Circadian probiotic systems

Abstract

Among the various aspects of the present disclosure is the provision of a circadian probiotic system. The present teachings include compositions for a probiotic supplement to deliver a drug to a subject. The supplement can be modified to include a synthetic gene circuit to drive a therapeutic controlled gene output that can be delivered to the gut of the subject on a prescribed time basis, and the synthetic gene circuit can include at least one circadian clock gene that drives expression of at least one prescribed therapeutic or diagnostic gene target. The present teachings also include therapeutic methods to deliver at least one diagnostic and therapeutic gene target to a subject time-sensitively. The method can include modifying a probiotic supplement to include a synthetic circadian gene circuit that drives expression of the at least one gene target. In another aspect, the method can include delivering the probiotic supplement to the subject.

Description

[0001] TITLE OF THE INVENTION

[0002] CIRCADIAN PROBIOTIC SYSTEMS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 509,698 filed on June 22, 2023, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] Not applicable.

[0007] MATERIAL INCORPORATED-BY-REFERENCE

[0008] Not applicable.

[0009] FIELD OF THE INVENTION

[0010] The present disclosure generally relates to circadian probiotic systems.

[0011] BACKGROUND OF THE INVENTION

[0012] The emerging field of “chronotherapy” is based on the idea that most drugs target circadian or clock-controlled genes and therefore the time of day that they are administered can significantly influence their effectiveness. In many cases, biologic drugs have been shown to be effective only if delivered intermittently or at specific times of the day. This process is very difficult to maintain since oftentimes it can require constant daily injections or delivery at inconvenient times, such as the middle of the night.

[0013] SUMMARY OF THE INVENTION

[0014] Among the various aspects of the present disclosure is the provision of a circadian probiotic system. The present disclosure describes compositions and methods that make use of the administration of a probiotic such as bacteria, yeast, or any other organism that has been modified to provide timed gene expression as a basis for the delivery of biologic drugs at prescribed times and frequencies. Once reprogrammed, the probiotic can be introduced to the body for this purpose. The genes controlling circadian rhythms may be naturally occurring genes in the probiotic or may be genes, original or modified, from other species (fruit fly, mammalian, etc.).

[0015] Briefly, therefore, the present disclosure is directed to probiotic supplements modified to include a synthetic circadian gene circuit to drive the expression of a prescribed diagnostic or therapeutic gene target and methods of use thereof.

[0016] The present teachings include compositions for a probiotic supplement to deliver a drug to a subject. In one aspect, the supplement can be modified to include a synthetic gene circuit to drive a therapeutic controlled gene output that can be delivered to the gut of the subject on a prescribed time basis. In another aspect, the synthetic gene circuit can include at least one circadian clock gene that drives the expression of at least one prescribed therapeutic or diagnostic gene target. In another aspect, the probiotic can be bacteria, yeast, or any combination thereof. In another aspect, the probiotic supplement can be endogenous or exogenous. In some embodiments, the probiotic supplement is genetically modified in situ. In other embodiments, a probiotic supplement can be genetically modified ex situ, and then reintroduced into the subject. In accordance with another aspect, the probiotic can be administered to the subject by oral supplement, gavage, fecal transplant, or by being engineered into an implantable living tissue. In another aspect, the at least one circadian clock gene can be selected from BMAL1, PER1 / 2 / 3, CRY1 / 2, NPAS2, RORa / b, REV-ERBa, RORE, Db, NFIL3, GSK3b, CK1e / d, D box regions, E box regions, RRE regions, and any combinations thereof. In some embodiments, the at least one circadian clock gene can include controlled mutations to modify and control the phase, amplitude, or period of gene expression. In some aspects, the synthetic gene circuit is created using plasmid or viral transduction, CRISPR-Cas9 gene editing, TALENs, ZFNs, or any other method of genetic modification. In some embodiments, the overall duration and dynamics of the response can be controlled using a synthetic genetic counter that counts clock cycles in cells. In yet another aspect, the at least one therapeutic or diagnostic gene target is anti- catabolic, anti-inflammatory, pro-anabolic, pro-regenerative, pro-circadian, antimicrobial, anti-pain, a morphogen, a growth factor, anti-cancer, or any combination thereof.

[0017] The present teachings also include therapeutic methods to deliver at least one diagnostic and therapeutic gene target to a subject time-sensitively. In one aspect, the method can include modifying a probiotic supplement to include a synthetic circadian gene circuit that drives expression of the at least one gene target. In another aspect, the method can include delivering the probiotic supplement to the subject. In another aspect, the at least one gene target is anti- catabolic, anti-inflammatory, pro-anabolic, pro-regenerative, pro-circadian, antimicrobial, anti-pain, a morphogen, a growth factor, anti-cancer, and any combinations thereof. In accordance with another aspect, synthetic circadian gene circuits can be selected from BMAL1, PER1 / 2 / 3, CRY1 / 2, NPAS2, RORa / b, REV-ERBa, RORE, Db, NFIL3, GSK3b, CK1e / d, D box regions, E box regions, RRE regions, and any combination thereof. In some embodiments, the probiotic supplement can be delivered to the gut and used to deliver an antiinflammatory drug in a circadian manner with peak drug concentrations at midnight using the expression of the circadian gene PER2 as a driver to coincide with the initiation of an inflammatory flair. In another aspect, the at least one synthetic circadian gene circuit can be mutated to control the time of drug delivery. In some embodiments, the circadian gene circuit drives intermittent expression of parathyroid hormone (PTH). In one aspect, the intermittent expression of PTH can promote bone growth in an osteoporosis patient. In some embodiments, the probiotic supplement can deliver specific nutrients or nutrient precursors / modifiers on a timed basis that improves nutrient absorption, nutrient retention, general health benefits, and any combination thereof. In some embodiments, two or more out-of-phase circadian clock genes can be used to drive the delivery of pain-modifying drugs that are delivered every 6, 12, or 24 hours automatically.

[0018] Other objects and features will be in part apparent and in part pointed out hereinafter. DESCRIPTION OF THE DRAWINGS

[0019] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0020] FIG. 1A is an image of a healthy joint (left) and a rheumatoid arthritis (RA) joint (right). Normal synovial joints consist of a joint capsule surrounding the joint, with a synovial membrane lining the inner surface and synovial fluid that reduces friction in the articular cartilage. Joints in RA exhibit chronic inflammation resulting in cartilage destruction, pannus invasion, synovial hyperplasia, and bone erosion.

[0021] FIG. 1 B is a schematic of an RA joint showing chronic inflammation results in feedback of inflammatory cells recruited to the joint and dysregulating growth of the synovial tissue, resulting in synovial hyperplasia and pannus invasion that contributes to cartilage destruction & bone erosion, mediated through the production of proinflam matory cytokines and catabolic protein.

[0022] FIG. 1 C is a timeline of inflammation depicting periodic inflammatory flares that begin prior to clinical diagnosis of chronic disease (UA: undifferentiated arthritis, RA: rheumatoid arthritis).

[0023] FIG. 2A is a timeline of inflammation showing inflammation in RA flares over long-term and daily periods.

[0024] FIG. 2B is a graph of cytokines (IFNy, IL10, TNFa, IL6) that mediate inflammation in RA peak in the early morning hours.

[0025] FIG. 3 is a schematic of the bidirectional connections between circadian and NFKB pathways. Interactions between the circadian and NFKB pathways can be context-dependent, and the proteins of these pathways directly interact with each other, leading to up and down-regulation depending on the context. Interactions between BMAL1 and NFKB subunits can lead to reduced circadian cycles, such as when inflammatory cytokines lead to a drop off in the circadian rhythms. NFKB subunits can act as repressors of BMAL1 / CLOCK complexes bound to e-box elements, demonstrating a transcriptionally repressive role in contrast to the typical role of these subunits in promoting transcription. Interactions between CLOCK and NFKB subunits can positively regulate the NFKB pathway, showing multiple interactions between these pathways at the protein level, which positively regulates some genes while negatively regulating others.

[0026] FIG. 4A is a schematic of a circadian-driven gene circuit using the Per2 promoter to produce the gene 111m.

[0027] FIG. 4B is an image of the Per2 promoter of FIG. 9A showing the e-box (black box) and NFKB response elements (grey boxes), which regulate separate pathways and can be context-dependent, resulting in up or down-regulation of circadian and inflammation pathways (as shown in FIG. 3).

[0028] FIG. 4C is a set of graphs of PDiPSCs expressing the Per2-111 ra luciferase vector showing circadian reporter activity over time when treated with control (left) and IL-1 a (right). Circadian reporter activity is maintained following the addition of IL-1 a.

[0029] FIG. 4D is a graph of PDiPSCs expressing the Per2-ll1 ra luciferase vector showing 111 rn gene expression overtime, demonstrating 111m is regulated in a circadian manner.

[0030] FIG. 5A is a set of graphs depicting transcriptional activation through the NFKB (left) promoter, E-box (middle) protomer, or both (right) promoters. This demonstrates the use of different gene circuits to respond to on-demand signals (NFKB), a gene circuit that has a cyclical circadian output with daily fluctuation (E-box), and a gene circuit that combines both elements (both). The arrow represents when an inflammatory stimulus is added.

[0031] FIG. 5B is a schematic of an “OR” gene circuit designed with additive-like behavior that responds to both circadian and inflammatory cues.

[0032] FIG. 5C is a schematic of an “AND” gene circuit designed which would drive an output when there is inflammation and circadian transcriptional activity but not when there is neither or either alone.

[0033] FIG. 5D is a schematic of an “OR” gene circuit incorporating an E-box promoter and an NFKB response element (left) to respond to both circadian and inflammatory cues. An example is the addition of IL113 (right), which would increase the expression of NFKB-responsive circuits but impede a circadian- driven output. With both promoters present there is a baseline circadian output under non-inflammatory conditions and an increase in amplitude under inflammatory conditions.

[0034] FIG. 6A is a Per2 plasmid that drives GFP and luciferase or IL1 Ra production. This plasmid was modified to develop a synthetic gene circuit that responds to both circadian and inflammatory cues.

[0035] FIG. 6B is the Per2 plasmid of FIG. 6A which has undergone a restriction enzyme digest to remove the Per2 promoter.

[0036] FIG. 6C is a region of synthesized DNA of 3x E’-boxes (top) and minimal CMV (bottom), which include the restriction enzyme sites Spel and Asci or Asci and BamHI.

[0037] FIG. 6D is a 3xE’Box plasmid which is the ligation product of FIG. 6B and FIG. 6C. The plasmid contains 3x E’boxes which drives GFP and luciferase or IL1 Ra production.

[0038] FIG. 7A is an NRE (NFKB response element) plasmid that contains the minimal CMV element and drives a luciferase IL1 Ra output.

[0039] FIG. 7B a PCR product (top, left) of the NFKB RE from the NRE plasmid (FIG. 7A). The PCR product is digested (bottom, left) with the restriction enzymes Spel and Asci to create sticky ends with a nucleotide overhang. The digested NFKB product undergoes PCR to add 3xE’box homology and mCMV homology (top, right) or backbone homology and 3xE’box homology (bottom, right).

[0040] FIG. 7C is the 3xE’Box plasmid (FIG. 6D) digested with the restriction enzyme Asci which allows for the insertion of NFKB RE with 3xE’Box and mCMV homology (FIG. 7B).

[0041] FIG. 7D is a 3xE’Box-5x NFKB plasmid which is the production of 3xE’Box plasmid and NFKB RE (FIG. 7C). This plasmid contains both circadian and inflammatory response elements.

[0042] FIG. 7E is the 3xE’Box plasmid (FIG. 6D) digested with the restriction enzyme Spel which allows for the insertion of NFKB RE with backbone and 3xE’Box homology (FIG. 7B).

