows: plasmid (1), codes for a optogenetic system driven by a CAG promoter, to enable constitutive expression of the optogentic system, e.g., production of opsin SOUL; plasmid (2) codes for therapeutic protein (i.e., GLP-1 or Orexin A) linked with GFP* via a linker such as P2A, all driven by pNFAT (activated by NFAT), plasmid (3) codes for dCas9 modification of protein expression levels and can include a unique pNFAT driving transcription of a dCas9 coding region fused to copies of the transcription activation domains p65 or HSF1, or to the human p300 acetyltransferase (p300), plasmid (4) codes for iCaspase 9 being driven by a CAG promoter. [00123] The components of plasmid (3) can be non-virally-derived domains found in human proteins that activate gene expression and will be modulated in copy number to elicit desired amounts of expression. Downstream of plasmid (4) is a synthetic 3’UTR and a U6 promoter driving transcription of the gRNA to target the therapeutic gene promoter for activation. Each plasmid can have a different selection marker (e.g., puromycin, neomycin, blasticidin, and zeocin) and be engineered to have the backbone to allow for lipofectamine transfection with PiggyBac transposase genomic integration.

[00124] For cell engineering, in one embodiment, an allogenic human cell line, ARPE-19 (retinal pigment epithelium, or RPE), was chosen because it is non-tumorigenic, displays contact inhibited growth characteristics, is amenable to genetic modification, and has been shown safe in previous human trials. Genetic components can be introduced using the standard piggyBac transposase system to the engineered cells. Other transfection method commonly known in the art can also be used.

[00125] In vitro validation and optimization is also performed via fluorescence output and kinetics. For example, the system can measure GFP* after stimulation by blue light and orange light via a live-cell plate reader over the duration of expression. Using this as the basis for further engineering, expression is tuned to be stronger by modifying the dCas9 system as follows: 1) adding more copies of transactivation domains; 2) using stronger activators (e.g., p300); 3) adding more NFAT binding sites to the promoter region; 4) and/or tuning the Kozak sequence. In one embodiment, synthetic 3’UTR variants and degradation tags are used to control stability of the mRNA transcript and protein, respectively.

[00126] In vitro validation and optimization of therapeutic outputs is also performed. Therapeutic outputs can be monitored via qPCR, RNA-seq, ELISA, and Western blot across fixed intervals following stimulation by varying durations of blue light and orange light. GFP* production can also be determined via fluorescence reading and compared to GLP-1 and Orexin A production by way of ELISA measurements to confirm a 1 : 1 stoichiometric ratio. Small molecule kill switch validation can also be performed. To show that the kill switch functions as expected, cells can be cultured with API 903 (the trigger molecule), and cell viability can be assayed via live-dead staining at various time points after culturing. To determine apoptotic and senescence resistance, the system can also screen for senescence and apoptosis resistant cells using CRISPR guide RNA (gRNA) knockout libraries in combination with doxorubicin, cisplatin, and/or DMSO challenge for a total of 4 different screens (using DMSO as a control). Cells harboring resistance genotyped and iCaspase9 are administered to ensure that the kill switch retains function. Cell fitness, proliferation, viability, and expression levels can be validated through morphological evaluation, BrdU incorporation, MTT assay, and ELISA, respectively.

[00127] In alternative embodiments, an optogenetic system other than the above-discussed systems to perform cell activation may be used. Other optogenetic system that can be used include melanopsin, EL222 and PhyB/PIF6, which, while they do not have the trigger benefit, but are more established and are shown to work in multiple situations. Fig. 6 depicts preliminary data showing that ARPE-19 cells can be made to express luciferase with high on/off ratio in response to blue light using an EL222 optogenetic system in accordance with an illustrative embodiment.

Precision Control of Dosing based on Optical Feedback

[00128] To enable precise and controllable drug production levels despite changes in cell health, stress, and metabolism, a hybrid bioelectronic feedback control system can be created and used. This control system exploits synthetic biology to produce bioactive peptide therapies, and a bioelectronic layer for precise feedback control of production levels. Fig. 7 shows a biohybrid precision control scheme based on co-production of therapeutic peptide and proxy reporter fluorophore (for example, GFP*) in accordance with an illustrative embodiment. As shown, optoelectronics such as photodiode are used to sense and adjust optical stimulation periods to maintain a given setpoint for delivery of therapeutic agents.

[00129] To implement this hybrid feedback control system, light source of stimulation system (LEDs) can be integrated into the wearable device to drive optogenetic channels which regulate therapeutic agents production in the engineered cells. To provide this control signal with minimal power consumption, step-function opsins that are activated and inactivated by different color LEDs are used. Specifically, below each cell housing/well in the wearable device are bonded Individual Cree UltraThin blue LED and Rohm semiconductor PicoLED series orange LEDs. In alternative embodiments, different types and/or wavelengths of light sources may be used. The blue LEDs provide the optical “ON” signal (e.g., 2s pulse) that turns on the step-function opsin, e.g., SOUL, leading to the elevated calcium levels in the engineered cells, as illustrates in plasmid (1) of Fig. 5, which in turn lead to the production of the therapeutic agents by the engineered cell, as illustrated in plasmid (2) of Fig. 2. The orange LED will provide an optical “OFF” signal (e.g., 2s pulse) that closes the step-function opsin. By tuning the interval between the ON and OFF signal (Dΐ), one can control the intracellular Ca++ levels and thus the therapeutic dose with a power savings of approximately 50X compared to traditional optogenetic control, as shown in Fig. 8. To make the dose levels precise, optoelectronics are integrated in the carrier and used to track the fluorescent reporters associated with each therapy. In an illustrative embodiment, the same blue LED used for the ON signal can be used as the excitation light source to track GFP* fluorescence.

