Programmable recruitment of transcription factors to endogenous genes

Abstract

The present disclosure provides methods of modulating gene expression in a cell-specific manner in response to intracellular or extracellular stimulation. The present disclosure provides a platform, named PROTEGE platform, comprising a DNA binding module and a transcription factor binding module, referred to herein as PGM. The PGM binds to a promoter region of a target gene in sequence specificity by a DNA binding module and also binds to TF by a TF binding module. When TF is activated in response to an intracellular or extracellular stimulus, it binds to PGM and modulates the expression of the target gene in response to the stimulus as it is very close to the gene promoter.

Description

Technical Field

[0001] The present disclosure is in the field of programmable regulation of gene expression, wherein programmable regulation of gene expression is performed in a cell-specific manner by recruiting transcription factors to one or more genes in response to intracellular and / or extracellular stimuli. The present disclosure provides a platform designated herein as the Protege platform.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 341,820, filed on May 13, 2022, the contents of which are incorporated herein by reference in their entirety.

[0004] Reference to electronically submitted sequence listing

[0005] This application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. The .XML copy is 73,779 KB in size. Background of the Invention

[0007] Organisms respond to disease and injury by regulating the expression of specific genes to promote recovery, healing, or disease resistance. Cells sense external signals generated by disease or injury and respond by activating transcription factors to regulate the expression of genes under their control. However, genes that may be beneficial for healing, recovery, or disease resistance are often not regulated to achieve their beneficial effects. This defect may be due to the gene not being controlled by the relevant transcription factor, or to insufficient activation or repression of the gene by the relevant transcription factor.

[0008] The conventional solution to this problem is to administer the product encoded by the potentially beneficial gene as a pharmaceutical agent. For example, recombinant human bone morphogenetic protein-2 (rh-BMP-2) is used to promote recovery after spinal surgery. Similarly, recombinant human platelet-derived growth factor (rhPDGF) is used to treat diabetic ulcers to improve wound healing. This approach is often unsuccessful or of limited effect because it does not restrict the activity of the added gene product to the time and location where it is needed, resulting in compensatory effects or adverse side effects.

[0009] Another method is achieved through the latest progress of gene editing, which modifies the genome to change the expression of therapeutic gene products. For example, mutations or polymorphisms that cause specific gene product defects can be changed to establish beneficial gene expression levels. Such modifications use gene editors, which include sequence-specific DNA binding components and endonucleases that cleave DNA at or near the binding site. Changes in cleavage sites can be performed by homology-directed repair (HDR), where exogenous DNA containing the desired editing sequence is used as a repair template.

[0010] Several types of sequence-specific nucleases have been used for gene editing, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR endonucleases. For example, a ribonucleoprotein complex containing the Cas9 (CRISPR-associated protein 9) endonuclease and a guide RNA can bind to and cleave a DNA genomic sequence specified by the guide RNA. Gene editing carries the risk of off-target editing, and the edits made are permanent. Therefore, harmful off-target edits or intended edits with deleterious effects are irreversible.

[0011] Gene expression can be reversibly regulated by synthetic transcription factors. Like naturally occurring transcription factors, synthetic transcription factors bind to specific sequences in the promoter or enhancer regions of genes, and deliver or recruit endogenous factors to promote or interfere with the assembly of transcription initiation complexes or promote the chromatin modification of regulation transcription. Synthetic transcription factors are created using zinc fingers, TALEs and CRISPR-related (Cas) proteins modified to eliminate their endonuclease activity. For example, dead Cas9 (deadCas9) (dCas9) is a mutant form of Cas9, which causes its endonuclease activity to be disabled due to mutations in its endonuclease domain. It can still be bound to its guide RNA and target DNA chain. The transcription factor connected to dCas9 or its combined guide RNA can be delivered to the target DNA sequence in the gene promoter or enhancer region and regulate its transcription. The transcription factor used in this situation includes Vp64, p65, Hsf1 and EB virus R transactivator (Rta). Transcription factors previously used for this purpose are either non-native to the treated cells (e.g., viral transcription factors in mammalian cells) and / or artificially covalently fused to dCas9 or other proteins that mediate its binding to dCas9. Therefore, they are not endogenously produced transcription factors whose activity depends on physiological signals affecting the cell.

[0012] Nucleic acid therapies for regulating gene expression (e.g., ASOs, antagomirs, siRNAs, therapeutic mRNAs) may pose a significant risk of irreversible, harmful gene modifications. Furthermore, the effects of these therapies are not limited to the physiological conditions in which they are needed. With the exception of mRNA, the ability of these approaches to increase the expression of beneficial genes is limited. Summary of the invention

[0013] Cells respond to disease and injury by expressing genes in response to environmental cues that they are needed. But not all genes that could promote healing and recovery are expressed at optimal levels, or not expressed at all. Current medical treatments that aim to artificially provide beneficial gene products often do not work or are harmful because they do not restrict their effects to where and when they are needed in the body. We need a way to turn genes on (or off) in response to physiological signals that indicate the benefits of gene regulation.

[0014] The activity of artificial transcription factors previously reported does not respond to environmental signals caused by disease or injury. This response can be achieved by directing the activity of endogenous transcription factors that are activated by environmental signals associated with disease or injury to target genes. Reversibly regulating the expression of target genes in a way that makes the expression levels of these genes dependent on environmental signals caused by disease or injury will limit the time during which changes in gene expression levels have a therapeutic effect on cells, cell locations, and altered gene expression. Therefore, it will avoid gene expression in cells, cell locations, and changes in gene expression over time that can produce adverse side effects.

[0015] Previously described gene expression regulators do not directly incorporate natural, endogenously produced transcription factors into their designs. For example, the previous design based on dCas9 was directly fused to the dCas9 protein, fused to the phage capsid protein (MS2) that can bind to the RNA sequence incorporated into the guide RNA, or coupled to antibodies that can bind to the polypeptide sequence fused to the dCas9 protein, and the transcriptional regulatory domain was connected to the dCas9 guide RNA ribonucleoprotein complex. In various of these methods, the transcriptional regulatory domain needs to be covalently coupled to another protein (e.g., dCas9, MS2 or antibody), so it is necessary to deliver the transcriptional regulatory domain exogenously or deliver the transcriptional regulatory domain by transfection with an expression vector of the fusion protein. This requirement prevents the DNA binder based on CRISPR from directly delivering or recruiting endogenous transcription factors to the gene of interest.