[0043] FIG. 7F is a 5XNFKB-3XE’BOX plasmid which is the production of 3xE’Box plasmid and NFKB RE (FIG. 7C). This plasmid contains both an inflammatory response element and circadian response element.

[0044] FIG. 8A is a timeline and experimental setup to test the protective effects of the circuits responsive to inflammatory cues. HEK293T cells are transduced with the NFKB plasmid that generates a luciferase reporter. The cells are synchronized with 1 ,5h serum shock (50% serum) or 1 ,5h Dex treatment (100nM). Then 1 ng / mL of IL-1 a is added to the cells. Baseline luminescence is recorded at 24 hours and experimental luminescence is recorded at 72 hours.

[0045] FIG. 8B is a graph of luminescence of HEK293T cells transduced with the NFKB luciferase reporter. The cells were not synchronized and were not treated with IL-1.

[0046] FIG. 8C is a graph of luminescence of HEK293T cells transduced with the NFKB luciferase reporter. The cells were treated with IL-1 but not synchronized. The circuit became activated with the addition of an inflammatory signal.

[0047] FIG. 8D is a graph of luminescence of HEK293T cells transduced with the NFKB luciferase reporter. The cells were treated with IL-1 and synchronized with a 1 ,5h Dex treatment. The addition of Dex for synchronization 24h prior to adding IL1 showed slightly lower initial output but overall, a very similar response to FIG. 80.

[0048] FIG. 8E is a graph of luminescence of HEK293T cells transduced with the NFKB luciferase reporter. The cells were treated with IL-1 and synchronized with a 1 ,5h serum treatment. The serum shock synchronization increased the circuit response over time compared to the other conditions.

[0049] FIG. 9A is a set of graphs of cells transduced with the Per2 promoter driving luciferase expression and synchronized with a 1.5h Dex treatment (100nM) to determine the protective effects of circuits responsive to circadian cues. The left graph shows bioluminescence measured over 72 hours for (bottom) HEK293T cells plated at 5k per well and treated with 0.1 mM luciferin, (middle) PDiPSCs plated at 20k per well and treated with 0.1 mM luciferin, and (top) PDiPSCs plated at 20k per well and treated with 1 mM luciferin.

[0050] FIG. 9B is a magnified graph of the HEK293T cells plated at 5k per well and treated with 0.1 mM luciferin from FIG. 9A, demonstrating a low amplitude rhythmic circadian output of luciferase over a 24-hour cycle.

[0051] FIG. 90 is a set of magnified graphs of PDiPSCs plated at 20k cells per well and treated with 0.1 mM luciferin (top) and 1 mM luciferin (bottom) from FIG. 9A. The PDiPSCs demonstrate relatively-circadian repeating outputs over 72h that varied in the baseline level of output depending on the concentration of luciferin added.

[0052] FIG. 9D is a set of images of PDiPSCs plated at 20k cells per well and transduced with the Per2 promoter driving GFP expression. The top images are bright field, and the bottom images show GFP expression of cells transduced with 1x virus (left) and 2x virus (right).

[0053] FIG. 10A is a timeline and experimental setup to test the protective effects of the circuits responsive to circadian and inflammatory cues. PDiPSCs are plated at 20k cells per well and transduced with Per2, NFKB, or both, synchronized, treated with 0.01 ng / mL IL-1 a, and the luminescence is recorded after 72 hours.

[0054] FIG. 10B is a graph of bioluminescence from PDiPSCs transduced with the NFKB promoter and treated with or without IL-1.

[0055] FIG. 10C is a graph of bioluminescence from PDiPSCs transduced with the Per2 promoter and treated with or without IL-1 .

[0056] FIG. 10D is a graph of bioluminescence from PDiPSCs transduced with both the NFKB and Per2 promoter and treated with or without IL-1 . Overall bioluminescence was higher in the cell transduced with both promoters and the addition of IL-1 increased the response.

[0057] FIG. 10E is a graph of bioluminescence from PDiPSCs transduced with only NFKB or both NFKB and Per2, then treated with or without IL-1 . The double transduction increases the amplitude of bioluminescence which is expected from an additive output and may lend to the ability of the circuits to increase response over time when IL-1 is added. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present disclosure is based, at least in part, on the discovery that a probiotic supplement can be modified to include a synthetic gene circuit to drive a controlled gene output. As shown herein, circadian systems are described.

[0059] This disclosure covers the use of circadian rhythms for drug delivery using mammalian cells and the use of circadian “clock genes” to modify probiotic supplements (e.g., bacteria) as a therapeutic modality that is delivered to the gut on a prescribed timed basis.

[0060] The present disclosure involves the creation of a probiotic supplement that has been modified to include a synthetic gene circuit to drive a controlled gene output. The circuit may involve any combination of the genes involved in regulating or responding to circadian rhythms to drive the expression of a prescribed therapeutic or diagnostic gene target. The supplement may be taken orally or otherwise to colonize the gut, thus providing safe and controlled production of prescribed therapeutic molecules or nutrients to the recipient.

[0061] This invention involves genetic alteration to a probiotic to allow timed delivery of a therapeutic biologic drug in a prescribed timed intermittent or circadian manner.

[0062] In various aspects, the probiotic compositions including a probiotic supplement including, but not limited to, a yeast or a bacteria modified to incorporate a synthetic circuit which comprises, consists essentially of, or consists of an engineered nucleic acid sequence having at least one transcriptional regulatory region (e.g. a promoter or repressor) of one or more oscillating genes ( e.g ., BmaH, GSK3, NPAS2, Clocks, Cry1 / 2, Per1 / 2 / 3, Dec1 / 2, Rors ( RORcc / b ), Rev-erbs (REV-ERBa), Dbp, CK1s / 5, Nfil3, D box regions, E box regions, RRE regions, or any other oscillating genes downstream or upstream of these targets, including tissue-specific clocks-controlled genes) operably linked to a nucleic acid sequence encoding a biologic therapeutic {e.g., a therapeutic polypeptide or RNA molecule) in a circadian mediated manner.

[0063] In some embodiments, the synthetic circuit includes a regulatory region of an oscillating gene operably linked to a nucleic acid sequence encoding the biologic therapeutic. In one embodiment, the regulatory region nucleic acid molecule and the nucleic acid sequence encoding the biologic therapeutic are recombinant. In another embodiment, the regulatory region nucleic acid molecule and the nucleic acid sequence encoding the biologic therapeutic are heterologous to each other and / or to the cell in which they are incorporated. For example, the synthetic circuit can be created using a plasmid, viral transduction, or gene editing. In some embodiments, the disclosure provides the synthetic circuit incorporated into a plasmid or vector, such as an expression vector and / or viral vector. Alternatively, the regulatory region of an oscillating gene may be an endogenous nucleic acid molecule to the target cell and through targeted gene editing (e.g., CRISPR-Cas gene editing, TALENs, ZFNs, or any other method of genetic modification) the nucleic acid sequence encoding the biologic therapeutic becomes operably linked to the endogenous regulatory region of an oscillating gene such that transcriptional activation of the oscillating gene leads to expression of the therapeutic biologic in a circadian mediated manner.

[0064] In various aspects, the synthetic circuit of the disclosure can be regulated using a central clock system (i.e. suprachiasmatic nucleus, SCN) or a peripheral clock in the body. For example, the period / frequency / phase of the expression of the therapeutic biologic via a synthetic circuit of the disclosure can be modulated through regulation of the circadian genes or through the use of combinations of genes that are off-cycle from one another. In some embodiments, the period / frequency / phase of the gene oscillation can be modulated through mutation of any of the circadian genes. The overall duration and dynamics of expression of the therapeutic biologic can be controlled using a synthetic genetic counter that counts clock cycles in the cell. Methods of measuring clock cycles in a cell are known in the art and discussed in the examples below.

[0065] As used herein, a “synthetic circuit” refers to a nucleic acid sequence comprising at least one transcriptional regulatory region from one or more circadian-responsive genes such that the regulatory region is operably linked to a nucleic acid sequence encoding a therapeutic biologic. As used herein, a “circadian- responsive gene” or “clock gene” refers to a reference endogenous gene that is transcriptionally regulated (completely or partially) by a cell circadian clock comprising the endogenous gene, and results in modulated expression of the coding region of the gene. Non-limiting examples of clock genes and there corresponding transcriptional regulatory nucleic acid sequences are Bmall, GSK3, NPAS2, Clocks, Cry1 / 2, Per1 / 2 / 3, Ded / 2, Rors ( RORa / b), Rev-erbs ( REV-ERBa ), Dbp, CK / d, Nfil3, D box regions, E box regions, RRE regions and any combination thereof.

[0066] In various aspects, clock genes are components of the circadian which interact with each other, in part, through their regulatory sequences in an intricate manner generating oscillations of gene expression. The underlying principle of circadian clocks is successive gene activation in the form of a cycle: the initial activation of a gene is regulated by the last one in the sequence, making up an auto-regulatory feedback loop for which one cycle takes about 24 hours. Positive regulatory elements activate the expression of negative regulatory elements, which in turn stop the activity of the positive elements. This system moves away from equilibrium before returning and hence, perpetual cycling is the consequence. For example, two transcriptional activators Bmall (brain and muscle ARNT-like protein 1 ) and Clock (or Npas2 in neuronal tissue) form heterodimers in the cytoplasm and enter the nucleus where they bind to E- box sequences in the promoters of Period {Perl, 2) and Cryptochrome (Cry1 , Cry2 ) genes contributing to the activation of their expression. In the cytoplasm various combinations of Per and Cry proteins interact with each other, enter the nucleus, and inhibit the activity of Bmall / Clock or Bmall ZNpas2 complexes. Without these complexes activating transcription of the Per and Cry genes, levels of Per and Cry transcripts and their respective protein products decline, hence Per and Cry genes shut off their transcription.

[0067] As another example, a second loop regulates the expression of the BmaH gene. In the nucleus, Bmall / Clock or Bmal1 / Npas2 heterodimers bind to E- boxes present in the promoters of genes that encode the retinoic acid-related orphan nuclear receptors Rev-erba and Rora, which compete for the ROR element (RORE) in the BmaH promoter. Rora activates BmaH expression, while Rev-erba represses it. As a consequence oscillations of BrnaM and Rora / Rev- erba are out of phase. If activation wins over expression BrnaM protein is produced and it forms heterodimers in the cytoplasm with Clock or Npas2 depending on the tissue. These heterodimers enter the nucleus and initiate the next cycle of gene activation of both loops. In various aspects, the transcriptional activation of other clock genes may be facilitated by the histone acetyl transferase (HAT) activity of the Clock protein. Histone acetylation promotes transcription through the modification of histones and allows the opening of the condensed chromatin. This provides access to the transcriptional machinery. The HAT activity of Clock is associated with the transcriptional activation of the clock genes Per and Cry and therefore influences the generation and maintenance of endogenous circadian rhythms in mammals. Transcriptional repression is mediated by several events. Per and Cry bind to the Bmall / Clock complex. This results in the loss of HAT activity of Clock by promoting Clock phosphorylation (P) and / or inducing a conformational change of Bmall / Clock. Loss of Clock HAT activity promotes histone deacetylation. This prevents the general transcription machinery from binding to DNA and hence transcription is repressed. Upon degradation of Per and Cry, Clock is either dephosphorylated or degraded and resynthesized. It then interacts again with Bmall and acetylates histones to activate a new transcription cycle.

[0068] The major targets of CLOCK / BMAL1 include other core clock genes that encode the mammalian Period ortholog (Perl, Per2, and Per3) and CRYPTOCHROME (Cry1 and Cry2) repressor proteins. These negative regulators heterodimerize and then translocate into the nucleus where they repress their gene transcription by interacting directly with CLOCK / BMAL1 . In addition to this direct transcriptional feedback, the mRNA expression of Per1 / 2 / 3 and Cry1 / 2 is also regulated by various other mechanisms. The degradation of PER and CRY proteins is also regulated by the serine / threonine kinases including, but not limited to, casein kinase 1d (CK16) and CK1e, the F-box proteins, FBXL3 and FBXL21 , and other proteins. Once negative transcriptional feedback and post-transcriptional and post-translational regulation become sufficient to decrease PER / CRY protein levels in the nucleus, repression is relieved and CLOCK / BMAL1 starts a new cycle of Per / Cry gene transcription.