[00130] Fig. 8 shows a comparison of traditional optogenetic control strategies that use constant illumination to activate the ion channels for the proposed step-function opsin control strategy that utilizes a blue LED to open the light-gated channels and an orange LED to close the channels in accordance with an illustrative embodiment. As discussed, by varying the interval At between the ON and OFF, one can control the intracellular Ca++ levels, which in turn determine production levels. As a result, the proposed techniques significantly reduce the average power consumption from > 5 mW to 0.1 mW. [00131] In another illustrative embodiment, fluorescence measurements can be made by integrating a green emission light collected by the photodiode over the blue light stimulation block. The LED and photodiode performance can be measured in vitro by comparing fluorometry data to ground truth microscopy data that will measure LED timing, intensity, and fluorescence. In one implementation, lifetime testing can include soaking the encapsulated LEDs in phosphate buffered saline at 37°C for two months. For in vivo experiments to test photometry, fluorescent microspheres are encapsulated in the chassis and the fluorescence levels from the carrier can be measured.

[00132] The GFP* emission is not expected to interfere with the optogenetic system activation state since the emission light is approximately 10 ⁶ times weaker than the LEDs. Additionally, the feedback controller will account for any non-idealities by adjusting At to maintain a desired production setpoint. In an alternative embodiment, an alternative destabilized fluorescent protein such as DsRed-Express that can be excited using the orange “OFF” LED is used, such that any issues regarding cross-talk between the fluorophore and the control signals can be solved.

[00133] Figs. 9A-9B show an alternative embodiment of the optical system for the wearable device. Fig. 9A shows the design of the top side of a circuit board with the LEDs and photodiodes. In this embodiment, the light source of the stimulating system and the photodiode of the sensing system are integrated. Fig. 9B shows the LEDs integrating the blue and orange light into one wired prototype LED device with the wires extending out the right side of the LED device.

[00134] In Fig. 10, the LEDs device as shown in Fig. 9 were used in hybrid bioelectronic device having one or more cell wells/housings 31, as shown in Fig. 3A-D. One cell well/housing 31 contains an alginate capsule filled with non-GFP producing ARPE cells, while the other cell well/housing contains an alginate capsule filled with GFP producing cells. In this embodiment of the device, the LEDs color was alternated between blue and orange during the “ON” period. As shown in Fig. 10 below, this alternating color between blue and orange showed that when the blue LEDs were illuminated, there was a clear difference in the photodiode readings that was not seen when the orange LEDs were on, as seen in panel B of Fig. 10. This difference is expected to be from the green fluorescent light that is present in the cell well/housing containing GFP producing cell. To further support this claim, a control test was performed by filling the housings with PBS only. As seen in panel A of Fig. 10 reflecting the control test, there was nearly no difference between the photodiode readings of the two cell housings even when the blue LEDs were on. This illustrates the notion that the difference in the readings was caused by the green fluorescence produced by the engineered cells.

[00135] Fig. 11 shows the testing of the integrity of the encapsulation of LEDs device and to ensure that the LEDs and photodiodes would be able to withstand conditions similar to the body. Specifically, a carrier containing the electrical components was placed into a saline solution for 14 days during which time it was continuously powered. If any of the saline solution were to reach the circuitry, the board would short and the LEDs would turn off. The board was visually inspected daily to make sure that the LEDs were still illuminated.

[00136] The photodiode performance cannot be directly inspected by the visual inspection of LED light. However, if the saline solution penetrating the carrier, the photodiodes would be shorted due to the saline solution, and thus the LEDs would have turned off. Since the LEDs stayed lit throughout the experiment, it was assumed that the photodiodes did not fail during testing.

[00137] The results of the test reflects that the optical source of the stimulating system is capable for proving long term use, and the board showed no decrease in performance during or after the 14-day test, as the picture in Fig. 11 was taken after the 14-days period. No images of the board at the beginning of the test are provided since they look the exact same as the images in Fig. 11. This test not only proved that the encapsulation method was successful, but that the LEDs and photodiodes were able to work after encapsulation and even when submerged into a saline solution.

[00138] In one embodiment of the invention, similarly sized photodiodes and LEDs were selected for the device so that the LED light would not be blocked by the photodiode. Additionally, the photodiodes are in series with a 100 kQ resistor to create a measurable voltage. It should be noted that placing the LEDs directly next to the photodiode did lead to noise in the fluorescent photovoltage readings. When the blue LEDs were on, the photovoltage readings averaged approximately 960 mV. The orange LEDs generated a small photovoltage, but since these LEDs will not be illuminated during fluorescent readings, the photovoltage is inconsequential.