[0016] We describe herein compositions and methods for transcriptional regulation of a specific gene of interest using an artificial transcription factor and one or more endogenously produced natural transcription factors activated in response to an external signal, wherein the artificial transcription factor can simultaneously bind to a specific sequence of genomic DNA in or near the target gene. The principle is shown in Figure 1. The target gene is programmed by the sequence of the crRNA component of the guide RNA, and the one or more transcription factors to which the transcriptional regulation responds are programmed by one or more transcription factor response elements incorporated into the guide nucleic acid. The following embodiments are non-limiting examples of the invention provided by the present disclosure:

[0017] 1. An engineered non-naturally occurring system comprising a programmable gene regulator (PGM) for reversibly altering the expression of a target gene of interest in a cell in response to one or more intracellular or extracellular environmental signals, or a sgCNA subcomponent thereof, the system comprising the following subcomponents:

[0018] an endonuclease-defective DNA-binding polypeptide (preferably, a dCas polypeptide); and

[0019] a chimeric nucleic acid (sgCNA) comprising a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), and at least one nucleic acid segment comprising at least one transcription factor binding site;

[0020] The crRNA comprises a sequence complementary to a nucleic acid sequence in the promoter region of a target gene of interest, and the transcription factor binding sites in the PGM are each capable of binding to at least one endogenous transcription factor, which is activated in response to one or more environmental signals in a cell containing the PGM, and then recognizes and binds to the transcription factor binding site of the PGM, which binds to the promoter of the gene of interest through the crRNA, thereby bringing the transcription factor close to the gene of interest and responding to one or more environmental signals to activate or inhibit the expression of the gene of interest.

[0021] 2. The engineered non-naturally occurring system or sgCNA subcomponent thereof according to embodiment 1, wherein (i) at least one transcription factor binding site in the PGM is also present in the target gene and / or (ii) at least one transcription factor binding site in the PGM is not an endogenous transcription factor binding site in the target gene.

[0022] 3. The engineered non-naturally occurring system of any one of embodiments 1 and 2, or a sgCNA subcomponent thereof, wherein when one or more endogenous transcription factors are activated in response to one or more environmental signals, wherein the PGM recruits the one or more endogenous transcription factors to a gene of interest, thereby activating gene expression in response to the one or more environmental signals in a cell-specific manner.

[0023] 4. The engineered non-naturally occurring system according to any one of embodiments 1 to 3, or a sgCNA subcomponent thereof, wherein the one or more transcription factors are identified as transcription factors that (i) are known to be activated in response to environmental signals and (ii) are known or unknown to activate / repress expression of a target gene of interest.

[0024] 5. The engineered non-naturally occurring system of any one of embodiments 1 to 4, or a sgCNA subcomponent thereof, wherein the DNA binding polypeptide is a nuclease-deficient cas polypeptide.

[0025] 6. The engineered non-naturally occurring system of any one of embodiments 1 to 5, or a sgCNA subcomponent thereof, wherein a gene of interest is identified as a gene whose expression (a) produces a beneficial cellular response in response to one or more environmental signals, but whose expression is undetectable or whose expression is increased by the PGM relative to the gene expression level in the absence of the PGM; or (ii) produces a deleterious effect on the cell and whose expression is decreased in response to one or more environmental signals relative to the gene expression level in the absence of the PGM.

[0026] 7. The engineered non-naturally occurring system according to any one of embodiments 1 to 6, or a sgCNA subcomponent thereof, wherein the gene of interest encodes a protein, a microRNA, or a long non-coding RNA.

[0027] 8. The engineered non-naturally occurring system of any one of embodiments 1 to 7, or a sgCNA subcomponent thereof, wherein the signal is any physical signal such as a light signal (e.g., UV light), ionizing radiation, heat / temperature, hypertonic or hypotonic conditions; a mechanical signal such as pressure (e.g., touch), acoustic motion, and / or blood pressure; and / or any chemical signal such as a growth factor, a cytokine, a chemokine, cyclic AMP, a hormone, a neurotransmitter, an extracellular matrix component, a bacterial antigen, a viral antigen, a lipid, a lipopolysaccharide, a gas level (e.g., an oxygen level, a nitric oxide level), an ion level (e.g., a calcium level, a sodium level), pH, a reactive oxygen species, a heavy metal, oxidized LDL and / or a free radical, a cell-cell signal (e.g., T-cell binding, cell-cell contact), or a combination thereof.

[0028] 9. The engineered non-naturally occurring system according to any one of embodiments 1 to 8, or a sgCNA subcomponent thereof, wherein the transcription factor is selected from a forkhead transcription factor, a nuclear receptor, a POU-domain protein, a SMAD, preferably Nrf2, FOX01, NF-kB, USF2, NFAT, EGR1, STAT3 and / or SREBP.

[0029] 10. The engineered non-naturally occurring system, or a sgCNA subcomponent thereof, or a sgCNA subcomponent thereof according to any one of embodiments 1 to 9, wherein the TF binding module comprises (i) at least one TF binding segment (TFBS), wherein the TF binding segment comprises DNA and / or RNA or (ii) wherein the TF binding module comprises at least one TF binding segment (TFBS), wherein the TF binding segment comprises a DNA aptamer or an RNA aptamer selected for binding to an endogenous transcription factor.

[0030] 11. The engineered non-naturally occurring system according to any one of embodiments 1 to 10, or a sgCNA subcomponent thereof, wherein the TF binding module comprises a sequence derived from a naturally occurring RNA.

[0031] 12. The engineered non-naturally occurring system according to any one of embodiments 1 to 11, or a sgCNA subcomponent thereof, wherein the TF binding segment (TFBS) comprises a double-stranded DNA segment comprising at least one TF response element.

[0032] 13. The engineered non-naturally occurring system according to any one of embodiments 1 to 12, or a sgCNA subcomponent thereof, wherein the strands of the DNA portion of the sgRNA form a duplex and are joined by a loop sequence of arbitrary length.

[0033] 14. The engineered non-naturally occurring system of embodiment 13, or a sgCNA subcomponent thereof, wherein the loop comprises four nucleotides.

[0034] 15. The engineered non-naturally occurring system according to any one of embodiments 13 and 14, or a sgCNA subcomponent thereof, wherein the sequence of the loop comprises 5'-guanosine-adenosine-adenosine-adenosine-3'.

[0035] 16. The engineered non-naturally occurring system of any one of embodiments 1 to 15, or a sgCNA subcomponent thereof, wherein the crRNA comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleobases that are at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a target nucleic acid sequence of a target gene of interest.

[0036] 17. The engineered non-naturally occurring system according to any one of embodiments 1 to 16, or a sgCNA subcomponent thereof, wherein the endonuclease-defective sequence-specific DNA binding protein (preferably, a dCas polypeptide) is fused to at least one copy of a nuclear localization signal.

[0037] 18. The engineered non-naturally occurring system according to any one of embodiments 1 to 17, or a sgCNA subcomponent thereof, wherein the crRNA comprises any one of the following sequences: SEQ ID NO: 6 to SEQ ID NO: 34.

[0038] 19. The engineered non-naturally occurring system according to any one of embodiments 1 to 18, or a sgCNA subcomponent thereof, wherein the transcription factor binding sequence comprises one or more sequences selected from SEQ ID NO: 1, 2, 3, 5 and those in Table 2.

[0039] 20. The engineered non-naturally occurring system of any one of embodiments 1 to 19, or a sgCNA subcomponent thereof, wherein the tracrRNA is capable of binding to dCas9.