[0069] In various additional aspects, additional genes and feedback loops may be involved in additional transcriptional loops including, but not limited to, clock genes such as CLOCK / BMAL1 that activate transcription of genes for the nuclear receptors REV-ERBa and REV-ERBp. These proteins compete with the retinoic acid- related orphan receptors RORa, RORp, and RORy for binding sites (ROR-binding elements) on the BmaH gene, providing both positive (ROR) and negative (REV-ERB) regulation of transcription, and provide a link between the circadian clock and metabolism. An additional non-limiting example of a feedback loop includes a D-box binding protein (DBP) and a nuclear factor, interleukin-3-regulated protein (NFIL3, also known as E4BP4) both of which are regulated by CLOCK / BMAL1 and CRY1 and bind to D-box elements on circadian promoters such as Rora and Rorb. Together, these feedback loops that make up the ‘molecular clock’ are governed by transcriptional, post- transcriptional, and post- translational regulatory mechanisms that are sufficient to maintain circadian rhythms; however, external cues are still important for synchronizing rhythms of cells within and across tissues.

[0070] In various aspects, the probiotics of the present disclosure include synthetic circuits comprising one or more transcriptional regulatory regions from one or more circadian-responsive genes (e.g. promoters). A “promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Transcriptional repressors are proteins that bind to specific sites on DNA (i.e. repressor elements) and prevent transcription of nearby genes. Enhancers are short regulatory elements of accessible DNA that help establish the transcriptional program of cells by increasing the transcription of target genes. They are bound by transcription factors, co-regulators, and RNA polymerase II (RNAP II)

[0071] In one embodiment, the transcriptional regulatory region of the disclosed probiotics includes one or more nucleic acid sequences corresponding to a promoter, repressor, and / or enhancer region of a circadian-responsive gene. In one aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of an E-box domain to which the transcription factor BMAL1 and / or CLOCK bind. In another aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of one or more PER and or CRY repressor elements. In another aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of one or more ROR response elements. In another aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of one or more RRE elements. In another aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of one or more D-box elements upon which DBP and NFIL3 dimers bind. In another aspect, the transcriptional regulatory region of a synthetic circuit of the disclosure comprises, consists essentially of, or consists of one or more E-box domains, PER repressor element, CRY repressor element, RRE element, D-box element, ROR response element or any combination thereof.

[0072] In one embodiment, the transcriptional regulatory region nucleic acid molecule of a synthetic circuit of the disclosure has one or more of a nucleic acid sequence which comprises, consists essentially of, or consists of the Perl gene promoter (Period Circadian Regulator 1 )(e.g., a promoter as designated by GeneCard ID: GC17M010119; chr17 :8, 140,472-8, 156, 506 (GRCh38 / hg38); GeneHancer Identifier: GH17J008149, GH17J008142, GH17J008172, GH17J007571 , GH17J007233, GH17J008220, GH17J007474, GH17J007306, GH17J007715, GH17J007434, GH17J007612, GH17J007880, GH17J008185, GH17J007853, GH17J007347, GH17J007328, GH17J008117, GH17J008093, GH17J007684, GH17J007247, GH17J008375, GH17J007279, GH17J008379, GH17J008137, GH17J008079, GH17J007176, GH17J007173, GH17J008975, GH17J008300, GH17J007450, GH17J008419, GH17J009052, GH17J008994, GH17J007175, GH17J008181 , GH17J008182, GH17J008417, GH17J008412, GH17J007825, GH17J008133, GH17J008145, GH17J008143, GH17J008128, and / or GH 17 J008144); a nucleic acid sequence which comprises, consists essentially of, or consists of the Per2 gene promoter (Period Circadian Regulator 2)(e.g., a promoter as provided by GeneCard ID: GC02M238244; chr2:238, 244, 038-238, 290, 102 (GRCh38 / hg38); GeneHancer Identifier: GH02J238283, GH02J238299, GH02J238239, GH02J237588, GH02J237922, GH02J238369, GH02J238713, GH02J238246, GH02J237894, GH02J238247, GH02J238888, GH02J237903, GH02J238642, GH02J238294, GH02J238261 , GH02J238271 , GH02J238256, GH02J238279, GH02J238267, GH02J238302, GH02J238306, GH02J238304, GH02J238259, GH02J238282, GH02J238303, GH02J238260, GH02J238298, GH02J238281 , GH02J238270, GH02J238309, and / or GH02J238254); a nucleic acid sequence which comprises, consists essentially of, or consists of the Per3 gene promoter (Period Circadian Regulator 3)( e.g., a promoter as designated by GeneCard ID: GH01J007782, GH01J008437, GH01J008700, GH01 J007703, GH01J007297, GH01J008002, GH01J007678, GH01J007826, GH01 J007993, GH01J007752, GH01J007902, GH01J007498, GH01J007912, GH01 J007831 , GH01J007893, GH01J008377, GH01J007722, GH01J007717, GH01 J008362, GH01 J007991 , GH01J007755, GH01J007749, GH01J008365, GH01 J007899, GH01J008036, GH01 J007681 , GH01J007740, GH01J008014, GH01 J007761 , GH01J007746, GH01J007667, GH01J007915, GH01J007921 , GH01 J007914, GH01J007706, GH01 J007781 , GH01J007810, GH01J007853, GH01 J007847, GH01J007856, GH01J007885, GH01J007888, GH01J007889, GH01 J007890, and / or GH01 J007887); a nucleic acid sequence which comprises, consists essentially of, or consists of the BMAL1 gene promoter (aka ARNTL, Aryl Hydrocarbon Receptor Nuclear Translocator Like)( e.g., a promoter as designated by GeneCard ID: GC11 P013276; chr11 : 13, 276, 552-13, 387, 268 (GRCh38 / hg38); GeneHancer Identifier: GH11 J013276, GH11 J013311 , GH11 J013337, GH11 J013213, GH1 1 J013258, GH11J013331 , GH11J013271 , GH11J013329, GH11J013324, GH1 1 J013303, GH11J013335, GH11J013210, GH11J013250, GH11J013327, GH1 1 J013284, GH11J013336, GH11J013221 , GH11J013294, GH11J013347, GH1 1 J013367, GH11J013306, GH11J013219, GH11J013253, GH11J013364, GH1 1 J013228, GH11J013322, GH11J013383, GH11J013297, GH11J013239, GH1 1 J013207, GH11J013283, GH11J013287, GH11J013288, GH11J013318, GH1 1 J013345, GH11J013138, GH11J013461 , GH11J013267, GH11J013319, GH1 1 J013134, GH11J013117, GH11J013104, GH11J013108, GH11 J013101 , GH1 1 J013114, GH11J013091 , GH11J013137, GH11J013439, GH11J013159, GH1 1 J013007, GH11J013125, GH11J013448, GH11J013097, GH11J013264, GH1 1 J013073, GH11J013168, GH11J013064, GH11J013257, GH11J013434, GH1 1 J013052, GH1 1 J013227, GH11 J013242, GH11 J013243, GH11 J013425, GH1 1 J013144, GH1 1 J013167, GH11 J013043, GH11 J013030, GH11 J013436, GH1 1 J013445, GH1 1 J013040, GH11J013375, GH11 J013150, GH11 J013077, GH1 1 J013015, GH1 1 J013109, GH11 J013443, GH11 J013444, GH11 J013076, GH1 1 J013079, and / or GH11 J013023); a nucleic acid sequence which comprises, consists essentially of, or consists of the CRY1 gene promoter (Cryptochrome Circadian Regulator 1 )( e.g., a promoter as designated by GeneCard ID: GC12M106991 ; chr12: 106,991 ,364-107,093,872 (GRCh38 / hg38); GeneHancer Identifier: GH12J107090, GH12J106130, GH12J106299, GH12J107057, GH12J107025, GH12J106581 , GH12J107514, GH12J107079, GH12J106345, GH12J107109, GH12J106111 , GH12J106984, GH12J107460, GH12J107903, GH12J107042, GH12J107912, GH12J107033, GH12J107014, GH12J106328, GH12J106336, GH12J107086, GH12J107121 , GH12J106586, GH12J107385, GH12J107351 , GH12J107948, GH12J107104, GH12J107134, GH12J107131 , GH12J107126, GH12J107073, GH12J107030, GH12J107214, GH12J107200, GH12J107225, GH12J107032, GH12J106989, GH12J107236, GH12J107035, GH12J106994, GH12J107241 , GH12J107076, GH12J107066, GH12J107166, GH12J107296, GH12J107055, GH12J107056, GH12J106990, GH12J107295, GH12J107287, GH12J107036, GH12J107228, GH12J107277, GH12J107178, GH12J107007, GH12J107298, GH12J107234, GH12J107307, GH12J107311 , GH12J107299, GH12J107282, GH12J107302, and / or GH12J107305); a nucleic acid sequence which comprises, consists essentially of, or consists of the Cry2 gene promoter (Cryptochrome Circadian Regulator 2)( e.g., a promoter as designated by GeneCard ID: GC1 1 P046397; chr11 :45,847, 118-45, 883, 248 (GRCh38 / hg38); GeneHancer Identifier: GH1 1 J045846, GH11J045872, GH11J045826, GH11J045821 , GH11J045818, GH1 1 J045374, GH11J045385, GH11J045868, GH11J045842, GH11J045854, GH1 1 J045803, GH11J045851 , GH11J045879, GH11J045833, GH11J045861 , GH1 1 J045832, GH11J045875, GH11J045862, GH11J045812, and / or GH11J045865); a nucleic acid sequence which comprises, consists essentially of, or consists of the NPAS2 gene promoter (Neuronal PAS Domain Protein 2)( e.g., a promoter as designated by GeneCard ID: GC02P100820; chr2: 100,820, 139-100,996,829 (GRCh38 / hg38);

[0073] GeneHancer Identifier: GH02J100817, GH02J101001 , GH02J100779, GH02J100741 , GH02J100875, GH02J100880, GH02J100891 , GH02J100921 , GH02J100766, GH02J100784, GH02J100751 , GH02J100730, GH02J100731 , GH02J100885, GH02J100757, GH02J100857, GH02J101012, GH02J100923, GH02J101007, GH02J100853, GH02J101080, GH02J100644, GH02J100877, GH02J100594, GH02J100825, GH02J101026, GH02J100610, GH02J101047, GH02J100745, GH02J100856, GH02J100941 , GH02J101163, GH02J100649, GH02J101162, GH02J101011 , GH02J100949, GH02J100902, GH02J100889, GH02J100749, GH02J101160, GH02J100831 , GH02J100776, GH02J100747, GH02J100861 , GH02J100717, GH02J101157, GH02J101050, GH02J100823, GH02J101053, GH02J100862, GH02J100945, GH02J100726, GH02J100720, GH02J100721 , GH02J100673, GH02J101161 , GH02J101077, GH02J101075, GH02J100812, GH02J100796, GH02J100865, GH02J100800, GH02J100901 , GH02J100845, GH02J100873, GH02J100909, GH02J100809, GH02J100844, GH02J100588, GH02J100739, GH02J100976, GH02J100984, GH02J100869, GH02J100934, GH02J100957, GH02J100706, GH02J100606, GH02J100579, GH02J100840, GH02J100980, GH02J100985, GH02J100603, GH02J100907, GH02J100992, GH02J100571 , GH02J100872, GH02J100752, GH02J100690, GH02J100963, GH02J100953, GH02J100905, GH02J100703, GH02J100692, GH02J100683, GH02J100629, GH02J100982, GH02J100971 , GH02J100685, GH02J100964, GH02J100592, GH02J100584, and / or GH02J100601 ); a nucleic acid sequence which comprises, consists essentially of, or consists of the RORA gene promoter (RAR Related Orphan Receptor A)( e.g., a promoter as designated by GeneCard ID: GC15M060488; chr15:60,488,284-61 ,229,302 (GRCh38 / hg38,); GeneHancer Identifier: GH15J060579, GH15J061227, GH15J060525, GH15J060538, GH15J060562, GH15J060567, GH15J061835, GH15J061032, GH15J061116, GH15J061156, GH15J061173, GH15J061834, GH15J061168, GH15J061157, GH15J061167, GH15J061336, GH15J060839, GH15J061146, GH15J061198, GH15J061364, GH15J061118, GH15J060797, GH15J061141 , GH15J061379, GH15J061644, GH15J060719, GH15J061186, GH15J061838, GH15J061204, GH15J061178, GH15J061182, GH15J061179, GH15J061143, GH15J061145, GH15J061155, GH15J061841 , GH15J061647, GH15J060697, GH15J061212, GH15J061216, GH15J061231 , GH15J061210, GH15J061084, GH15J061050, GH15J061063, GH15J061069, GH15J061010, GH15J061238, GH15J061239, GH15J061219, GH15J061112, GH15J061107,