[00139] In order to prevent the blue light from reaching the photodiode, the LED light is filtered. The chosen filter was the Wratten 12 filter by Kodak. The Wratten 12 filter is a thin photography filter that acts as a long pass filter with a cut-on wavelength of approximately 500 nm. According to the absorption spectra, the filter should block 99.8% of the blue excitation light. As shown in Fig. 12A, based on the measured absorption curve transmission of the GFP emission light was expected to be 20%. In other embodiment, other filters having approximately same cut-on wavelength may be used.

[00140] In one embodiment, the thin film filter was bent into a box shape using a 3D printed mold. The filter was first laser cut into a cross shape and then the flaps were bent with the mold to form the box. This box was then laid on top of the photodiode and adhered to the photodiode using optical adhesive (NOA 84). This procedure is shown in Fig. 12B. In other embodiment, the filter can be formed into other shapes, and cutting and adhering of the filter can be achieved with any known tools in the field of arts.

[00141] Photovoltage measurements were taken again after the addition of the filter and the results shows that the filter effectively block the blue light. In particular, as shown in Fig. 12C, comparing to the blue light source without being encapsulated in the filter, the blue light encapsulated in the filter no longer generates any photovoltage, while the orange light still generated the same low photovoltage as before.

Device Mold and Materials

[00142] In one embodiment, the structure of the prototype is formed by pouring liquid PDMS into a mold and then letting it cure. Many iterations of mold designs were tested, and once the best design was found, the mold was machined out of aluminum, as shown in Fig.13 A. This was necessary to prevent the carrier from sticking to the mold which was seen with all 3D printed molds.

[00143] The shape of the mold allowed for bubbles to become trapped at the top, which would form voids in the device. These voids are points of failure for the device and therefore needed to be removed. A large vacuum glove box was purchased so that the PDMS could be poured into the mold under vacuum. When the mold is returned to atmospheric pressure, many of the bubbles pop and any that do not rise to the surface where they are manually popped with a heat gun. This molding technique has led to bubble-free devices and has expedited the molding process.

[00144] The mold design was made so that the circuit board designed in that subtask would fit into the mold and be encapsulated inside of the PDMS. Encapsulation of the circuitry protects the board during use in a saline solution and thus in the user’s body. The mold design also took into account the need for cell housings/wells that will be located in the wearable device. The cell wells/housings are formed during molding by placing a comb, similar to electrophoresis gel combs, into the liquid PDMS. The cell wells/housings were successfully formed and are located directly over the photodiodes on the circuit board as shown in Fig.13B.

In Vivo Testing of Drug Delivery

[00145] It is important to validate engineered cells in vivo to verify that they display or execute to the proper engineered functions. In one implementation, an experimental group (e.g., mice) is fluidly connected to a wearable device housing encapsulated engineered cells, and subsequently have the engineered cells turned on/off using the optogenetic system. Control groups include a first group fluidly connected to a wearable device housing encapsulated engineered cells with no optogenetic activation, a second group fluidly connected to a wearable device with no cells.

[00146] When compared to the experimental group, the group fluidly connected to a wearable device housing engineered cells with no optogenetic activation allows one to verify the ability of the system to trigger production of the therapeutic protein. Also, when compared to the experimental group and first control group, the second group fluidly connected to only wearable device with no cells allows one to determine whether the optogentic system is “leaking” and producing the therapeutics without being triggered.

The above-discussed procedure is used to validate the ability of the proposed system to controllably deliver therapeutics, e.g., GLP-1 and Orexin-A, in vivo. Specifically, engineered cells are housed in a wearable device which is fluidly connected to the test subjects. In vivo light exposure and assaying for protein production is performed. The therapeutics (e.g., GLP-1 and Orexin A in one embodiment) are assayed for via ELISA, while GFP* is assayed via microscopic imaging (e.g., simple fluorescence microscopy, or in vivo imaging system (IVIS)). The engineered cells are exposed to activating light in varying patterns to demonstrate control over expression patterns. Various time points after light exposure are taken to determine the rate that the therapeutics are secreted once the cells are turned on. At each time point, blood samples were taken, along with IVIS fluorescence images. Blood samples are assayed for therapeutics, and the IVIS images are used to quantify GFP* production. ELISA and fluorescence data is compared to calibrate how much fluorescence correlates to a quantity of therapeutic produced.

Power and Data Communication [00147] As discussed herein, in one embodiment, the wearable device has an on-board battery and is battery powered. In one embodiment, the battery is a replaceable battery or a rechargeable battery. In one embodiment, the wearable device may include a backup battery for providing power to the device when a main battery is removed from the device or being recharged. In other embodiment, other commercial power supplying methods can be integrated into the wearable device.

[00148] The wearable device are in communication with a terminal, e.g., smartphone, tablet, and etc., wirelessly. The wireless communication is based on Bluetooth, radio frequency (RF), electromagnetics, magnetic induction, electromagnetics, ultrasound, or etc.

External Sensing and Communications

[00149] As discussed herein, in one embodiment, the wearable device clicks securely into a socket in a comfortable, adjustable harness positioned on the abdomen or other area on the host. Alternatively, a different mounting technique may be used. The wearable device includes a gateway for all data and control, and includes a multi modal sensor suite designed to understand human behavior- such as sleep rhythms. The wearable device provides controls and a display on the device itself, in a protected pocket, or by connecting to a phone application. The phone application, beyond providing a mechanism for user input and control, also provides a way for the user to localize the wearable device and perform initial setup and system configuration. In alternative embodiments, a phone application may not be used, and all control and functionality can be performed through the user interface of the wearable device, which can include a touchscreen display, one or more buttons or other controls, etc. The wearable device is designed to enable long wear time, provide bio-sensing ability, enable intuitive controls, and be reconfigurable for diverse applications, including supporting the proposed entrainment therapies and future therapies.