[0040] 21. The engineered non-naturally occurring system according to any one of embodiments 1 to 20, or a sgCNA subcomponent thereof, wherein the crRNA, TFBS and tracrRNA of the PGM are assembled in any one of the following molecular arrangements:

[0041] 5′-crRNA-TFBS-tracrRNA-3′;

[0042] 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-3' (wherein the TFBS is integrated anywhere within the tracrRNA sequence, including as an extension of one or more hairpin structures thereof);

[0043] 5′-crRNA-tracrRNA-TFBS-3′;

[0044] 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-3’;

[0045] 5'-crRNA-TFBS-tracrRNA-TFBS-3';

[0046] 5’-crRNA-tracrRNA’-TFBS-tracrRNA”-TFBS-3’;

[0047] 5’-crRNA-TFBS-tracrRNA’-TFBS-tracrRNA”-TFBS-3’;

[0048] 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’;

[0049] 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’;

[0050] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-3’;

[0051] 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-TFBS-tracrRNA"'-TFBS-tracrRNA""-TFBS-3';

[0052] 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3';

[0053] 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3';

[0054] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’;

[0055] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’;

[0056] wherein tracrRNA', tracrRNA", tracrRNA"' and tracrRNA"' are consecutive segments of the complete tracrRNA sequence.

[0057] 22. The engineered non-naturally occurring system of any one of embodiments 1 to 21, or a sgCNA subcomponent thereof, wherein the PGM comprises one or more different TFBSs including response elements for multiple different transcription factors.

[0058] 23. The engineered non-naturally occurring system of any one of embodiments 1 to 22, or a sgCNA subcomponent thereof, wherein the PGM comprises a nucleic acid backbone having one or more different TFBSs, wherein the continuity of the nucleic acid backbone is disrupted at one or more positions and the complete nucleic acid sequence is assembled by base pairing of nucleotides from different chains.

[0059] 24. The engineered non-naturally occurring system of embodiment 23, or a sgCNA subcomponent thereof, wherein the discontinuity in the nucleic acid backbone is located within one or more TFBSs.

[0060] 25. The engineered non-naturally occurring system of any one of embodiments 1 to 24, or a sgCNA subcomponent thereof, wherein the TFBS is separated from the crRNA or tracrRNA by a linker having at least 1, 5, 10, 20 or 30 DNA, RNA or modified nucleotides.

[0061] 26. An isolated nucleic acid comprising any one or more of the sgCNA, crRNA, tracrRNA, transcription factor binding site, or any other segment of the sgCNA of the PGM of any one of embodiments 1 to 25; or encoding a DNA binding protein of the PGM of any one of embodiments 1 to 25.

[0062] 27. An isolated nucleic acid according to embodiment 26, wherein the nucleic acid contains one or more modified or non-natural nucleotides.

[0063] 28. The isolated nucleic acid of any one of embodiments 26 and 27, wherein the nucleic acid is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-200, 200-300, 300-400, or 400-500 bases in length.

[0064] 29. A vector comprising the isolated nucleic acid of any one of embodiments 26 to 28 under the control of a heterologous promoter, preferably wherein the vector is an AAV vector or another vector.

[0065] 30. A virus comprising the isolated nucleic acid of any one of embodiments 26 to 28, preferably wherein the virus is a lentivirus or an adenovirus.

[0066] 31. A cell comprising the PGM or sgCNA subcomponent thereof as described in any one of embodiments 1 to 25, and / or the nucleic acid as described in any one of embodiments 26 to 28, and / or the vector as described in embodiment 29, and / or the virus as described in embodiment 30.

[0067] 32. A cell according to embodiment 31, wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell.

[0068] 33. A composition comprising the PGM or sgCNA subcomponent thereof as described in any one of embodiments 1 to 25, the nucleic acid as described in any one of embodiments 26 to 28, the vector as described in embodiment 29, the virus as described in embodiment 30, the cell as described in embodiment 31, or a combination thereof.

[0069] 34. The composition according to embodiment 33, further comprising a cationic or ionizable lipid or a cationic or ionizable polymer, preferably in the form of nanoparticles.

[0070] 35. The composition according to any one of embodiments 33 and 34, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0071] 36. A method for reversibly altering the expression of a target gene of interest in a cell in response to one or more intracellular or extracellular environmental signals, comprising contacting the cell with a PGM or sgCNA subcomponent thereof as described in any one of embodiments 1 to 25, a nucleic acid as described in any one of embodiments 26 to 28, a vector as described in embodiment 29, a virus as described in embodiment 30, a composition as described in any one of embodiments 33 to 35, or a combination thereof.

[0072] 37. A method according to embodiment 36, wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell.

[0073] 38. A method of treating a disease, disorder or injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a PGM or sgCNA subfraction thereof as described in any one of embodiments 1 to 25, an isolated nucleic acid as described in any one of embodiments 26 to 28, a vector as described in embodiment 29, a virus as described in embodiment 30, a cell as described in any one of embodiments 31 and 32, a composition as described in any one of embodiments 33 to 35, or a combination thereof.

[0074] 39. The method of embodiment 38, wherein the disease, disorder or injury is selected from cellular stress, excisional or incisional wounds, radiation exposure, viral or bacterial infection, sepsis, diabetic nephropathy, atherosclerosis, cystic fibrosis, Alzheimer's disease, oxidative stress, ischemia-reperfusion injury, inflammation, cancer, anticancer resistance, genetic disease or any other proliferative disease or disorder, inflammatory disease or disorder, autoimmune disease or disorder, liver disease or disorder, spleen disease or disorder, lung disease or disorder, blood disease or disorder, nervous system disease or disorder, gastrointestinal (GI) tract disease or disorder, genitourinary system disease or disorder, infectious disease or disorder, musculoskeletal disease or disorder, endocrine disease or disorder, metabolic disease or disorder, immune disease or disorder, central nervous system (CNS) disease or disorder, nervous system disease or disorder, ophthalmic disease or disorder or cardiovascular disease or disorder.

[0075] 40. The method of embodiment 38, wherein the disease, disorder or injury is selected from excisional or incisional wounds, radiation exposure, viral or bacterial infection, sepsis, diabetic nephropathy, atherosclerosis, cystic fibrosis, Alzheimer's disease, oxidative stress, ischemia-reperfusion injury, inflammation and cancer.

[0076] 41. A kit comprising the PGM or sgCNA subcomponent thereof of any one of embodiments 1 to 25, the nucleic acid of any one of embodiments 26 to 28, the vector of embodiment 29, the virus of embodiment 30, the composition of any one of embodiments 33 to 35, or a combination thereof, and a container and / or instructions for using the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0078] Figure 1A : The principle of physiologically responsive gene expression regulators. Transcription factors (TFs) are activated via physiological stimuli. Examples of physiological stimuli include, but are not limited to, oxidative stress or growth factor signaling. Responsive gene expression regulators are ribonucleoprotein complexes comprising disabled CRISPR-associated proteins (such as dCas9) and chimeric guide nucleic acids. The chimeric guide nucleic acid comprises a DNA hairpin that incorporates a binding site, a crRNA sequence, and a tracrRNA sequence for the activated TF. The complex binds to the genomic DNA sequence adjacent to the target gene, causing the target gene to respond to physiological signals. The binding site is programmed by the crRNA sequence in the guide nucleic acid. The association of the activated TF with the bound dCas9 complex brings the TF close to the target gene, thereby regulating target gene transcription. Figure 1B :Schematic diagram of PGM action. Figure 1C : Description of the crRNA module, tracrRNA module and transcription factor binding site module.