[0074] GH15J061052, GH15J061383, GH15J061038, GH15J060970, GH15J061213,

[0075] GH15J061083, GH15J061088, , GH15J060916, GH15J060758, GH15J061003,

[0076] GH15J061079, GH15J061393, GH15J061096, GH15J060751 , GH15J060913,

[0077] GH15J062059, GH15J060740, GH15J060905, GH15J060899, GH15J061501 ,

[0078] GH15J060803, GH15J060941 , GH15J060795, GH15J060932, GH15J061090,

[0079] GH15J061024, GH15J060648, GH15J061680, GH15J060858, GH15J060634,

[0080] GH15J060760, GH15J060965, GH15J060741 , GH15J061460, GH15J060718,

[0081] GH15J060518, GH15J060507, GH15J060786, GH15J061528, GH15J061046,

[0082] GH15J061424, GH15J061101 , GH15J061028, GH15J061075, GH15J061098,

[0083] GH15J060928, GH15J060778, GH15J060774, GH15J060776, GH15J060859,

[0084] GH15J060773, GH15J061535, GH15J061691 , GH15J060765, GH15J061702,

[0085] GH15J061008, GH15J060964, GH15J060615, GH15J060604, GH15J060842,

[0086] GH15J060744, GH15J061715, GH15J060727, GH15J060822, GH15J060818,

[0087] GH15J060554, GH15J061907, GH15J060813, GH15J060705, GH15J060708,

[0088] GH15J061748, GH15J060944, GH15J060700, GH15J060695, GH15J060523,

[0089] GH15J061653, GH15J060678, GH15J060682, GH15J060673, GH15J060493,

[0090] GH15J060660, GH15J060779, GH15J061073, GH15J061070, GH15J061058,

[0091] GH15J061027, GH15J061438, GH15J061436, GH15J061054, GH15J061037,

[0092] GH15J061017, GH15J060865, GH15J061598, GH15J060646, GH15J060772,

[0093] GH15J060642, GH15J061448, GH15J060855, GH15J061537, GH15J060764,

[0094] GH15J062026, GH15J060763, GH15J061697, GH15J060626, GH15J060850,

[0095] GH15J060851 , GH15J060849, GH15J062035, GH15J060620, GH15J061005,

[0096] GH15J062046, GH15J060747, , GH15J061856, GH15J060961 , GH15J060999,

[0097] GH15J060834, GH15J060835, GH15J060836, GH15J060909, GH15J061718,

[0098] GH15J060829, GH15J061879, GH15J060731 , GH15J061558, GH15J060902,

[0099] GH15J061499, GH15J060994, GH15J061892, GH15J061893, GH15J060566,

[0100] GH15J060821 , GH15J060712, GH15J060817, GH15J061639, GH15J060993,

[0101] GH15J060706, GH15J060949, GH15J061649, GH15J061648, GH15J060533,

[0102] GH15J060535, GH15J060703, GH15J060701 , GH15J060889, GH15J060531 ,

[0103] GH15J060696, GH15J060694, GH15J061651 , GH15J060698, GH15J061764,

[0104] GH15J061515, GH15J061518, GH15J061655, GH15J060511 , GH15J060681 ,

[0105] GH15J061659, GH15J061661 , GH15J060504, GH15J060875, GH15J060935, GH15J061780, GH15J061779, GH15J060494, GH15J060982, GH15J060983, GH15J060672, GH15J060490, GH15J060491 , GH15J060666, GH15J060486, GH15J060783, GH15J061974, GH15J061524, GH15J060784, GH15J060656, GH15J060653, GH15J060637, GH15J060624, GH15J061829, GH15J060610, GH15J060596, GH15J060726, GH15J061890, GH15J061736, GH15J061743, GH15J061915, GH15J061923, GH15J060530, GH15J061776, GH15J060500, GH15J060485, GH15J061795, and / or GH15J060654); a nucleic acid sequence which comprises, consists essentially of, or consists of the RORB gene promoter (RAR Related Orphan Receptor B)( e.g., a promoter as designated by GeneCard ID: GC09P074497; chr9:74,497 ,335-74,693,177 (GRCh38 / hg38); GeneHancer Identifier: GH09J074496, GH09J074495, GH09J074556, GH09J074333,