[00150] In one embodiment, the wearable device can be built iteratively, verifying and testing novel functionality as more advanced prototypes are designed. As one example, a desktop, wall powered system with essential components can be designed, so that low level software development can begin at speed. The wearable device can be developed for testing and validating sensors to monitor circadian rhythms, where the wearable device includes only the external sensors, and a data acquisition unit/ microcontroller with built-in telemetry functions for data offload to a nearby desktop, thereby facilitating circadian rhythm sensing for NHPs. Concurrently, power circuitry and battery lifetime management circuitry can be handled in a more portable prototype holding all functionality beyond just sensing. This also enables harness and enclosure design to begin, and for the external hub to be further miniaturized until the final device with all functionality and user controls is delivered. This sequence of hardware designs enable the handling of various device challenges, which are described below.

[00151] In commercial off the shelf wearables, battery lifetime can vary dramatically based on the actions of the user, the software running, the signals environment, and the internal components that are activated. While running out of battery is merely an inconvenience for the average person, for a user of the proposed system it may severely hamper mission/work readiness. Reliability and predictability of battery lifetime for complex cyber-physical systems is critical but challenging because of the intersection of software, hardware, and user non determinism. Therefore, a robust energy model is embedded in the firmware of the wearable device that is circadian rhythm and environment aware, enabling prediction of the wearable device lifetime with high accuracy, such that completion of therapies and the mission/work is ensured. The energy modeling can include complex cyber and physical components, a physical signals environment, sensing algorithms, user interaction modeling, and therapy delivery. This static model can be augmented by in-situ power measurements and execution traces such that the static model is continuously refined. This energy model is a core portion of the wearable device operating system, and can leverage embedded energy models to enable ultra-long (e.g., nine month plus) battery lifetime.

[00152] In an illustrative embodiment, the wearable device uses non-contact COTS sensors to measure and track diurnal variations in body temperature, heart rate and variability, and physical activity, which are inputs to a model of circadian control that estimates the circadian phase of the wearer.. The system can also use well studied sensors for sleep and activity monitoring, such as ballistocardiography based heart rate sensing and accelerometer based actigraphy. Ballistocardiography (BCG) is a measure of ballistic forces generated by the heart, enabling measurement of R-R interval. Accelerometers can measure these forces even if not directly above the heart, or even attached to the body (for example, R-R interval was captured when an accelerometer was placed under the mattress of a sleeping person). As the system will continuously sense circadian phase, estimates of heart rate will occur even when the wearer is active. As such, a 9-axis inertial measurement unit is used that allows for removal of gravity effects and motion artifacts, and provides orientation of the wearer (from the magnetic sensor), to understand posture. Actigraphy methods can be used to separate sleep activities from confounding activities such as exercise, eating, or walking. Infrared based skin temperature sensors are also included to validate/calibrate internal temperature from the implant, and provide a coarse estimate of heat flux based on known values of thermal conductivity of human skin, which can be used to estimate core body temperature. The wearable device captures all raw signals from these sensors, cleans the signals, and extracts relevant biomarkers for sensing circadian phase. Software machine learned models that reduce noise are developed for each sensor.

[00153] The user of the proposed system needs mechanisms to control therapies no matter the situation (before a mission, when traveling, while working, in the field, etc.). Physical controls of the system are designed with visual feedback on the wearable device to program, stop, and start therapies in the field. In an illustrative embodiment, the controls lie in a protected compartment in the enclosure to ensure no accidental actions. The controls can be mirrored on a smartphone application with the same capabilities in one embodiment. This provides seamless control no matter what situation the user is in. The smartphone application connects with the wearable device using an encrypted Bluetooth LE channel in one embodiment, and allows for richer visualization of entrainment progress. As a result, the wearer can understand the effect of designed therapies in real time. This innovative, mirrored, multi-context interaction approach provides a new way to think about and visualize on the go applications for users in high stress, highly mobile environments.

[00154] The enclosure for the wearable device will protect the circuitry in a slim profile watertight package. Controls can be in a protected pocket to prevent accidental button presses. The enclosure can be designed iteratively, in a large functional form factor, then miniaturized, hardened, and waterproofed. On first use, the wearable device can be placed in the harness, and the localization procedure is initiated from the smartphone app (or alternatively on a user interface of the wearable device). The application (or the wearable device itself) guides the user on which direction to move the wearable devicefor optimal power based on measuring received signal strength from the implant NFC uplink. Once the location is set, the harness is tightened to secure the placement. The harness is co-designed along with the wearable device based on existing abdomen harnesses that secure items like radio transmitters, etc.

[00155] Security of communication, and security of operation are critical for delivery of entrainment therapies. This problem is addressed by the proposed design. Specifically, ME communication is highly localized, making it difficult to emulate control commands sent from the wearable device to the terminal. The smartphone application, if used, can connect with the wearable device using an encrypted Bluetooth LE channel, reducing possibility of malicious data exfiltration. Alternatively, a different secure communication channel may be used to perform communication between the user device (e.g., smartphone, tablet, laptop, etc.) upon which the application is located and the wearable device. Security checks can also be performed at each layer of the software/hardware stack in the wearable device, which will further reduce the possibility of tampering and data exfiltration.