[0079] Figure 2A : Schematic diagram of the conventional structure of sgRNA. Figure 2B An exemplary embodiment of a chimeric guide nucleic acid (sgCNA) synthetic annotation module included in accordance with the present disclosure is shown.

[0080] Figure 3 Various non-limiting examples of different applications of the PROTEGE platform are provided.

[0081] Figure 4A This exemplifies the need to turn on therapeutic genes at the right time and location. Figure 4B .Examples of how the PROTEGE platform enables controlled, reversible expression of therapeutic genes in response to injury or disease without altering the genome content.

[0082] Figures 5A-5D: Show that a physiologically responsive programmable gene regulator (PGM) recruits activated transcription factors to a target DNA sequence. 5A. Experimental design. The target DNA sequence is a 20 base pair sequence (pink and gold) contained in a DNA duplex fixed in a well of a multi-well plate. The gene regulator comprises a dCas9 (yellow circle) complexed with a single guide nucleic acid, which comprises a crRNA module (turquoise) complementary to the target sequence, a tracrRNA module (teal), and a DNA module forming a hairpin structure, the stem of which contains an Nrf2 response element (red). After the gene regulator binds to the fixed target DNA, nuclear extracts of HEK293 cells treated with tert-butylhydroquinone (tBHQ) are added to stimulate the activation and nuclear localization of Nrf2. After washing the wells to remove unbound Nrf2, bound Nrf2 was detected with an anti-Nrf2 antibody, treated with an HRP-conjugated anti-rabbit secondary antibody and developed with an HRP substrate, and visualized by absorbance at 450 nm. In Figures 5B-5D, each value is the average of three replicates in separate wells. The error bars are the standard deviation of the mean. Figure 5B. Dependence of Nrf2 binding on the presence of PGM. For the "-PGM" wells, PBS was added instead of the PGM solution. Figure 5C. Dependence of Nrf2 binding on the presence of a fixed target DNA sequence in the wells. In the "-target DNA sequence" wells, the target sequence was replaced with a fixed duplex containing a scrambled form of the target sequence (same sequence composition, different sequence). Figure 5D. Dependence of Nrf2 binding on Nrf2 activation. Nuclear extracts added to the "-Nrf2" wells were from cells that were not treated with tBHQ.

[0083] Figures 6A and 6B: Nrf2-dependent regulation of klotho transcription in cultured cells. Figure 6A. Human embryonic kidney cells were treated with PGMs targeting the klotho promoter and containing Nrf2 response elements. After 16 hours, Nrf2 was activated by tBHQ. Total RNA was isolated after another 24 hours, and klotho expression relative to GAPDH expression was measured by RT-qPCR. Figure 6B. Relative expression of klotho was normalized to expression without addition of PGM or tBHQ. Values ​​are means of three biological replicates and error bars are standard deviations. P values ​​were calculated from one-way ANOVA. DETAILED DESCRIPTION

[0084] In order that the present disclosure may be better understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0085] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0086] Unless stated otherwise or obvious from the context, as used herein, the term "or" should be construed as inclusive and encompasses both "or" and "and."

[0087] As used herein, the term "and / or" should be considered to specifically disclose each of the two features or components, with or without the other. Thus, the term "and / or" used in a phrase such as "A and / or B" is intended to include A and B; A or B; A (alone) and B (alone). Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0088] As used herein, the terms "such as" and "ie" are used by way of example only and not limitation, and should not be construed to refer to only those items explicitly listed in the specification.

[0089] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、1 03, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more than the values ​​shown. Also included are any larger numbers or fractions therebetween.

[0090] Conversely, the term "no more than" includes individual values ​​that are less than the indicated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0091] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included are any larger numbers or fractions therebetween.

[0092] Throughout the specification, the word "comprise" or variations thereof, such as "comprises / comprising" shall be understood to mean the inclusion of the indicated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. It shall be understood that any instances in which aspects are described as "comprising" are also provided for similar aspects described as "consisting of" and / or "consisting essentially of". "Consisting of" is a closed term and excludes any element, step or component not specified in the claim. In re Gray, 53 F.2d 520, 11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448, 450 (Bd.App. 1948) (“consisting of” is defined as “limiting the claim to materials other than the enumerated materials, except for impurities normally associated therewith”). The term “consisting essentially of” limits the scope of a claim to the specified substances or steps and “those which do not materially affect the basic and novel characteristics” of the claimed invention.

[0093] Unless otherwise specified or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend on the measurement or determination of the value or composition, i.e., the limits of the measurement system. For example, according to the practice of the art, "about" or "approximately" may refer to one or more than one standard deviation. "About" or "approximately" may represent a range of up to 10% (i.e., ±10%). Therefore, "about" may be understood as being greater than or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the value shown. For example, about 5 mg may include any amount from 4.5 mg to 5.5 mg. In addition, particularly with respect to biological systems or processes, these terms may represent up to an order of magnitude or up to 5 times a value. When a particular value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that "about" or "approximately" means within an acceptable error range for the particular value or composition.

[0094] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0095] The units, prefixes and symbols used herein are provided in their International System of Units (SI) accepted forms. Numerical ranges are inclusive of the numbers defining the range.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same common meanings as understood by those of ordinary skill in the art to which the present disclosure belongs. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd edition, (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th edition, (2013), Academic Press; and "Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al., eds., 2nd edition, (2006), Oxford University Press, provide a common dictionary of many terms used in the present disclosure to those skilled in the art.

[0097] The terms "transduction" and "transduced" refer to the process of introducing foreign DNA into cells by viral vectors (see Jones et al., "Genetics: principles and analysis" Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0098] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an "agent," a small molecule, a PGM described in the specification) is any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes regression of a disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of damage or disability caused by disease affliction. These terms can be used interchangeably. The ability of a therapeutic agent to promote regression of a disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by measuring the activity of the agent in in vitro assays. Therapeutically effective amounts and dosage regimens can be determined empirically by testing in known in vitro or in vivo (e.g., animal model) systems.

[0099] The term "combination" refers to a fixed combination in the form of a dosage unit, or a compound of the invention and a combination partner (e.g., another drug, also referred to as a "therapeutic agent" or "agent"), as explained below, can be administered independently simultaneously or separately within certain time intervals, particularly when these time intervals allow the combination partners to exhibit a synergistic effect (e.g., a synergistic effect). The individual components can be packaged as a kit or packaged separately. One or both components (e.g., powders or liquids) can be reconstituted or diluted to the desired dose prior to administration. The terms "co-administration" or "combined administration" and the like as used herein are intended to cover the administration of selected combination partners to a single subject (e.g., a patient) in need thereof, and are intended to include treatment regimens in which the agents need not be administered by the same route of administration or at the same time.

[0100] The term "genetically engineered" or "engineered" refers to a process of modifying the genome of a cell including, but not limited to, deletion of coding or non-coding regions or portions thereof, or insertion of coding regions or portions thereof.