[0106] GH09J074544, GH09J074309, GH09J074245, GH09J074459, GH09J074463,

[0107] GH09J074564, GH09J074444, GH09J074304, GH09J074471 , GH09J073565,

[0108] GH09J074557, GH09J073548, GH09J074445, GH09J074270, GH09J074239,

[0109] GH09J074250, GH09J074263, GH09J074279, GH09J074492, GH09J074501 ,

[0110] GH09J074886, GH09J074573, GH09J074571 , GH09J074379, GH09J074486,

[0111] GH09J074277, GH09J074480, GH09J074578, GH09J074293, GH09J074609,

[0112] GH09J074322, GH09J074802, GH09J074025, GH09J074759, GH09J074778,

[0113] GH09J074777, GH09J074447, GH09J074575, GH09J074284, GH09J074855,

[0114] GH09J074860, GH09J074280, GH09J073869, GH09J073845, GH09J074773,

[0115] GH09J074083, GH09J074741 , GH09J074644, GH09J074670, GH09J074611 ,

[0116] GH09J074374, GH09J074630, GH09J074235, GH09J074092, GH09J074091 ,

[0117] GH09J074085, GH09J074078, GH09J074783, GH09J074847, GH09J074326,

[0118] GH09J074692, GH09J074653, GH09J074296, GH09J074308, GH09J074288,

[0119] GH09J074674, GH09J074306, GH09J074690, GH09J074044, GH09J073916,

[0120] GH09J074039, GH09J074869, GH09J074125, GH09J073906, GH09J074241 ,

[0121] GH09J074036, GH09J074037, GH09J074035, GH09J074031 , GH09J073891 ,

[0122] GH09J074874, GH09J074117, GH09J073683, GH09J074272, GH09J074877,

[0123] GH09J074881 , GH09J074880, GH09J074884, GH09J073871 , GH09J073643,

[0124] GH09J073996, GH09J074825, GH09J073617, GH09J073615, GH09J073611 ,

[0125] GH09J074087, GH09J074220, GH09J073595, GH09J073979, GH09J073577,

[0126] GH09J074735, GH09J073573, GH09J073569, GH09J074217, GH09J073566,

[0127] GH09J074784, GH09J073539, GH09J073515, GH09J074200, GH09J073918, GH09J073905, GH09J073680, GH09J073657, GH09J073843, GH09J073824, GH09J073791 , and / or GH09J073530); a nucleic acid sequence which comprises, consists essentially of, or consists of the REV-ERB alpha gene promoter (aka NR1 D1 ; Nuclear Receptor Subfamily 1 Group D Member 1 )(e.g., a promoter as designated by GeneCard ID: GC17M040092; chr17:40, 092, 793- 40,100,589 (GRCh38 / hg38); GeneHancer Identifier: GH17J040102, GH17J040088, GH17J040061 , GH17J040217, GH17J039183, GH17J040086, GH17J040117, GH17J040062, GH17J040083, and / or GH17J040077); a nucleic acid sequence which comprises, consists essentially of, or consists of the DBP gene promoter (D-Box Binding PAR BZIP Transcription Factor)(e.g., a promoter as designated by GeneCard ID: GC19M048630; chr19:48, 630, 030-48, 637 ,379 (GRCh38 / hg38); GeneHancer Identifier: GH19J048632, GH19J048736, GH19J048897, GH19J048724, GH19J048674, GH19J048977, GH19J048768, GH19J048976, GH19J048680, GH19J048842, GH19J048764, GH19J048775, GH19J048588, GH19J048644, GH19J048622, GH19J048628, GH19J048626, and / or GH19J048621 ); a nucleic acid sequence which comprises, consists essentially of, or consists of the NFIL3 gene promoter (Nuclear Factor, Interleukin 3 Regulated)(e.g., a promoter as designated by GeneCard ID: GC09M091409; chr9:91 ,409,045-91 ,425,063 (GRCh38 / hg38); GeneHancer Identifier: GH09J091417, GH09J091482, GH09J091426, GH09J091191 , GH09J091098, GH09J091415, GH09J091070, GH09J091391 , GH09J090964, GH09J091394, GH09J091300, GH09J091085, GH09J091089, GH09J090985, GH09J091177, GH09J091126, GH09J091174, GH09J091374, GH09J091378, GH09J090979, GH09J090969, GH09J091186, GH09J091392, GH09J090987, GH09J090950, GH09J090906, GH09J091059, GH09J090769, GH09J091257, GH09J091148, GH09J091265, GH09J091150, GH09J090986, GH09J091407, GH09J091371 , GH09J091067, GH09J090983, GH09J091297, GH09J091082 GH09J091416, GH09J091390, GH09J090929, GH09J090951 , GH09J091071 , GH09J091147, GH09J091428, GH09J091432, GH09J091427, GH09J091402, GH09J091500, GH09J091410, GH09J091406, GH09J091514, GH09J091486, GH09J091503, GH09J091494, GH09J091535, GH09J091434, GH09J091530, GH09J091484, GH09J091895, GH09J091711 , GH09J091448, GH09J091560, GH09J091564, GH09J091589, GH09J091795, GH09J091884, GH09J091905, GH09J091816, GH09J091823, GH09J091837, GH09J091944, GH09J091714, GH09J091775, GH09J091856, GH09J091465, GH09J091577, GH09J091734, GH09J091681 , GH09J091814, GH09J091746, GH09J091659, GH09J091720, GH09J091786, GH09J091540, GH09J091580, GH09J091546, GH09J091727, GH09J091728, GH09J091798, GH09J091802, GH09J091740, GH09J091758, GH09J091831 , GH09J091834, GH09J091842, GH09J091846, GH09J091710, GH09J091715, GH09J091770, GH09J091780, GH09J091534, GH09J091572, GH09J091573, GH09J091611 , GH09J091548, GH09J091558, GH09J091677, GH09J091682, GH09J091680, GH09J091890, GH09J091894, GH09J091809, GH09J091741 , GH09J091812, GH09J091810, GH09J091907, GH09J091693, GH09J091690, GH09J091820, GH09J091751 , GH09J091934, GH09J091935, GH09J091760, GH09J091661 , GH09J091840, GH09J091772, GH09J091863, GH09J091784, GH09J091866, GH09J091873, GH09J091606, GH09J091603, GH09J091729, GH09J091875, GH09J091878, GH09J091877, GH09J091793, GH09J091643, GH09J091813, GH09J091918, GH09J091920, GH09J091925, GH09J091924, GH09J091755, GH09J091939, GH09J091765, and / or GH09J091854); a nucleic acid sequence which comprises, consists essentially of, or consists of the GSK3B gene promoter (Glycogen Synthase Kinase 3 Beta)(e.g., a promoter as designated by GeneCard ID: GC03M119821 \ chr3:119, 821 , 321-120, 095, 823 (GRCh38 / hg38); GeneHancer Identifier: GH03J120088, GH03J120048, GH03J120067, GH03J120072, GH03J119892, GH03J120157, GH03J119809, GH03J120143, GH03J119841 , GH03J120079, GH03J119973, GH03J120151 , GH03J119993, GH03J120175, GH03J120057, GH03J119236, GH03J119872, GH03J120169, GH03J120101 , GH03J119827, GH03J119911 , GH03J120202, GH03J119821 , GH03J120135, GH03J119893, GH03J120017, GH03J120035, GH03J120024, GH03J120111 , GH03J120033, GH03J119825, GH03J119635, GH03J120003, GH03J120422, GH03J119831 , GH03J120063, GH03J119989, GH03J119917, GH03J119548, GH03J119626, GH03J120013, GH03J120075, GH03J120031 , GH03J119931 , GH03J120010, GH03J119630, GH03J119950, GH03J119828, GH03J120100, GH03J120176, GH03J119742, GH03J119875, GH03J119969, GH03J120098, GH03J119627, GH03J120181 , GH03J120244, GH03J120129, GH03J120214, GH03J119937, GH03J120274, GH03J119794, GH03J120198, GH03J120284, GH03J119779, GH03J 120245, GH03J119944, GH03J120276, GH03J119786, GH03J120038, GH03J120036, GH03J120012, GH03J120032, GH03J120211 , GH03J120227, GH03J120275, GH03J120239, GH03J120263, GH03J119977, GH03J119913, GH03J120196, GH03J120195, GH03J120229, GH03J 120231 , GH03J119980, GH03J119789, GH03J119788, GH03J119834, GH03J119829, GH03J119823, GH03J119914, GH03J119939, GH03J120283, GH03J119935, GH03J120272, GH03J120264, GH03J119868); a nucleic acid sequence which comprises, consists essentially of, or consists of the CSNK1 E gene promoter (Casein Kinase 1 Epsilon)(e.g., a promoter as designated by GeneCard ID: GC22M055249; chr22:38,290,691 -38,318,084 (GRCh38 / hg38); GeneHancer Identifier: GH22J038308, GH22J038393, GH22J038294, GH22J038289, GH22J038335, GH22J038295, GH22J038174, GH22J038768, GH22J039315, GH22J038567, GH22J037683, GH22J037847, GH22J037558, GH22J037606, GH22J038691 , GH22J038422, GH22J038056, GH22J038984, GH22J038681 , GH22J039232, GH22J039087, GH22J038923, GH22J037556, GH22J037400, GH22J037392, GH22J038550, GH22J038025, GH22J038217, GH22J038227, GH22J038324, GH22J038282, GH22J038353, GH22J038350, GH22J038348, GH22J038286, GH22J038309, GH22J038278, GH22J038373, GH22J038338, GH22J038274, GH22J038391 , GH22J038333, GH22J038360, GH22J038390, GH22J038369, GH22J038363, GH22J038354, GH22J038394, GH22J038381 , GH22J038288, GH22J038342, GH22J038387, and / or GH22J038356); a nucleic acid sequence which comprises, consists essentially of, or consists of the CSNK1 D gene promoter (Casein Kinase 1 Delta)(e.g., a promoter as designated by GeneCard ID: GC17M082239; chr17.82,239,019-82, 273, 750 (GRCh38 / hg38); GeneHancer Identifier: GH17J082271 , GH17J082291 , GH17J082211 , GH17J081864, GH17J081384, GH17J081709, GH17J082492, GH17J081700, GH17J082227, GH17J082696, GH17J082456, GH17J082281 , GH17J082064, GH17J081348, GH17J082585, GH17J081507, GH17J082234, GH17J082671 , GH17J082734, GH17J081635, GH17J081682, GH17J081976, GH17J081308, GH17J082447, GH17J081551 , GH17J081990, GH17J082361 , GH17J082204, GH17J082417, GH17J081664, GH17J081999, GH17J081424, GH17J082715, GH17J082590, GH17J081814, GH17J082383, GH17J081432, GH17J082553, GH17J082567, GH17J082398, GH17J082558, GH17J082544, GH17J082635, GH17J082415, GH17J082414, GH17J082565, GH17J082552, GH17J082506, GH17J082313, GH17J082381 , GH17J082433, GH17J082407, GH17J082265, GH17J082332, GH17J081425, GH17J082256, GH17J082424, GH17J082175, GH17J082176, GH17J082286, GH17J082639, GH17J082477, GH17J083096, GH17J082173, GH17J082436, GH17J082547, GH17J082263, GH17J082222, GH17J082049, GH17J082246, GH17J082278, GH17J082260, GH17J082309, GH17J082225, GH17J082244, GH17J082245, GH17J082226, GH17J082276, GH17J082251 , GH17J082310, GH17J082252, GH17J082284, GH17J082249, GH17J082224, GH17J082223, GH17J082277, GH17J082255, GH17J082248, and / or GH17J082312); and / or any combination thereof.

[0128] In one embodiment, the probiotic cell is genetically modified by introducing into the genome of the cell a heterologous nucleic acid sequence comprising a regulatory nucleic acid sequence of a clock gene operably linked to a nucleic acid sequence encoding a therapeutic biologic. In one aspect, the regulatory sequence of a clock gene is a BmaH regulatory sequence, a Clocks regulatory sequence, a Cry1 / 2 regulatory sequence, a Per1 / 2 / 3 regulatory sequence, a Dec1 / 2 regulatory sequence, a Rors regulatory sequence, a Reverbs regulatory sequence, a Dbp regulatory sequence, or a Nfil3 regulatory sequence. In another aspect, the regulatory sequence of a clock gene is selected from any other oscillating genes downstream or upstream of these targets, including tissue-specific clocks-controlled genes. In one aspect, the regulatory sequence is a positive element, that leads to increased transcription of the therapeutic biologic. In an exemplary embodiment, the positive element is a promoter or enhancer sequence (e.g., an e-box sequence). In another aspect, the regulatory sequence is a negative element that leads to decreased transcription of the therapeutic biologic. In an exemplary embodiment, the negative element is a repressor sequence. In still another aspect, the regulatory sequence includes one or more positive elements from one clock gene and one or more negative elements from a different clock gene.

[0129] In another embodiment, the probiotic cell is genetically modified by introducing into the genome of the cell a heterologous nucleic acid sequence comprising a nucleic acid sequence encoding a therapeutic biologic. In one aspect, using a site-specific nuclease the heterologous nucleic acid sequence encoding a therapeutic biologic is targeted to the genome of the probiotic cell in a specific location such that it becomes operably linked to a regulatory sequence of the clock gene. Integration of the heterologous nucleic acid may or may not disrupt the expression of the endogenous corresponding clock gene. In one aspect, the regulatory sequence of the clock gene is a BmaH regulatory sequence, a Clocks regulatory sequence, a Cry1 / 2 regulatory sequence, a Per1 / 2 / 3 regulatory sequence, a Dec1 / 2 regulatory sequence, a Rors regulatory sequence, a Rev-erbs regulatory sequence, a Dbp regulatory sequence, or a Nfi I3 regulatory sequence. In another aspect, the regulatory sequence of clock genes is from any other oscillating genes downstream or upstream of these targets, including tissue-specific clocks-controlled genes. In one aspect, the regulatory sequence is a positive element such as a promoter or enhancer sequence. In another aspect, the regulatory sequence is a negative element such as a repressor sequence. In still another aspect, the heterologous nucleic acid sequence may further comprise a heterologous regulatory sequence thereby creating an engineered gene circuit that regulates the expression of the therapeutic biologic through both endogenous and heterologous regulatory sequences.

[0130] In each of the above embodiments, a promoter sequence may be an ortholog, paralog, or homolog of anyone of the above-referenced promoters. In one aspect, the transcriptional regulatory region is a fragment of any of the above promoter sequences. In another embodiment, the transcriptional regulatory region is any of the above promoter sequences having one or more mutations (i.e. insertion, deletions, point mutation, etc.) that modulate (e.g., increases or decreases) the phase, amplitude, or period of the biological therapeutic expression. In one embodiment, the transcriptional regulatory region is a promoter nucleic acid sequence from two or more distinct circadian- responsive genes.

[0131] In addition, the transcriptional regulatory region from a circadian- responsive gene (and combinations thereof) can be selected based on the desired temporal delivery of the biologic therapeutic. Thus, a synthetic circuit according to the present disclosure provides the use of distinct circadian signaling networks for defining the specificity, timing, and dose-response for the expression of therapeutic biologic drugs. Using an engineered, living tissue construct for coordinated drug delivery obviates many of the traditional limitations of “smart” materials, such as long-term integration, rapid dynamic responses, and extended drug delivery without the need for replacement or reimplantation of the drug delivery system. The modular approach of using chronogenetic synthetic circuits provides cells within engineered tissues that allow circadian clock-controlled biologic drug delivery at prescribed times of the day.

[0132] In some embodiments, the transcriptional regulatory region is placed upstream of a minimal promoter element and a nucleic acid sequence encoding the therapeutic biologic. For example, a TATA box derived from, in a non-limiting example, the minimal CMV promoter can be placed between the transcriptional regulatory region and therapeutic biologic such that binding of one or more transcription factors to the transcriptional regulatory region leads to transcription of the therapeutic biologic. In each of the above embodiments, additional regulatory elements can be included in the synthetic circuit using common knowledge and methods in the art.

[0133] In one embodiment, the therapeutic biologic may include in non-limiting examples any range of endogenous or exogenous (e.g., non-mammalian) genes with therapeutic or diagnostic applications, including anti-catabolic, antiinflammatory, pro-anabolic, pro-regenerative, pro-circadian, anti-microbial, antipain outputs, morphogens, growth factors, anti-cancer, etc. The proper therapeutic biologic will vary depending upon the host treated and the particular condition, disease, or disorder to be treated. In an exemplary embodiment, the therapeutic biologic is the cytokine antagonist IL-1 receptor antagonist (IL-1 ra) or the type I soluble TNF receptor (sTNFRI). In some embodiments, the therapeutic biologic can be, miRNA, a shRNA, a therapeutic growth factor, a transcriptional regulator, an extracellular matrix (ECM) protein, an antiinflammatory protein, or a biomarker used to monitor treatment efficacy or disease progression. For example, the transgene may be sTNFRI, IL-1 Ra, IL-4, IL6, IkB-alpha, TGF, IL-10, IGF, CTGF, FGF, PDGF, TNF, IL-7, IL-15, IL-12, IFN, NOS, PTGIS, Decorin, TGFb-receptor, ALDH2, NR3C1 , melatonin, melatonin receptor, adenosine, adenosine receptor, a suicide gene, or a matrix degrading enzyme, such as a matrix metalloproteinase (MMP).