[00156] In the event of issues with battery lifetime being shorter than anticipated due to ME power costs, the size and/or shape of the wearable device can be adjusted to support a larger battery. Further, if it is determined that the non-contact sensor selection is not sensing heart rate accurately enough for the circadian phase sensing algorithm, electrodes for EKG can be placed on the harness itself, utilizing skin contact.

Buffer/Nutrient Solution for Cell Wells/Housings

[00157] In one embodiment, RZA 15 is used as a suspension solution to suspend engineered cells in the wells/housings. RZA15 is a molecule that will help protect the cells from fibrosis in vivo promoting cell viability. RZA15 was synthesized in the following manner as shown in Fig. 14 A.

[00158] In particular, in one embodiment for synthesizing the RZA15, 4- Propargylthiomorpholine 1,1 -Dioxide (1 eq.) was added to a 250 mL round bottom flask and dissolved in methanol: water mixture (5:1). Consequently, Tris[(l-benzyl-lH-l,2,3-triazol-4- yl)methyl]amine (0.25 eq.), Triethylamine (0.25 eq.), and copper iodide (0.1 eq.) was added. The reaction mixture was purged with argon for 15 mins and cooled to 0°C following which 11- azido-3,6,9-trioxaundecan-l-amine (1 eq., 6.30 g, 28.86 mmol) was added. The reaction mixture was stirred at room temperature for 15 mins and afterward heated to 55°C for overnight. The reaction was cooled to room temperature and filtered through celite to remove any insoluble part. The filtrate was dried using rotavap under reduced pressure with silica. The crude reaction was then purified by liquid chromatography with dichloromethane: ultra (22% MeOH in DCM with 3% NH40H) mixture 0% to 40% on a 120 gm ISCO silica column. The final product was further characterized with ESI mass and NMR mass spectroscopy according to Fig. 14B.

[00159] Once the RZA5 is synthesized, it is conjugated to UPVLVG alginate to be used as the hydrogel to suspend cells in the carrier. The conjugation was then carried out in the following manner as shown in Fig. 15 A.

[00160] In one embodiment for conjugation, in a round bottom flask, 2g (leq) of UPVLVG (BP-1903-04; Novamatrix) was dissolved in water (75 mL). Then RZA15 small molecule (3.99 g, 10.20 mmol, leq) was dissolved in water by vortexing and the pH was adjusted to 7.4 using HC1. Then RZA15 aqueous solution was slowly added to the UPVLVG while stirring. Subsequently a solution of (4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) (DMTMM, 0.5 eq.) was added dropwise to the mixture of UPVLVG and RZA15. The reaction was heated to 55°C and stirred overnight. The solution was filtered through a cyano- silica and dialyzed in a 40cm long 10-12K pretreated dialysis tubing in a beaker using saline (2 days) and mili-Q water (3 days). The dialyzed solution was frozen at -80 degrees and lyophilized until dry. The final product was then characterized by NMR and elemental analysis with elemental analysis showing a 16% modification of the alginate material. NMR and elemental characterization of the conjugates is shown in Fig. 15B.

Supporting and Controlling Cells in a Bioelectronic Carrier

[00161] One key challenge for implementing a feedback control on the bioelectronic carrier is to engineer sufficient energy efficiency to operate within the desired power budget. In one embodiment, a step-function control strategy is used to reduce the power needs. In one embodiment, the step-function control strategy reduces the power needs by approximately 50X. In addition to reducing the average power consumption, the inventors also designed the system to minimize the peak instantaneous power requirements, to avoid the need for large energy storage elements on the device. To reduce the need for large peak power, LED control sequences can be interleaved within 10-minute blocks so that only one LED will be active in any cell housing/well at any one time. Additionally, the seconds scale activation and inactivation pulses required to turn on and off the optogenetic system channels can be converted into high frequency (50 Hz) pulse trains with the same total energy. In one embodiment, these two strategies offer an approximately 10X reduction in peak power that allows for miniaturization of the charge storage elements. Light-leakage and optical cross talk between wells can be reduced in some embodiments by doping the PDMS with gold NPs that will render the walls opaque without compromising the permeability and elastomeric properties of the material. To ensure that the LED and photodiode remain functional inside the body, Parylene C, Parylene N, SiC (Silicon Carbide), and Medical grade epoxies can be used for effective bioencapsulation. In addition, the photodiode can be coated with a combination dielectric and absorption filter which has been shown to be effective for on-chip fluorescent imaging.

[00162] To enable the proposed high-density cell loading, oxygen can be supplied to the encapsulated cell well/housing. This can be accomplished via electrolysis of water at adjacent microelectrodes in one embodiment. In another embodiment, O2 gradients are tailored and maintained to optimize the performance of encapsulated cell well/housing by precisely tuning the platform’s electrode size, spacing, and power supply. The use of selective polymer membranes can be used to minimize reactive byproducts and protect the device from bio-fouling. A primary goal is to generate enhanced oxygen concentration on a small device footprint and with a low overall power budget. In alternative embodiments, depending on the type of cell(s) being used or the cell density, O2 may be supplied with oxygen producing CaO nanoparticles, or O2 may not be supplied to the cell well/housing at all.