[0101] As used herein, the terms "homologous", "homology" or "percent homology" refer to the degree of sequence identity between an amino acid or polynucleotide sequence and a corresponding reference sequence. "Homology" can refer to polymer sequences, such as polypeptides or DNA sequences, that are similar. Homology can refer, for example, to nucleic acid sequences having at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In other embodiments, a "homologous sequence" of a nucleic acid sequence can exhibit 93%, 95% or 98% sequence identity relative to a reference nucleic acid sequence. For example, a "region homologous to a genomic region" can be a DNA region having a similar sequence to a given genomic region in a genome. The homology region can have any length sufficient to promote the binding of the spacer or protospacer sequence to the genomic region. For example, the homology region can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 00, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases so that the homologous region has enough homology to bind to the corresponding genomic region. When stating the percentage of sequence homology or identity, in the case of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the comparison of two or more sequences over a portion of their length when the maximum correspondence is compared and compared. When a position in the compared sequence can be occupied by the same base or amino acid, the molecule can have homology at that position. Unless otherwise stated, sequence homology or identity is evaluated over a specified length of a nucleic acid, polypeptide or portion thereof. In some embodiments, homology or identity is assessed over a functional portion or a specified portion of length.Sequence alignments for assessing sequence homology can be performed by algorithms known in the art, such as the Base Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410, 1990.Internet interfaces for performing BLAST analyses are publicly available from the National Center for Biotechnology Information. Other known algorithms include those published in Smith and Waterman, "Comparison of Biosequences", Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol. 48:443, 1970; Pearson and Lipman "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci. USA 85:2444, 1988; or by automated implementations of these or similar algorithms. Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277)), and the GGSEARCH program fasta.bioch.virginia.edu / fasta_www2 / , which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both programs are based on the Needleman-Wunsch algorithm, which is used to find the best alignment of two sequences along their entire length (including gaps). A detailed discussion of sequence analysis is also found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology", John Wiley & Sons, 1994-1998, Chapter 15, 1998). The skilled artisan understands that the amino acid (or nucleotide) position in homologous sequences can be determined based on alignment.

[0102] As used herein, "patient" or "subject" includes anyone suffering from a disease or condition. The terms "subject" and "patient" are used interchangeably herein. The "subject" considered to be given refers to a person (i.e., a male or female of any age group, such as a pediatric subject (e.g., an infant, a child, or a teenager) or an adult subject (e.g., a youth, a middle-aged adult, or the elderly)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey) or a mouse). The term "patient" refers to an object that needs to be treated for a disease, condition, or injury. In some embodiments, the object is a person. In some embodiments, the patient is a person. The person can be a male or female at any stage of development. Objects or patients that "need" to treat a disease, condition, or injury include, but are not limited to, objects or patients that exhibit any risk factors or symptoms of a disease, condition, or injury. In some embodiments, the object is a non-human experimental animal (e.g., a mouse, a rat, a dog, or a pig).

[0103] As used herein, the term "in vitro cell" refers to any cell cultured in vitro. Specifically, the in vitro cell can be a eukaryotic cell or a prokaryotic cell. The term "in vivo" refers to within a patient's body.

[0104] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids comprising a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to two short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and also to long chains, which are commonly referred to in the art as proteins, including many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0105] As used herein, "tissue" is a group of cells and their extracellular matrix from the same source. Cells perform specific functions together. Various tissue types are linked together to form organs. Cells can be different cell types. In some embodiments, the tissue is epithelial tissue. Epithelial tissue is formed by cells covering the surface of organs (e.g., skin surface, respiratory tract, soft organs, reproductive tract and digestive tract lining). Epithelial tissue plays a protective function and is also involved in secretion, excretion and absorption. Examples of epithelial tissue include, but are not limited to, simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified epithelium, columnar epithelium and glandular epithelium. In some embodiments, the tissue is connective tissue. Connective tissue is fibrous tissue composed of cells separated by non-living substances (e.g., extracellular matrix). Connective tissue provides shape to organs and fixes them in place. Connective tissue includes fibrous connective tissue, skeletal connective tissue and liquid connective tissue. Examples of connective tissue include, but are not limited to, blood, bone, tendon, ligament, fat and areolar tissue. In some embodiments, the tissue is muscle tissue. Muscle tissue is an actively contractile tissue formed by muscle cells. The function of muscle tissue is to generate force and cause movement. Muscle tissue includes smooth muscle (e.g., present in the inner wall of an organ), skeletal muscle (e.g., usually attached to bones) and cardiac muscle (e.g., present in the heart, which contracts to pump blood to various parts of the organism). In some embodiments, the tissue is nervous tissue. Nervous tissue includes cells that make up the central nervous system and the peripheral nervous system. Nervous tissue forms the brain, spinal cord, cranial nerves and spinal nerves (e.g., motor neurons). In certain embodiments, the tissue is brain tissue. In certain embodiments, the tissue is placental tissue. In some embodiments, the tissue is cardiac tissue.

[0106] The terms "treat / treat", "treat / treat" and "treating / treating" refer to reversing, alleviating, delaying the onset of a disease described herein, or inhibiting the progression of a disease described herein. In some embodiments, treatment or treatment may be given after one or more signs or symptoms of a disease have appeared or been observed, or when suspected of being ill or at risk of a disease (e.g., prophylactically (as further described herein)). In other embodiments, treatment or treatment may be performed in the absence of signs or symptoms of the disease. For example, treatment may be given to a susceptible subject before the onset of symptoms (e.g., based on the subject's symptom history, or a family member of the subject). Treatment may also be continued after symptom relief, such as to delay or prevent its recurrence. In some embodiments, treatment may be given after using the methods disclosed herein and observing that the spatiotemporal gene expression of one or more nucleic acids of interest in a cell or tissue has changed compared to a healthy cell or tissue or a tissue not altered by the methods disclosed herein. The term "treat / treat" may also refer to the restoration of cells to a physiological state, and encompasses the reversal of cell stress, the prevention of cell death, the restoration to normal growth, and the like.

[0107] As used herein, the terms "tumor", "cancer" and "neoplasm" refer to abnormal tissue masses, wherein the growth of the mass exceeds that of normal tissue and is not coordinated with the growth of normal tissue. Tumors may be "benign" or "malignant", depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis. "Benign tumors" are usually well differentiated, characteristically grow slower than malignant tumors, and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade or metastasize to distal sites. Exemplary benign tumors include, but are not limited to, lipomas, chondromas, adenomas, skin tags (acrochordon), senile angiomas (senile angiomas), seborrheic keratoses (seborrheic keratoses), lentigos (lentigos) and sebaceous hyperplasia (sebaceous hyperplasias). In some cases, certain "benign" tumors may subsequently develop into malignant tumors, which may be caused by additional genetic changes in a subpopulation of tumor cells of the tumor, and these tumors are referred to as "premalignant tumors". An exemplary premalignant tumor is a teratoma. Differently, "malignant tumors" are usually poorly differentiated (degenerative) and have the characteristics of rapid growth, accompanied by progressive infiltration, invasion and destruction of surrounding tissues. In addition, malignant tumors usually have the ability to transfer to distant sites. The terms "metastasis", "metastatic" or "metastasis to" refer to the spread or migration of cancer cells from a primary or original tumor to another organ or tissue, and can usually be identified by the presence of "secondary tumors" or "secondary cell clusters" of the tissue type of the primary or original tumor, rather than the tissue type of the organ or tissue where the secondary (metastasis) tumor is located. For example, prostate cancer that has migrated to the bone is called metastatic prostate cancer, and includes cancerous prostate cancer cells that grow in bone tissue.