[0134] In one aspect, when the desired therapeutic is anti-inflammatory the therapeutic biologic nucleic acid sequence encodes for one or more of IL-1 Ra, sTNFRI / 2, IL-10, and IL-4. In another aspect, when the desired therapeutic response is pro-anabolic, the therapeutic biologic nucleic acid sequence encodes for one or more of a growth factor from the TGF superfamily, IGF, CTGF, FGF, PDGF, and the like. In another aspect, when the desired therapeutic response is anti-pain, the therapeutic biologic nucleic acid sequence encodes for one or more of a kappa opioid ligand pro peptide (prodynorphin), mu / delta opioid ligand pro-peptide (proenkephalin), delta / mu opioid ligand propeptide (proopiomelanocortin), and endocannabinoid ligand synthesis drivers. In another aspect, when the desired therapeutic response is anti-cancer, the therapeutic biologic nucleic acid sequence encodes for one or more of a TNF, IL- 7, IL-15, IL-12, IL-2, IFN, and the like. In another embodiment, when the therapeutic biologic nucleic acid molecule encodes for one or more of NOS and PTGIS. In still another embodiment, when the desired therapeutic response is anti-fibrotic, the therapeutic biologic molecule encodes one or more of Decorin, TGF -receptor, MMPs, ALDH2, and NR3C1. In still another embodiment, when the desired response is pro-circadian the therapeutic biologic molecule encodes one or more of melatonin, melatonin receptor, adenosine, adenosine receptor, and the like.

[0135] In various aspects, the probiotic cells of the present disclosure may be any microorganism suitable as a probiotic, including but not limited to bacteria or yeast. Non-limiting examples of suitable probiotic bacteria species include Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus. Other non-limiting examples of suitable probiotic bacteria species include Bacteroides species such as B. thetaiotaomicron, B. ovatus, B. fragilis, B. vulgatus, B. distasonis, B. uniformis, B. acidifaciens, B. barnesiaes,' B. caccae, B. caecicola, B. caecigallinarum, B. cellulosilyticus, B. cellulosolvens, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. dorei, B. eggerthii, B. gracilis, B. faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. galacturonicus, B. gallinaceum, B. gallinarum, B. goldsteinii, B. graminisolvens, B. helcogene, B. intestinalis, B. luti, B. massiliensis, B. nordii, B. oleiciplenus, B. oris, B. paurosaccharolyticus, B. plebeius, B. polypragmatus, B. propionicifaciens, B. putredinis, B. pyogenes, B. reticulotermitis, B. rodentium, B. salanitronis, B. salyersiae, B. sartorii, B. sedimenti, B. stercoris, B. suis, B. tectus, or B. xylanisolvens. Non-limiting examples of yeast cells suitable for use as a probiotic include Saccharomyces boulardii yeast cells.

[0136] In various aspects, the probiotic cell may be endogenous or exogenous, and may be genetically modified in situ. The probiotic may be genetically modified ex situ and then reintroduced in the body as an oral supplement, gavage, fecal transplant, or engineered into an implantable living tissue.

[0137] The genes used to drive the circadian expression of a therapeutic agent or behavior of the probiotic include circadian genes such as BMAL1, PER1 / 2 / 3, CRY1 / 2, NPAS2, RORa / b, REV-ERBa, RORE, Db, NFIL3, GSK3b, CK1e / d, D box regions, E box regions, and RRE regions. Additional non-limiting examples of suitable circadian genes are provided in PCT Application No.

[0138] PCT / US2022 / 022904, the content of which is incorporated by reference herein in its entirety.

[0139] The proposed genes may include controlled mutations of any of these genes to modify and control the phase, amplitude, or period of gene expression.

[0140] The synthetic gene circuit can be created using plasmid or viral transduction, CRISPR-Cas9 gene editing, TALENs, ZFNs, or any other method of genetic modification.

[0141] The period / frequency / phase of the gene oscillation can be modulated through mutation of any of the circadian genes or through the use of combinations of genes that are off-cycle from one another.

[0142] The overall duration and dynamics of the response can be controlled using a synthetic genetic counter that counts clock cycles in the cell.

[0143] The output may include any range of endogenous or exogenous (e.g., non-mammalian) genes with therapeutic or diagnostic applications, including anti-catabolic, anti-inflammatory, pro-anabolic, pro-regenerative, pro-circadian, anti-microbial, anti-pain outputs, morphogens, growth factors, anti-cancer, etc.

[0144] This approach may be used to improve health in humans or animals, including companion animals or livestock.

[0145] In one embodiment, the therapy involves the use of modified probiotics such as bacteria that are taken as a daily or weekly oral supplement. The probiotic then populates the gut and delivers an anti-inflammatory drug in a circadian manner with peak drug concentrations at midnight using the expression of PER2 as a driver to coincide with the initiation of an inflammatory flare as occurs in autoimmune diseases such as rheumatoid arthritis. By selecting clock genes that are synchronized with the host SCN clock, or alternatively, out of phase, the timing of delivery can be prescribed. By mutating the genes involved in the circadian rhythm of the probiotic, drug delivery may be shifted by hours one way or another.

[0146] In another embodiment, the therapy involves the use of circadian genes to drive the circadian expression of parathyroid hormone (PTH). PTH is an anabolic agent that drives bone formation, but only when delivered intermittently. In contrast, continuous delivery of PTH can cause activation of osteoclasts and bone loss. A probiotic system can be programmed to automatically deliver PTH in an intermittent manner over a long period as a treatment for bone loss, such as osteoporosis.

[0147] In another embodiment, the probiotic could deliver specific nutrients or nutrient precursors / modifiers on a timed basis that improves nutrient absorption, retention, or other health benefits.

[0148] In another approach, two or more out-of-phase clock genes could be used to drive the delivery of pain-modifying drugs that are delivered every 6, 12, or 24 hours automatically. The specific period can be modified by using known or novel mutations in the clock genes that can influence circadian rhythms.

[0149] GENE MODULA TION AGENTS

[0150] As described herein, gene expression has been implicated in various diseases, disorders, and conditions. As such, modulation of genes (e.g., modulation of circadian rhythm genes) can be used for the treatment of such conditions. A gene modulation agent can modulate gene response or induce or inhibit gene expression. Gene modulation can comprise modulating the expression of gene products on cells, modulating the quantity of cells that express a gene, or modulating the quality of the gene-expressing cells.

[0151] Gene modulation agents can be any composition or method that can modulate gene expression on cells (e.g., circadian rhythm genes). For example, a gene modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. As another example, the gene modulation can be the result of gene editing.

[0152] A gene modulation agent can be an antibody (e.g., a monoclonal antibody to a gene product).

[0153] A gene-modulating agent can be an agent that induces or inhibits progenitor cell differentiation into gene-expressing cells (e.g., cells that express circadian rhythm genes).

[0154] A gene modulation agent can be a modified probiotic that drives a controlled gene output.

[0155] Gene Signal Reduction / Enhancement, Elimination / Creation, or Inhibition / llprequlation by Small Molecule Inhibitors, shRNA, siRNA, or ASOs

[0156] As described herein, a gene modulation agent can be used for use in therapy for inflammatory diseases, including but not limited to autoimmune diseases, including but not limited to rheumatoid arthritis, as well as osteoporosis. A gene modulation agent can be used to reduce / eliminate or enhance / increase inflammatory or bone growth signals. For example, a gene modulation agent can be a small molecule inhibitor of an inflammatory gene. As another example, a gene modulation agent can be a short hairpin RNA (shRNA). As another example, a gene modulation agent can be a short interfering RNA (siRNA).

[0157] As another example, RNA (e.g., long noncoding RNA (IncRNA)) can be targeted with antisense oligonucleotides (ASOs) as a therapeutic. Processes for making ASOs targeted to RNAs are well known; see e.g. Zhou et al. 2016 Methods Mol Biol. 1402:199-213. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes. INFLAMMATION INHIBITING AGENT

[0158] One aspect of the present disclosure provides for targeting of inflammatory disease, associated receptors, or associated downstream signaling. The present disclosure provides methods of treating or preventing inflammatory disease and osteoporosis based on the discovery that modified probiotics can drive the production of a desired gene product that can treat a disease.

[0159] As described herein, inhibitors or activators of genes (e.g., antibodies, fusion proteins, small molecules) can reduce or prevent an inflammatory disease. A gene-inhibiting / enhancing agent can be any agent that can inhibit inflammation, downregulate inflammation, knockdown genes associated with inflammation, or activate anti-inflammatory genes.

[0160] As an example, a gene-inhibiting / enhancing agent can inhibit inflammatory signaling.

[0161] For example, the gene-inhibiting / enhancing agent can be an antiinflammatory antibody. As an example, the anti-inflammatory antibody can be an anti-PER2 antibody or an anti-inflammatory antibody with activity against any combination of inflammatory genes. Furthermore, the anti-inflammatory antibody can be a murine antibody, a humanized murine antibody, or a human antibody.

[0162] As another example, the gene inhibiting / enhancing agent can be an antiinflammatory antibody that can prevent binding of an inflammatory ligand to its receptor and prevent activation of inflammatory signals and associated downstream signaling.

[0163] As another example, the gene-inhibiting / enhancing agent can be a fusion protein. For example, the fusion protein can be a decoy receptor for an inflammatory ligand. Furthermore, the fusion protein can comprise a mouse or human Fc antibody domain fused to the ectodomain of an inflammatory receptor.

[0164] As another example, a gene inhibiting / enhancing agent can be a modified probiotic that drives production of a desired gene product that treats a disease.

[0165] As another example, a gene-inhibiting / enhancing agent can be an inhibitory protein that antagonizes an inflammatory receptor. For example, the gene-inhibiting / enhancing agent can be a viral protein, which has been shown to antagonize inflammatory signaling.

[0166] As another example, a gene inhibiting / enhancing agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA) targeting inflammatory signals.

[0167] As another example, a gene inhibiting / enhancing agent can be an sgRNA targeting inflammatory genes.

[0168] Methods for preparing a gene inhibiting / enhancing agent (e.g., an agent capable of inhibiting inflammatory signaling) can comprise the construction of a protein / Ab scaffold containing the natural inflammatory receptor as an inflammatory neutralizing agent; developing inhibitors of the inflammatory receptor “down-stream”; or developing inhibitors of the inflammatory receptor production “up-stream”.

[0169] Inhibiting inflammation can be performed by genetically modifying an inflammatory gene in a subject or genetically modifying a subject to reduce or prevent expression of the inflammatory gene, such as through the use of CRISPR-Cas9 or analogous technologies, wherein, such modification reduces or prevents inflammation.

[0170] CHEMICAL AGENT:

[0171] Examples of gene modulation agents are described herein.

[0172] R groups can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci -1 oalkyl hydroxyl; amine; Ci- carboxylic acid; Ci-wcarboxyl; straight chain or branched C1-1 oalkyl, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched Ci- walkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, 0, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci-walkyl hydroxyl; amine; Ci-wcarboxylic acid; Ci- wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C1-1 oalkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci-walkyl hydroxyl; amine; Ci- carboxylic acid; Ci-wcarboxyl; straight chain or branched C1-1 oalkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched Ci-walkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms. Any of the above can be further optionally substituted.

[0173] The term “imine” or “imino”, as used herein, unless otherwise indicated, can include a functional group or chemical compound containing a carbonnitrogen double bond. The expression “imino compound”, as used herein, unless otherwise indicated, refers to a compound that includes an “imine” or an “imino” group as defined herein. The “imine” or “imino” group can be optionally substituted.

[0174] The term “hydroxyl”, as used herein, unless otherwise indicated, can include -OH. The “hydroxyl” can be optionally substituted.

[0175] The terms “halogen” and “halo”, as used herein, unless otherwise indicated, include chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.

[0176] The term “acetamide”, as used herein, is an organic compound with the formula CH3CONH2. The “acetamide” can be optionally substituted.

[0177] The term “aryl”, as used herein, unless otherwise indicated, includes a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The “aryl” can be optionally substituted.

[0178] The terms “amine” and “amino”, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group. The “amine” or “amino” group can be optionally substituted.

[0179] The term “alkyl”, as used herein, unless otherwise indicated, can include saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, - isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3- methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5- dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated C1-10 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, - isobutylenyl, -1 -pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, - 2,3-dimethyl-2-butenyl, 1 -hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1- butynyl, -2-butynyl, -1 -pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated. The “alkyl” can be optionally substituted.