Cell Capture and Expansion in the Wearable External Device [00163] In some embodiments, the wearable device provides personalized on-site cell engineering utilizing cells collected from a user of the wearable device. As shown in Fig. 3D, the wearable device includes a cell housing/cartridge 51 and an intake cannula in fluid communication with the user wearing the wearable device on one end and with the cell housing/cartridge 51 on the other end. In some embodiments, the intake cannula may contain more than two ends, forming a fluid loop necessary for the processes described below.

[00164] The intake cannula receives body fluid, e.g., blood, interstitial fluid, and etc., from the user, and the body fluid enters into the cell housing/cartridge 51 therefrom. Immune cells in the body fluid of the user are captured, expanded, and/or primed for therapeutic applications in the cell housing/cartridge 51. In one embodiment, the cell housing/cartridge 51 receives blood from the user, and tumor specific effector lymphocytes (e.g., T cells, macrophages, NK cells, B cells) in the blood are captured in the cell housing/cartridge 51. The tumor specific effector lymphocytes are then expanded and primed through exposure to cytokines IL-2, IL-15, and/or IL-12. The primed tumor specific effector lymphocytes can be used for therapies for the user. In another embodiment, suppressive lymphocytes (e.g., T cells, macrophages) are captured from the body fluid of the user, and then expanded and primed in the cell housing/cartridge 51, through exposure to IL-10, IL-22, and/or IL-35. With such in-situ cell collection, expansion and priming processes, the wearable device provides personalized cell therapies specifically tailored to the user’s medical/physiological conditions.

Cells Loading Aid Device

[00165] Fig. 16 shows a cell-loading aid device 900 responsible for loading the engineered cells into the cell wells/housings of the hybrid bioelectronic device. The aid device 900 has a base 910 and a guide 940 attached to one longitude side of the base 910. The base 910 has a niche 920 for receiving the hybrid bioelectronic device. In the guide 940, there exists at least one hole 930 through which the engineered cells are injected into the cell wells/housings.

Computing System

[00166] In an illustrative embodiment, components of the proposed system such as the subcutaneous implant device, external hub, wearable device, and/or associated user device can include and/or be in communication with one or more computing systems that include a memory, processor, user interface, transceiver, and any other computing components. Additionally, any of the operations described herein may be performed by the computing system(s) of these components. The operations can be stored as computer-readable instructions on a computer- readable medium such as the computer memory. Upon execution by the processor, the computer- readable instructions are executed as described herein. As an example, Fig. 17 is a block diagram of a computing system to perform operations described herein in accordance with an illustrative embodiment.

[00167] Specifically, Fig. 17 depicts one embodiment of a computing device 1400 (e.g., an external hub or a wearable device) in direct or indirect communication with a network 1435, one or more user devices 1440, and a subcutaneous implant device 1445. The user device(s) 1440 can include a smartphone, tablet, laptop, smartwatch, activity tracker, or other user device that is in communication with the computing device 1400. As discussed, the user device(s) 1440 can include an application that interfaces with and controls the computing device 1400.

[00168] In this illustrative embodiment the computing device 1400 includes a processor 1405, an operating system 1410, a memory 1415, an input/output (I/O) system 1420, a network interface 1425, and power/sensing hardware and software 1430. In alternative embodiments, the computing device 1400 may include fewer, additional, and/or different components. The components of the computing device 1400 communicate with one another via one or more buses or any other interconnect system. Although not depicted in Fig. 17, it is to be understood that the subcutaneous implant device 1445 (alternatively, the wearable device) and the user device(s) 1440 can similarly include computing components such as a processor, an operating system, a memory, an input/output (I/O) system, a network interface, power/sensing hardware and software 1430, and/or any of the other computing components described herein.

[00169] The processor 1405 of the computing device 1400 can be in electrical communication with and used to control any of the external hub components (alternatively, the wearable device) described herein. The processor 1405 can be any type of computer processor known in the art, and can include a plurality of processors and/or a plurality of processing cores. The processor 1405 can include a controller, a microcontroller, an audio processor, a graphics processing unit, a hardware accelerator, a digital signal processor, etc. Additionally, the processor 1405 may be implemented as a complex instruction set computer processor, a reduced instruction set computer processor, an x86 instruction set computer processor, etc. The processor 1405 is used to run the operating system 1410, which, as discussed herein, can be a custom operating system specific to the requirements of the external hub (alternatively, the wearable device).

[00170] The operating system 1410 is stored in the memory 1415, which is also used to store programs, sensed patient data, algorithms, network and communications data, peripheral component data, and other operating instructions. The memory 1415 can be one or more memory systems that include various types of computer memory such as flash memory, random access memory (RAM), dynamic (RAM), static (RAM), a universal serial bus (USB) drive, an optical disk drive, a tape drive, an internal storage device, a non-volatile storage device, a hard disk drive (HDD), a volatile storage device, etc.

[00171] The I/O system 1420, or user interface, is the framework which enables users and peripheral devices to interact with the computing device 1400. The I/O system 1420 can include one or more keys or a keyboard, one or more buttons, one or more displays, a speaker, a microphone, etc. that allow the user to interact with and control the computing device 1400. The I/O system 1420 also includes circuitry and a bus structure to interface with peripheral computing components such as power sources, sensors, etc.