[0108] PROTEGE Platform: Programmable Gene Regulators

[0109] In one embodiment, the present disclosure provides a platform for rationally generating therapeutic measures that utilize beneficial genes that respond to disease or injury only in cells where a therapeutic response is desired. In one embodiment, the platform uses a molecular device, a programmable gene regulator ("PGM"), to recruit transcription factors that respond to the physiological conditions of the disease or injury of the selected therapeutic gene. PGMs for a given therapeutic target can be designed based on base pairing rules, known transcription factor binding DNA sequences, and known genomic sequences.

[0110] In one embodiment, by using PGMs, there is no need to edit the genome of the cell, as the systems and methods can utilize existing genetic material and metabolism and overcome safety issues associated with gene therapy and gene editing. Furthermore, the effects here are limited to the relevant cell population in a relevant physiological environment, so any off-target effects can be minimized. Furthermore, the modular programmability of the systems and methods can be applied to different gene targets and physiological actuators to address a range of injuries, diseases, and cell types. Examples of the use of this platform, referred to herein as Protege, can be found in Figure 1A and 1B The target gene is programmed by the sequence of the crRNA component of the guide nucleic acid, and the one or more transcription factors to which the transcriptional regulation responds are programmed by one or more transcription factor response elements incorporated into the guide nucleic acid.

[0111] In one embodiment, the novel PGM can function by recruiting endogenous transcription factors to the promoter region of the target gene. While existing designs of artificial transcription factors rely on the co-delivery of a module that affects gene transcription and a module that recognizes the promoter of the target gene, the disclosed method provides versatility and control by utilizing transcription factors already present in the cell. Figure 1A and 1B An overview of a possible embodiment of a programmable gene regulation platform is shown. As shown, transcription factors may be activated by physiological stimuli (such as oxidative stress or growth factor signaling). In some embodiments, the PGM comprises a ribonucleoprotein complex, which comprises a disabled CRISPR-associated protein (e.g., dCas9) and a single guide chimeric nucleic acid (sgCNA), which comprises a DNA hairpin, which incorporates a binding site for activating TF, a crispr ("cr") RNA sequence, and a trans-activating CRISPR ("tracr") RNA sequence. The crRNA sequence is a sequence complementary to the target DNA, which can generally be 17-20 nucleotides long. The tracrRNA sequence serves as a binding scaffold for the Cas protein. The complex binds to the genomic DNA sequence adjacent to the target gene, so that the target gene responds to physiological signals. The binding site is programmed by the crRNA sequence in the guide nucleic acid. The association of the activated TF with the bound dCas9 complex brings TF close to the target gene, thereby regulating target gene transcription. In one embodiment, transcription is activated or enhanced. In other embodiments, transcription can be inhibited or reduced. In one embodiment, the advantage of a design in which a DNA hairpin caps an existing hairpin structure in a parent guide RNA (rather than appending to the end) is that it provides a cohesive double-stranded site for ligation, facilitating modular synthesis as shown ( Figure 1C), which allows for easy “mixing and matching” of genomic targets (defined by the crRNA module) and transcription factors (defined by the transcription factor binding module). One does not have to resynthesize everything to change modules.

[0112] therefore, Figure 2A is a schematic diagram of the conventional structure of sgRNA, and Figure 2B 2C shows an exemplary embodiment of a chimeric guide nucleic acid synthesis, annotation module included in the present disclosure. Figure 2B In the sgRNA shown in FIG2C , the chimeric guide nucleic acid DNA hairpin caps a different hairpin than the sgRNA from which the sgCNA shown is derived.

[0113] Therefore, in one embodiment, the present disclosure provides a PGM comprising two modules: (i) a genomic DNA binding module defining a target gene and (ii) a transcription factor binding module defining a transcription factor to be recruited. In some embodiments, the transcription factor binding module is a DNA duplex comprising a consensus binding sequence of a transcription factor to be connected to a therapeutic gene. From the perspective of the entire molecule, the PGM comprises a Cas protein and a single guide chimeric nucleic acid (sgCNA) containing a crRNA sequence, a tracrRNA sequence, and a transcription factor binding site. For the purpose of synthesis, the crRNA and tracrRNA sequences can be flanked to facilitate T4 DNA ligase to connect the target DNA sequence. The crRNA / DNA fragment of sgCNA is referred to herein as the Cr RNA module. The tracrRNA / DNA fragment of sgCNA is referred to herein as the Tracr RNA module. The transcription factor binding site is also surrounded by an additional DNA sequence to form a hairpin duplex. The hairpin duplex fragment is referred to herein as a transcription factor binding module. See Figure 1B In one embodiment, each of the three modules is synthesized separately and then linked to form a chimeric sgCNA molecule.

[0114] Genomic DNA binding module

[0115] In one embodiment, the genomic DNA binding functional module comprises a nuclease-deficient Cas protein and a single guide chimeric nucleic acid (sgCNA) component that allows binding to the target DNA, especially the RNA element of the sgCNA. The genomic DNA binding module is designed to bind to the genomic DNA near the target therapeutic gene under natural conditions. When the transcription factor is stimulated (e.g., hypoxia) and activated, the PGM will bind to the activated transcription factor and deliver it to the target gene, thereby regulating the expression of the gene.

[0116] In one embodiment, the DNA binding functional module comprises two RNA parts of a chimeric guide nucleic acid, wherein the chimeric guide nucleic acid comprises a crRNA sequence and a tracrRNA sequence. In one embodiment, a transcription factor binding module, a DNA or RNA segment, or a modified nucleic acid folded into a hairpin duplex, can be inserted between the crRNA and tracrRNA sequences so that it does not interfere with the function of the guide RNA of the DNA binding module to recognize the target DNA. In one embodiment, the chimeric guide nucleic acid is synthesized by connecting three modules: a Trac module, a Cr module, and a TF binding module. See, for example Figure 1C RNA nucleotides are shown in bold blue and green, and DNA nucleotides are shown in black.

[0117] In one embodiment, the Trac and Cr modules comprise RNA and DNA segments. In one embodiment, the DNA segments are complementary to each other and to the 3' overhang of the TF binding module, and the 5' ends of the Trac and TF binding modules are phosphorylated to allow the Trac and Cr modules to be connected to the TF binding module. In one embodiment, the DNA segments on the Trac and Cr modules are long enough so that the three modules comprise a substrate for T4 DNA ligase.