[0180] The term “carboxyl”, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (-COOH). The “carboxyl” can be optionally substituted.

[0181] The term “alkenyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated. The “alkenyl” can be optionally substituted.

[0182] The term “alkynyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated. The “alkynyl” can be optionally substituted.

[0183] The term “acyl”, as used herein, unless otherwise indicated, can include a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (-OH) group. The “acyl” can be optionally substituted.

[0184] The term “alkoxyl”, as used herein, unless otherwise indicated, can include O-alkyl groups wherein alkyl is as defined above and 0 represents oxygen. Representative alkoxyl groups include, but are not limited to, -O-methyl, -O-ethyl, -O-n-propyl, -O-n-butyl, -O-n-pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, -O-sec-butyl, -O-isobutyl, -O-tert-butyl, -O-isopentyl, -0-2- methylbutyl, -0-2-methylpentyl, -0-3-methylpentyl, -0-2,2-dimethylbutyl, -0-2,3- dimethylbutyl, -0-2,2-dimethylpentyl, -0-2,3-dimethylpentyl, -0-3,3- dimethylpentyl, -0-2,3,4-trimethylpentyl, -0-3-methylhexyl, -0-2,2-dimethylhexyl, -0-2,4-dimethylhexyl, -0-2,5-dimethylhexyl, -0-3,5-dimethylhexyl, -0- 2,4dimethylpentyl, -0-2-methylheptyl, -0-3-methylheptyl, -O-vinyl, -O-allyl, -0-1 - butenyl, -0-2-butenyl, -O-isobutylenyl, -0-1 -pentenyl, -0-2-pentenyl, -0-3- methyl-1 -butenyl, -O-2-methyl-2-butenyl, -O-2,3-dimethyl-2-butenyl, -0-1 -hexyl, - 0-2-hexyl, -0-3-hexyl, -O-acetylenyl, -O-propynyl, -0-1 -butynyl, -0-2-butynyl, - 0-1 -pentynyl, -0-2-pentynyl and -0-3-methyl-1 -butynyl, -O-cyclopropyl, -0- cyclobutyl, -O-cyclopentyl, -O-cyclohexyl, -O-cycloheptyl, -O-cyclooctyl, -0- cyclononyl and -O-cyclodecyl, -O-CH2-cyclopropyl, -O-CH2-cyclobutyl, -O-CH2- cyclopentyl, -O-CH2-cyclohexyl, -O-CH2-cycloheptyl, -O-CH2-cyclooctyl, -0- CH2- cyclononyl, -O-CH2-cyclodecyl, -O-(CH2)2-cyclopropyl, -O-(CH2)2-cyclobutyl, -0- (CH2)2-cyclopentyl, -O-(CH2)2-cyclohexyl, -O-(CH2)2-cycloheptyl, -O-(CH2)2- cyclooctyl, -O-(CH2)2-cyclononyl, or -O-(CH2)2-cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated. The “alkoxyl” can be optionally substituted.

[0185] The term “cycloalkyl”, as used herein, unless otherwise indicated, can include an aromatic, non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 1 to 10 carbon atoms (e.g., 1 or 2 carbon atoms if there are other heteroatoms in the ring), preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, - cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1 ,3-cyclohexadienyl, -1 ,4- cyclohexadienyl, -cycloheptyl, -1 ,3-cycloheptadienyl, -1 ,3,5-cycloheptatrienyl, - cyclooctyl, and -cyclooctadienyl. The term “cycloalkyl” also can include -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alkyl-cycloalkyl groups include, but are not limited to, -CH2- cyclopropyl, -CH2-cyclobutyl, -CH2-cyclopentyl, -CH2-cyclopentadienyl, -CH2- cyclohexyl, -CH2-cycloheptyl, or -CH2-cyclooctyl. The “cycloalkyl” can be optionally substituted. A “cycloheteroalkyl”, as used herein, unless otherwise indicated, can include any of the above with a carbon substituted with a heteroatom (e.g., 0, S, N).

[0186] The term “heterocyclic” or “heteroaryl”, as used herein, unless otherwise indicated, can include an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of 0, S and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1 ,4)-dioxane, (1 ,3)- dioxolane, 4,5-dihydro-1 H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e. , at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated. The “hetreocyclic” can be optionally substituted.

[0187] The term “indole”, as used herein, is an aromatic heterocyclic organic compound with the formula C8H7N. It has a bicyclic structure, consisting of a sixmembered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The “indole” can be optionally substituted.

[0188] The term “cyano”, as used herein, unless otherwise indicated, can include a -CN group. The “cyano” can be optionally substituted.

[0189] The term “alcohol”, as used herein, unless otherwise indicated, can include a compound in which the hydroxyl functional group (-OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms. The “alcohol” can be optionally substituted.

[0190] The term “solvate” is intended to mean a solvate form of a specified compound that retains the effectiveness of such compounds. Examples of solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

[0191] The term “mmol”, as used herein, is intended to mean millimole. The term “equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “pg”, as used herein, is intended to mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term "pL", as used herein, is intended to mean microliter. The term “pM”, as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “°C”, as used herein, is intended to mean degree Celsius. The term “wt / wt”, as used herein, is intended to mean weight / weight. The term “v / v”, as used herein, is intended to mean volume / volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high-performance liquid chromatography. The term “RT”, as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term “N / A”, as used herein, is intended to mean not tested.

[0192] As used herein, the expression “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1 ,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterions. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound of the invention, or a salt thereof, that further can include a stoichiometric or non-stoichiometric amount of water bound by non- covalent intermolecular forces.

[0193] MOLECULAR ENGINEERING

[0194] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0195] The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0196] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0197] A “promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0198] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0199] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein-encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0200] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0201] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked. A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0202] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0203] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0204] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0205] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0206] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0207] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid that is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by lie, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. The amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon usage of the desired host cell.

[0208] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA: DNA sequence can be determined using the following formula: Tm = 81 .5 °C + 16.6(log [Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA: DNA hybrid is decreased by 1-1.5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0209] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated into the host cell genome.

[0210] Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0211] Host strains developed according to the approaches described herein can be evaluated by any number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0212] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0213] Genome Editing

[0214] As described herein, inflammatory and osteoporosis signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing. Processes for genome editing are well known; see e.g. Aldi 2018 Nature Communications 9(1911 ). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0215] For example, genome editing can comprise CRISPR / Cas9, CRISPR- Cpf1 , TALEN, or ZNFs. Adequate blockage of inflammatory signals by genome editing can result in protection from autoimmune or inflammatory diseases.

[0216] As an example, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N)2ONGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double-strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR / Cas systems could be a useful tool for therapeutic applications for a gene-driving probiotic to target cells by the removal of inflammatory signals or the activation of anti-inflammatory signals.

[0217] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

[0218] FORMULATION

[0219] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0220] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0221] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0222] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0223] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0224] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0225] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce the dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0226] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.

[0227] THERAPEUTIC METHODS

[0228] Also provided is a process of treating, preventing, or reversing a disease, including but not limited to inflammatory disease and osteoporosis in a subject in need of administration of a therapeutically effective amount of a modified probiotic, so as to provide safe and controlled production of prescribed therapeutic molecules or nutrients to the recipient.

[0229] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a disease, including but not limited to inflammatory disease and osteoporosis. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0230] Generally, a safe and effective amount of a gene modulation agent including a modified probiotic is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of the gene modulation agent described herein can substantially inhibit inflammation, slow the progress of an inflammatory disease, or limit the development of an inflammatory disease.

[0231] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0232] When used in the treatments described herein, a therapeutically effective amount of a gene modulation agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat a disease, including but not limited to inflammatory disease and osteoporosis.

[0233] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0234] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0235] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the seventy of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0236] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0237] Administration of a gene modulation agent such as the probiotics described herein can occur as a single event or over a time course of treatment. For example, a gene modulation agent can be administered daily, weekly, biweekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0238] Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease, including but not limited to inflammatory disease and osteoporosis.

[0239] A gene modulation agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a gene modulation agent, which can be a modified probiotic that drives production of therapeutic gene products can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of a gene modulation agent, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more of a gene modulation agent, an antibiotic, an antiinflammatory, or another agent. A gene modulation agent can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a gene modulation agent can be administered before or after the administration of an antibiotic, an anti-inflammatory, or another agent.

[0240] ADMINISTRATION

[0241] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body. As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0242] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0243] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0244] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.

[0245] SCREENING

[0246] Also provided are methods for screening.

[0247] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

[0248] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0249] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, LICSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0250] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0251] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “druglike”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0252] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0253] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and 0 atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0254] KITS

[0255] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to a probiotic and materials to facilitate oral administration in a pill form. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0256] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0257] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0258] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0259] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer programs include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0260] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0261] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0262] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0263] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0264] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0265] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0266] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0267] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0268] EXAMPLES

[0269] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0270] EXAMPLE 1 - CIRCADIAN PROBIOTIC SYSTEMS

[0271] To characterize the use of circadian rhythms for drug delivery using mammalian cells and the use of circadian “clock genes” to modify probiotic supplements (e.g., bacteria) as a therapeutic modality that is delivered to the gut on a prescribed timed basis.

[0272] Rheumatoid arthritis, or RA, is an autoimmune disease that can impact multiple joints in the body and can lead to severe pain and disability. The primary impact of RA is on the joints and impacts 0.5 - 1 % of people worldwide. Normal synovial joints consist of a joint capsule surrounding the joint, with a synovial membrane lining the inner surface and synovial fluid that reduces friction in the articular cartilage (FIG.1 A). While joints in RA exhibit chronic inflammation with periodic inflammatory flares that can begin even prior to clinical diagnosis (FIG. 1 C). This chronic inflammation results in a feedback of inflammatory cells recruited to the joint and dysregulated growth of the synovial tissue, resulting in synovial hyperplasia and pannus invasion that contributes to cartilage destruction & bone erosion, mediated through the production of proinflammatory cytokines and catabolic protein (FIG. 1 B).

[0273] First-line treatments for RA usually include NSAIDs and corticosteroids, which are anti-inflammatory and immuno-suppressive. Recently biologic DMARDs have become more common, these target many pathways in the immune system using biologies such as decoy receptors, antagonists, and monoclonal antibodies to prevent proinflammatory signaling. However, there are significant adverse side effects, a lack of effective sustained remission, and require high doses to achieve clinical impact. Most major current therapeutics don’t enable dynamic or localized treatment of these long-term or daily, circadian inflammatory flares (FIG. 2A). Several cytokines that mediate inflammation in RA peak in the early morning hours, coinciding with clinical symptoms (FIG. 2B). These factors demonstrated the benefits of timing medication with circadian flares, which is known a chronotherapy, where a low dose therapeutic can improved RA symptoms when administered prior to the daily flare. Designing a probiotic system using circadian genes as a clock mechanism will allow for the timed treatment of diseases like RA.

[0274] Circadian rhythms are largely driven by information from environmental cues organized in the suprachiasmatic nucleus (SCN), such as the 24-hour light / dark cycle. Tissue-level peripheral clocks are entrained by signals like glucocorticoids or neurohormones to synchronize the circadian rhythm of the whole body. Other circadian signal transduction mediators also regulate the immune response, such as the night neurohormone melatonin and the autonomic nervous system.