[00172] The network interface 1425 includes transceiver circuitry that allows the computing device 1400 to transmit and receive data to/from other devices such as the subcutaneous implant device 1445, the user device(s) 1440, remote computing systems, servers, websites, etc. The network interface 1425 enables communication through the network 1435, which can be one or more communication networks. The network 1435 can include a cable network, a fiber network, a cellular network, a Wi-Fi network, a landline telephone network, a microwave network, a satellite network, etc. The network interface 1425 also includes circuitry to allow device-to- device communication such as near field communication (NFC), Bluetooth® communication, etc.

[00173] The power/sensing hardware and software 1430 can include hardware, software, and algorithms (e.g., in the form of computer-readable instructions) which, upon activation or execution by the processor 1405, performs any of the various operations described herein such as sensing data, receiving sensed data, performing analyses of sensed data, generating control signals, generating power and controlling power usage, etc. The power/sensing hardware and software 1430 can utilize the processor 1405 and/or the memory 1415 as discussed above. [00174] In an illustrative embodiment, the subcutaneous implant device 1445 can be any of the implant devices (alternatively, the wearable device) described herein, and can include any of the functionality/components described herein. In one implementation, the subcutaneous implant device 1445 (alternatively, the wearable device) can include an electronic layer that can include one or more actuators to control cell production, one or more sensors, an ASIC, a processor, a memory, a battery, a transceiver, etc. Attached to the electronic layer is a biological cell layer that includes engineered cells. In an illustrative embodiment, the engineered cells are within a hydrogel that forms at least a portion of the biological cell layer. The hydrogel can be within a chamber that is accessible to the sensor(s) and/or actuator (e.g., a transparent bottom of the chamber can be used if the cells are actuated via optoelectronics).

Circadian Rhythm Control

[00175] In one exemplary implementation, the proposed system can be used to help control the circadian rhythm of the subject in which the system is implanted or worn. For example, the system can be used to accelerate human adaptation to a new time zone or work schedule by synergistically shifting central and peripheral circadian clocks. While various examples and implementation details are provided herein with respect to control of circadian rhythm, it is to be understood that the proposed system is not limited to circadian rhythm applications. Rather, as discussed herein, the proposed implantable cell generation system can be used to provide pain relief, fight diseases, cure disorders, provide immune response control, treat infertility, etc. [00176] It has been shown that desynchrony between multiple circadian clocks and the light/dark cycle can result in decrements in mental performance and health and act to hinder entrainment. Traditional methods to accelerate adaptation, or entrainment, focus on light therapies or fixed-dose, single-target medications that may not be ideally timed or efficacious. These approaches focus largely on central clock-driven rhythms such as the sleep/wake cycle, without considering adverse effects of misaligned internal clocks that entrain more slowly than central hypothalamic clocks. To most effectively accelerate entrainment, the proposed system engages the entire network of a host’s central and peripheral clocks with precise control of the timing and dose of peptide therapies. This level of precision exceeds what is possible with current therapies. Namely, the proposed system is able to achieve 1) specific biological action on select target receptors or molecules that cannot be accomplished with current bioelectronics, and 2) precise control of timing and dosage that cannot be accomplished with current synthetic biology.

Sensing Circadian Phase

[00177] In an illustrative embodiment, a multi-sensor fusion strategy is used to accurately measure the phases of the multiple 24-hour rhythms that are disrupted by long-distance travel and late-night operations. Specifically, biophysical, physiological, and behavioral markers are measured to track multiple rhythms and overcome confounding factors (like physical activity) that could mask a CR measurement based on any singular sensing modality. Real-time assessment of circadian phases of both central and peripheral clocks enables precise timing of therapeutics. Existing, robust sensor technologies, including internal and skin surface temperature, 9-axis inertial measurement units, and heart rate sensing techniques are used. These parameters exhibit robust circadian rhythms and are can be used to access the circadian timing. The timing of these rhythms are regulated by distinct SCN output pathways as well as by different local tissue physiology and environmental timing cues, thus together depicting the status of the hierarchical circadian system as a whole. In alternative embodiments in which the cell factory is used to treat pain, manage diseases, etc., sensor data may not be used. In other embodiments, different types of data may be sensed, specific to the non-circadian application. [00178] The sensor data is used as inputs to a well-established model of circadian control to produce an estimate of circadian phase and predict phase shifts in response to stimuli and delivery of therapies. The most common approach is to model circadian control as a limit cycle oscillator. These models simulate the rate of change in state variables (e.g., core body temperature, activity, heart rate) as functions of the current status of state variables plus “drives” from external stimulus (e.g., entraining agents such as light or therapeutic peptides). Using such models, the current internal timing of the animal (i.e., circadian phase) can be resolved from the values of the state variables, and predictions of phase-shifts induced by light and/or therapeutic peptides can be made at any phase of the cycle.

[00179] Since diurnal variations in each of the selected biomarkers reflect different facets of circadian control, a unified measure of circadian phase is generated that incorporates all 3 biomarkers as state variables in a single “limit sphere” model. Each state variable can have a different driving function in response to light or peptides to represent their different rates of entrainment. In this way, the model can also detect misalignment between the phase reference points of different state variables during re-entrainment. Estimates of circadian phase are measured as a percent error relative to reference measures obtained with a fully implanted sensor system that is the standard used in most sleep studies. In addition to data collected for model development and testing, data of peptide-induced phase shifts in mice can also be used to facilitate the selection of model framework. The model can be fine-tuned to direct daily peptide treatment schedule to achieve accelerated entrainment.