[0118] In one embodiment, the DNA segment comprises the sequence 5'-ACCCTGACTTGACGT-3' (SEQ ID NO: 75) for the crRNA module and the sequence 5'-AAGTCAGGGT-3' (SEQ ID NO: 76) for the tracrRNA module. In one embodiment, the module is prepared by conventional solid phase oligonucleotide synthesis and purified by polyacrylamide gel electrophoresis. Similarly, since T4 DNA ligase cannot efficiently connect RNA to DNA, the cr, tracr and transcription factor binding components of the sgCNA assembled by connecting with T4 DNA ligase may require adapter / joint segments attached to the cr and tracr components. It is obvious to those skilled in the art that many different joint segment sequences will be effective. The DNA joint segment should be at least partially complementary and should form a duplex with an overhang of at least one nucleotide, preferably at least four nucleotides, when hybridized. The overhang can be paired with a complementary overhang base in the DNA duplex at the connection site with the DNA transcription factor binding component of the sgCNA. The 5' or 3' end of the transcription factor binding component can be a concave end of the overhang. Any transcription factor binding module sequence with an overhang complementary to the overhang formed by the DNA segment of the Cr and Tracr modules can be connected, so that the same Cr and Tracr modules can be used together with different TF binding modules. The connection site on each chain can be at least 5 nucleotides, preferably at least 10 nucleotides, of the RNA nucleotides of the cr and tracr components from the sgCNA ligation reaction. Although many sequences can be used for DNA linker segments, the selected sequences should ensure that they do not have significant internal base pairing or form other internal structures (such as G quadruplexes) within a linker segment or between the crRNA or tracrRNA components to which they are attached. This requirement can be determined by inspection or by using nucleic acid folding tools known to those skilled in the art. The mfold program is an example of such a tool.

[0119] In some embodiments, the crRNA comprises an RNA sequence complementary to a nucleic acid sequence in a promoter region of a gene of interest, and the transcription factor binding sites in the PGM are each capable of binding to at least one endogenous transcription factor, which is activated in response to one or more environmental signals in the cell, and then recognizes and binds to the transcription factor binding site of the PGM, which is bound to the promoter of the gene of interest by the crRNA, thereby bringing the transcription factor close to the gene of interest and responding to one or more environmental signals to activate or inhibit the expression of the gene of interest. In one embodiment, the target gene and crRNA sequences are selected from those in Table 1.

[0120] Table 1: Exemplary target genes and corresponding exemplary crRNA sequences

[0121]

[0122]

[0123]

[0124] In one embodiment, the DNA binding module comprises a ribonucleoprotein complex, which further comprises a CRISPR-associated protein (such as Cas9) that has been mutated to eliminate its DNA cleavage activity. In one embodiment, tracrRNA binds to dCas9. In another embodiment, tracrRNA binds to any other nuclease-deficient DNA binding protein (DNAbp). In some embodiments, DNAbp is selected from nuclease-deficient Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Argu Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j and Argonaute.

[0125] Transcription factor binding module

[0126] In one embodiment, when endogenous transcription factors are activated in response to environmental signals, the PGM recruits endogenous transcription factors to genes of interest, thereby regulating gene expression in response to environmental signals in a cell-specific manner. In one embodiment, the PGM comprises at least one TFBM / TFBS. The use of two or more TFBS in the same PGM can be used to increase specificity or activity. In one embodiment, the TF binding module is a DNA hairpin incorporating one or more TF binding sequences (TFBS) into the double-stranded sequence of DNA. In one embodiment, the loop sequence of the hairpin is an abnormally stable GAAA tetraloop, which promotes the correct folding of the hairpin and the full guide nucleic acid. In one embodiment, the 3' overhang and 5' phosphate (5'P) allow the module to be connected to the Trac and Cr modules.

[0127] In one embodiment, at least one TFBS in the PGM is also present in the target gene. In one embodiment, at least one TFBS in the PGM is not an endogenous TFBS in the target gene. In one embodiment, the transcription factor is selected from forkhead transcription factors, nuclear receptors, POU domain proteins, SMADs, preferably Nrf2, FOX01, NF-kB, USF2, NFAT, EGR1, STAT3 and SREBP. In one embodiment, the transcription factor is Nrf2. In one embodiment, the transcription factor is selected from those listed in Table 2.

[0128] Table 2: Exemplary transcription factor binding sites

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] In one embodiment, the transcription factor is selected from those listed in public transcription factor databases (such as TRRUST database and Dorothea database). In one embodiment, the specific sequence (also referred to as TF motif) bound by TF can be selected from TF motif databases such as JASPAR, HOCOMOCO, CIS-BP, etc. (see, e.g., Stormo, GD (2015). DNA motif databases and their uses. Current Protocols in Bioinformatics, 51, 2.15.1–2.15.6). These motifs can also be used to predict TFBS in the genome using tools such as PWMscan (Ambrosini, G., Groux, R., and Bucher, P. (2018). PWMScan: A fast tool for scanning entire genomes with a position-specific weight matrix. Bioinformatics, 34, 2483–2484), or MOODS (Korhonen, J., Martinmaki, P., Pizzi, C., Rastas, P., and Ukkonen, E. (2009). MOODS: fast search for positionweight matrix matches in DNA sequences. Bioinformatics, 25(23), 3181–3182). In addition, recent advances in TF mapping techniques combined with deep learning algorithms for predicting TF binding sites have been successfully used to predict direct binding of some TFs (Avsec, Weilert, M., Shrikumar, A., Krueger, S., Alexandari, A., Dalal, K., Fropf, R., McAnany, C., Gagneur, J., Kundaje, A., and Zeitlinger, J. (2021). Base-resolution models of transcription-factor binding reveal soft motif syntax. Nature Genetics, 53, 354–366. In addition, TF profiles for thousands of cell types are available (e.g., Moore, JE, Purcaro, MJ, Pratt, HE, Epstein, CB, Shoresh, N., Adrian, J., Kawli, T., Davis, CA, Dobin, A., Kaul, R., Halow, J., van Nostrand, EL, Freese, P., Gorkin, DU, Shen, Y., He, Y., Mackiewicz, M., Pauli-Behn, F., Williams, BA…Weng, Z. (2020). Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature, 583, 699–710), and databases collecting experimentally measured TF binding sites (e.g., REMAP, ChIP-Atlas, or GTRD) can be used to select TF binding in specific cell types.

[0136] In one embodiment, the TF is selected from those listed in Table 3.

[0137] Table 3: Exemplary transcription factors of PGMs of the present disclosure

[0138]

[0139]

[0140] In one embodiment, the TF binding site (TFB / TFBS) is separated from the loop by eight base pairs to ensure that the structure of the TF binding site does not deviate from its native TF binding conformation. In some embodiments, the PGM comprises more than one TF binding site.