[0275] These circadian rhythms that are synchronized on a tissue level act via the cellular level by interactions of autoregulated transcriptional and translational feedback loops. The core loop is driven by a series of interacting proteins that form a positive arm and a negative arm of this loop. In the positive arm, the proteins BMAL1 and CLOCK are transcribed, translated, and dimerized. The BMAL1 -CLOCK dimer translocates to the nucleus, where it binds E-boxes and DNA sequences in the promoter region, and promotes the transcription of circadian genes. The period and cryptochrome genes, Per1 / 2 and Cry1 / 2, are regulated by E-box elements and are transcribed and then translated. Per and Cry proteins accumulate in the cytoplasm, dimerize, and then translocate back to the nucleus, where they interact with BMAL1 and CLOCK to prevent their transcriptional activity. This inhibits their transcription, forming the negative arm of this loop. Per and Cry are eventually degraded, and this cycle begins again, with an approximately 24-hour period (FIG. 3). Overall, the interactions between the circadian and NFKB pathways can be context-dependent and can lead to up and down-regulation. Interactions between clock and NFKB subunits can positively regulate the NFKB pathway. In contrast, the interactions between BMAL1 and NFKB subunits can lead to reduced circadian cycles, where the NFKB subunits can act as repressors of BMAL1 / CLOCK complexes bound to E- box elements, demonstrating a transcriptionally repressive role. Designing tools to better understand these interactions may be able to help explain the interaction in the context of cartilage and arthritis, and could be used to help generate therapeutic gene circuits for RA.

[0276] The Per2 promoter can be used to drive a circadian gene circuit that can produce a gene of interest, such as a luciferase reporter or the therapeutic IL1 RA gene. The gene circuit includes NFKB response elements and E-boxes to regulate circadian rhythm pathways (FIG. 4A). Integrating this gene circuit into PDiPSCs and using a pellet model demonstrates that circadian reporter activity can be maintained following the addition of IL1 a (FIG. 4B), and IL1 Rn was regulated in a circadian manner (FIG. 4C). These elements are useful tools in designing a circadian-specific circuits which can be driven by E-box elements.

[0277] EXAMPLE 2 - DESIGNING AND INVESTIGATING GENE CIRCUITS FOR CIRCADIAN AND INFLAMMATORY CUES

[0278] To design and investigate gene circuits used for circadian and inflammatory cues.

[0279] The NFKB synthetic gene circuit responds to inflammatory signaling through the NFKB pathway, leading to transcriptional activation based on the promoter with 5 NFKB response elements. This pathway has shown promising results in protecting cartilage pellets from inflammatory degradation by driving the production of IL-1 Ra. Similar to a Per2 -driven circuit, the NFKB circuit produces therapeutics in both an inflammation-dependent and circadian-driven manner. In general, the NFKB-responsive circuits increase expression proportionally to the level of inflammatory signaling, which would activate on- demand if an inflammatory signal like IL-1 was added (FIG. 5A, left). Similarly, we saw that circadian output is cyclical until an inflammatory stimulus is added, which can disrupt the amplitude and phase of outputs (FIG. 5A, middle). However, existing gene circuits have limitations in addressing the complexity of RA. First, inflammatory-driven circuits were designed to respond on-demand to inflammatory signaling but lack the ability to preemptively address daily fluctuations, which was shown to reduce RA symptoms when applied to corticosteroids, and other DMARDs. Excessive inflammation can also dampen and disrupt circadian rhythms, which would impede therapeutic production from circadian-driven circuits during highly inflammatory, non-circadian flares. Combining these two methods into a single tool could target inflammation in RA at multiple levels. By constructing a promoter with elements from both pathways, it generates a therapeutic output that is circadian under “normal” or noninflammatory conditions but can respond with an increase in amplitude when an inflammatory stimulus is added to further mitigate excessive inflammation and promote the return to a normal circadian output (FIG. 5A, right).

[0280] There are several different elements to consider when selecting a promoter sequence, including the intrinsic capacity of the promoter and its role in potential activity in different cells. Selecting different combinations of promoters and the number of repeats of promoter elements can affect the amplitude and phase of the output, with some being more transcriptionally active than others. Additionally, if a specific cell type, like macrophages, experiences a high baseline level of inflammatory activity, this will result in a higher level of transcriptional activity with the NFKB-driven circuit. Another important aspect can depend on the physical design of the gene circuit, including the spacing of elements and distance from the transcriptional start site, which can affect the transcriptional level and be used to tailor the output.

[0281] To develop a synthetic gene circuit that responds to both circadian and inflammatory cues, a promoter is chosen that contains basic circadian clock- controlled elements as well as E-boxes, and NFKB response elements. A gene circuit with additive-like behavior, an “or” like response, will generate an output when either circadian or inflammatory cues are present and has the potential to produce an additive response when both cues are present (FIG. 5B). While an “and” response would drive an output when there is inflammation and circadian transcriptional activity but not when there is neither or either alone. This type of circuit would be specific to a situation where a circadian output is desired only in inflammatory condition (FIG. 5C). The gene circuit can be designed with NFKB RE preceding the E-box and vise-versa, allowing for various levels of transcriptional activation (FIG. 5D).

[0282] The circuits were designed to include a promoter that drives GFP and luciferase or IL1 Ra production and the NFkB promoter that contains the minimal CMV element and a luciferase of IL1 ra output. The Per2 plasmid (FIG. 6A) was used as the backbone and the Per2 promoter was removed through restriction enzyme digest (FIG. 6B) and the ligate regions of synthesized DNA of the E- boxes and minimal CMV (FIG. 6C, FIG. 6D). This plasmid is used as the starting point to move the NFkB response elements (FIG. 7A) into the same circuit to include the GFP report for better selection. The NFkB RE is isolated by PCR and ligated to E-box, mCMV, or backbone plasmid homology for later insertion into a plasmid (FIG.7B). The plasmid 3xE’Box is digested with Asci (FIG. 7C) or Spel (FIG. 7E) and ligated to the corresponding PCR product to form the final plasmids which contains both circadian and inflammatory response elements (FIG. 7D, FIG. 7F).

[0283] To determine the protective effects of the circuit's response to circadian and inflammatory cues, HEK293T cells were transduced with the NFKB circuit which generates a luciferase reporter. Cells were synchronized prior to obtaining a baseline reading through a 1 ,5-hour serum shock or a 1 ,5-hour Dex treatment. Cells can then be treated with IL-1 a and bioluminescence can be recorded (FIG. 8A). Under control conditions, luminescence remained low over the 72h testing period but with the addition of IL-1 , the circuit became activated. The addition of Dex for synchronization 24h prior to adding IL1 showed slightly lower initial output but a serum shock synchronization slightly increased the circuit response over time (FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E). To determine the protective effects of the circuit response to circadian cues, the kinetics and amplitude of the Per2 promoter were examined to gauge how the E-box-driven promoter responds. HEK293T and PDiPSCs were transduced with the Per2 plasmid and treated with Dex to synchronize the cells, bioluminescence was assessed over 72h without the addition of any inflammatory cytokines (FIG. 9A). The HEK293T cells showed limited circadian- looking output, but when examined closer there was a low amplitude but rhythmic output of luciferase over 24h periods (FIG. 9B). In comparison, the PDiPSCs showed the best response at higher cell densities and demonstrated relatively circadian repeating outputs over 72h that varied in the baseline level of output depending on the concentration of luciferin added (FIG. 9C).

[0284] The circuits were also tested in combination to generate circadian-driven therapeutics while also responding on demand to excess inflammation. PDiPSCs were transduced with Per2, NFkB, or both circuits, cells were synchronized, IL- 1a was added to the cell, and bioluminescence recorded for 72h with 0.1 mM luciferin (FIG. 10A). In the cells containing both the NFkB and Per2 promoter, the cells demonstrated increased amplitude, which is expected from an additive output, and may lead to the ability of the circuits to increase response over time when IL1 is added to activate the circuit (FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E). The addition of the therapeutic 11-1 RN gene to produce 11-1 RA is a great system to use for a circadian-driven therapeutic since it has a half-life that can be used on a daily scale and has minimal off-target effects while providing an effective daily therapeutic.

Claims

ClaimsWhat is claimed is:1 . A probiotic supplement to deliver a drug to a subject, the supplement modified to include a synthetic gene circuit to drive a therapeutic controlled gene output that is delivered to the gut of the subject on a prescribed time basis, the synthetic gene circuit comprising at least one circadian clock gene that drives expression of at least one prescribed therapeutic or diagnostic gene target.

2. The probiotic supplement of claim 1 , wherein the probiotic is selected from a bacteria, a yeast, and any combination thereof.

3. The probiotic supplement of any previous claim, wherein the probiotic supplement is endogenous or exogenous.

4. The probiotic supplement of any previous claim, wherein the probiotic supplement is genetically modified in situ.

5. The probiotic supplement of any previous claim, wherein the probiotic supplement is genetically modified ex situ, and then reintroduced into the subject.

6. The probiotic supplement of any previous claim, wherein the probiotic is administered to the subject by a method selected from an oral supplement, a gavage, a fecal transplant, and an implantable living tissue.

7. The probiotic supplement of any previous claim, wherein the at least one circadian clock gene is selected from BMAL1, PER1 / 2 / 3, CRY1 / 2, NPAS2, RORa / b, REV-ERBa, RORE, Db, NFIL3, GSK3b, CK1e / d, D box regions, E box regions, RRE regions, and any combinations thereof.

8. The probiotic supplement of any previous claim, wherein the at least one circadian clock gene comprises controlled mutations to modify and control the phase, amplitude, or period of gene expression.

9. The probiotic supplement of any previous claim, wherein the synthetic gene circuit is created using a genetic modification method selectedfrom plasmid transduction, viral transduction, CRISPR-Cas9 gene editing, TALENs, and ZFNs.

10. The probiotic supplement of any previous claim, wherein an overall duration and dynamics of the response are controlled using a synthetic genetic counter that counts clock cycles in cells.11 . The probiotic supplement of any previous claims, wherein the at least one therapeutic or diagnostic gene target is selected from an anti- catabolic target, an anti-inflammatory target, a pro-anabolic target, a pro-regenerative target, a pro-circadian target, an anti-microbial target, an anti-pain target, a morphogen target, a growth factor target, an anticancer target, and any combination thereof.

12. A therapeutic method to deliver at least one diagnostic and therapeutic gene target to a subject time-sensitively, the method comprising: a. modifying a probiotic supplement to include a synthetic circadian gene circuit that drives expression of the at least one gene target, and b. delivering the probiotic supplement to the subject.

13. The therapeutic method of claim 12, wherein the at least one gene target is selected from an anti-catabolic target, an anti-inflammatory target, a pro-anabolic target, a pro-regenerative target, a pro-circadian target, an anti-microbial target, an anti-pain target, a morphogen target, a growth factor target, an anti-cancer target, and any combination thereof.

14. The therapeutic method of any one of claims 12-13, wherein the synthetic circadian gene circuit is selected from BMAL1, PER1 / 2 / 3, CRY1 / 2, NPAS2, RORa / b, REV-ERBa, RORE, Db, NFIL3, GSK3b, CK1e / d, D box regions, E box regions, RRE regions, and any combination thereof.

15. The therapeutic method of any one of claims 12-14, wherein the probiotic supplement is delivered to the gut and delivers an antiinflammatory drug in a circadian manner with peak drug concentrations at midnight using the expression of the circadian gene PER2 as a driver to coincide with the initiation of an inflammatory flair.

16. The therapeutic method of any one of claims 12-15, wherein the at least one synthetic circadian gene circuit is mutated to control the time of drug delivery.

17. The therapeutic method of any one of claims 12-16, wherein the circadian gene circuit drives intermittent expression of parathyroid hormone (PTH).

18. The therapeutic method of any one of claims 12-17, wherein the intermittent expression of PTH promotes bone growth in an osteoporosis patient.

19. The therapeutic method of any one of claims 12-18, wherein the probiotic supplement delivers specific nutrients or nutrient precursors / modifiers on a timed basis that improves nutrient absorption, nutrient retention, general health benefits, and any combination thereof.

20. The therapeutic method of any one of claims 12-19, wherein two or more out-of-phase circadian clock genes are used to drive delivery of pain-modifying drugs that are delivered every 6, 12, or 24 hours automatically.

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