[00180] Testing can be performed in subjects instrumented with a standard suite of sensors used in sleep research, including electroencephalography (EEG), electrooculography (EOG, eye- movements), and electromyography (EMG, neck muscles) that will provide gold-standard reference measures of circadian phase for comparison with the NTRAIN sensor suite. Data can be collected continuously around the clock via COTS implantable telemetry devices that integrate all of the required sensing functions in a fully implantable, battery-powered package that can transmit data continuously for long periods of time. An initial prototype of the NTRAIN sensor set (external hub) will be implanted to verify sensors in vivo prior to full integration of the external hub. In addition to performing continuous monitoring of physical and physiological biomarkers, established behavioral assays of cognitive function (e.g., working memory, attention) are also implemented to measure changes in cognitive performance throughout the circadian cycle. These tests can be incorporated into the daily enrichment schedule for the test subjects, which minimizes stress and improves psychological well-being. The enrichment schedule includes social interaction, physical activity, sensory stimulation, food, and cognitive/occupational activities. Thus, the cognitive testing protocols provide enrichment in all five categories, and the testing data generates operationally-relevant, performance-based measures for evaluating the effects of CR-entrainment therapies.

[00181] Circadian phase-sensing is used for determining the type, timing, and dose of therapies to deliver. Successful completion of this task depends on 3 key factors of low to moderate risk and are discussed in order of decreasing risk. First, obtaining reliable measures of circadian biomarkers is moderately risky, particularly in large animals. An established COTS system (DSI telemetry) that has been used for similar long-term monitoring studies in many species, including non-human primates, can be used. Second, the circadian control model is essential for interpreting the biomarker data. Accurate phase predictions are essential and there is a low risk that the algorithm will not generalize across all conditions. However, this risk is thought to be low to very low.

Circadian Rhythm Therapeutics Delivery

[00182] In one exemplary embodiment of multiple therapeutic delivery, multi-clock targeting with precision timing for circadian rhythm regulation can be performed by the proposed system. For example, in the circadian rhythm example, by targeting both the central and peripheral clocks, the hybrid bioelectronics system of the present invention provides synergistic effects towards enhanced entrainment, as shown in Fig. 1. However, because the same therapy applied during different phases of a circadian rhythm can have both phase-advancing or phase-delaying effects, it is important to validate therapeutic efficacy in terms of its administration window. The system uses phase response curves (i.e., the phase shift induced by therapy as a function of the phase of delivery), combined with real time sensing of internal body temperature and/or commercial off the shelf (COTS) wearable sensors to inform actuation-timing for most effective delivery of therapies. Fig. 1 depicts phases of peripheral and central clocks in response to an 8 hr shift, for normal entrainment (left), providing therapy affecting only the central clock (middle), and the approach of the hybrid bioelectronics system of the present invention (right) with therapy targeting both central and peripheral clocks in accordance with an illustrative embodiment. In Fig. 1, the fill color green represents normal phase relationship, and red represents misaligned phases. The system of the present invention rises above the current state-of-the-art in circadian rhythm management because it delivers a personalized therapy with precision dosing and timing for maximum efficacy. This is not possible with single-dose approaches that act only on sleep/wake rhythms.

[00183] In the illustrative embodiment for the circadian rhythm application, the therapeutics targeted for production and delivery by the engineered cells are GLP-1 and Orexin A. In alternative embodiments, different types of therapeutics may be produced. Production of such peptides presents an inherent advantage, especially in the application of circadian management. The peptides are produced by mammalian cells and thus are native, non-recombinant peptides. Unlike recombinant variants, GLP-1 and Orexin A have short metabolic half-lives (GLP-1, 4.6- 7.1 min; Orexin A, 27 min), making their use for entrainment more effective. Such half-lives are long enough to reach target tissues, short enough to have a precisely timed phase-shifting action, and are known to readily cross the blood-brain barrier, exhibiting potent actions on the brain when peripherally administered.

[00184] Precision timing and dosing is paramount to the therapeutic approach. As such, the wearable device can be used to determine current state of the patient. In one embodiment the relevant state is the patient’s circadian phase, and with knowledge of target shift magnitude (e.g., how many time zones will the user traverse) and ideal timing of both therapies (from phase response curves), will determine the optimal dose/timing schedule, which may be initiated by the user. As such, each therapy will be administered at most once per day in one embodiment. This routine can be repeated daily, per suggestion of the system, until the entrainment is achieved. Delivery of each therapy is expected to occur within seconds of illumination of the light source, and actively regulated to a fixed level and duration by the dosing control system. Therapy production is expected to stop within about 2 minutes of turning off the light source, and presence in the blood stream is dictated by the metabolic half-life of the peptide (5-30 min). The daily timing and dosing schedule can be generated and stored in the wearable device, and initiated by the user via pressing a button, voice command, etc.

[00185] The foregoing description of illustrative embodiments of the invention has been presented for purposes of illustration and of description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invention and as practical applications of the invention to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.