[0141] In one embodiment, the PGM modules are assembled into any of the following configurations:

[0142] 5′-crRNA-TFBS-tracrRNA-3′;

[0143] 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-3' (wherein the TFBSD is integrated anywhere within the tracrRNA sequence, including as an extension of one or more hairpin structures thereof);

[0144] 5′-crRNA-tracrRNA-TFBS-3′;

[0145] 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-3’;

[0146] 5'-crRNA-TFBS-tracrRNA-TFBS-3';

[0147] 5’-crRNA-tracrRNA’-TFBS-tracrRNA”-TFBS-3’;

[0148] 5’-crRNA-TFBS-tracrRNA’-TFBS-tracrRNA”-TFBS-3’;

[0149] 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’; 5’-crRNA-TFBS-tracrRNA’-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’;

[0150] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-3’;

[0151] 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-TFBS-tracrRNA"'-TFBS-tracrRNA""-TFBS-3';

[0152] 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3';

[0153] 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3';

[0154] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’;

[0155] 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’;

[0156] In these configurations, tracRNA', tracrRNA", tracrRNA"' and tracrRNA"' are contiguous segments of the complete tracrRNA sequence. The terms TB binding site and TFBS and TFB are used interchangeably.

[0157] PGM Delivery

[0158] In one embodiment, PGM or its individual components (i.e., protein components, sgRNA components) are delivered to the subject via the intestine. In one embodiment, PGM or its individual components are delivered to the subject parenterally. In one embodiment, PGM or its individual components are delivered locally. In one embodiment, PGM or its individual components are delivered locally, subcutaneously, intraocularly, intravitreally, subretinaly, intravenously (IV), intraventricularly (intracerebro-ventricularly), intramuscularly, intrathecally (IT), intracisternally, intraperitoneally by inhalation or direct injection into one or more cells, tissues or organs. In some embodiments, PGM delivery targets specific tissues or cell types. In one embodiment, PGM is delivered to cells via nucleic acid transfection (including electroporation, liposome delivery, etc.) or viral transduction. In some embodiments, PGM is delivered with lipid nanoparticles. In other embodiments, PGM is delivered with liposomes. In other embodiments, PGM is delivered with polymer nanoparticles (such as polymers, dendrimers, polymer micelles or polymer nanospheres). In other embodiments, the PGM is delivered using inorganic nanoparticles, such as silica nanoparticles, iron oxide nanoparticles, or gold nanoparticles.

[0159] In one embodiment, PGM is delivered by cell-penetrating peptides, chemical moieties or cell type-specific peptide delivery agents (including antibodies and peptides derived from combinatorial libraries, and peptides for selective internalization and / or subcellular localization found by phage display bio-panning) that mediate cellular uptake by binding to one or more receptors on the cell surface. In one embodiment, PGM is delivered together with peptides for selective internalization and / or subcellular localization found by phage display bio-panning, wherein the molecular guidance system platform described in PCT International Publications WO2019014199, WO2019014190, and WO2021066931 is used. In one embodiment, a peptide or peptide derivative that mediates cell-specific uptake of a binding carrier, such as a peptide found by phage display bio-panning for selective internalization and / or subcellular localization, is delivered to the relevant cell type. In one such embodiment, PGM is encapsulated in a lipid nanoparticle or liposome, which displays a cell-selective peptide or peptide derivative on its surface. Lipid nanoparticles or liposomes of various preparations can be used in this embodiment, including carrying polyethylene glycol on its surface to minimize immunogenicity. In one embodiment, the lipid nanoparticles can contain cationic or ionizable lipid compounds to complex with negatively charged PGM and help endosomal escape. In another embodiment, the cell type selective peptide or peptide derivative is directly coupled to the PGM by coupling with a protein component or a guide RNA component.

[0160] Exemplary applications of the PROTEGE platform

[0161] In addition to being useful for modulating gene expression in vitro, the PROTEGE platform can be used to treat any disease or condition that would benefit from upregulation or downregulation of specific target gene expression. Figure 3 Various non-limiting examples of different applications of the PROTEGE platform are provided. PGMs are designed to regulate gene expression in response to one or more intracellular or extracellular environmental signals. In one embodiment, the environmental signal is a physiological signal. In one embodiment, the environmental signal is associated with a pathological condition of disease, cell stress and / or injury. In one embodiment, the signal is an intrinsic signal such as a signal associated with development and differentiation.

[0162] In one embodiment, the signal is a physiological signal. In one embodiment, the signal is a light signal (e.g., UV light), ionizing radiation, heat / temperature, hypertonic or hypotonic conditions. In some embodiments, the signal is a mechanical signal. In some embodiments, the signal is selected from pressure (e.g., touch), acoustic motion, and blood pressure. In one embodiment, the signal is a chemical signal. In some embodiments, the chemical signal is a growth factor, a cytokine, a chemokine, cyclic AMP, a hormone, a neurotransmitter, an extracellular matrix component, a bacterial antigen, a viral antigen, a lipopolysaccharide, a gas level (e.g., oxygen level, nitric oxide level), an ion level (e.g., calcium level, sodium level), pH, reactive oxygen species, heavy metals, oxidized LDL, free radicals. In one embodiment, the signal is sensed by a receptor. In some embodiments, the receptor is an intracellular receptor (e.g., cytoplasm, nucleus). In some embodiments, the receptor is a cell surface / extracellular / transmembrane receptor. In some embodiments, the membrane receptor is selected from a G protein-coupled receptor, an ion channel receptor, and an enzyme-linked receptor. In some embodiments, the signal triggers a signal transduction cascade. In some embodiments, the signal transduction cascade triggers the activation of transcription factors to regulate gene expression. In some embodiments, the receptor is the transcription factor itself, such as the nuclear receptor of a fat-soluble ligand (e.g., steroid hormone). In an example, the receptor / transcription factor is an estrogen receptor or a glucocorticoid receptor, which resides in the cytoplasm until it is combined with its ligand to allow translocation to the nucleus and express the target gene.

[0163] The non-limiting examples of the well-known signal cascades leading to TF activation are TGFβ signaling leading to SMAD family TF activation, Jak-STAT signaling of STAT TF activation, Erbb2 signaling of Jun and Myc activation, Hippo signaling targeting TF families (TEAD1-TEAD4) containing TEA domains (TEAD), and Notch signaling of RBPJ inducing DNA binding to dissociate from the corepressor complex and recruit coactivator factor complexes. The example of TF inactivated by signaling includes the FOXO family, which is a subclass of forkhead box TF. In the absence of insulin, FOXO TF binds to DNA and activates gene expression. When insulin is present, FOXO TF is phosphorylated by kinases downstream of the PI3K-AKT signaling pathway, causing TF to be excluded from the nucleus, thereby inhibiting its target genes.

[0164] In one embodiment, the signal is associated with a physiological condition. In one embodiment, the signal is associated with a disease, cellular stress and / or injury such as wound healing, radiation exposure, viral or bacterial infection, sepsis, diabetic nephropathy, atherosclerosis, cystic fibrosis, Alzheimer's disease, oxidative stress, ischemia-reperfusion injury, inflammation, cancer, anticancer agent resistance, genetic disease, or any other proliferative disease or condition, inflammatory disease or condition, autoimmune disease or condition, liver disease or condition, spleen disease or condition, lung disease or condition, blood disease or condition, nervous system disease or condition, gastrointestinal (GI) tract disease or condition, genitourinary disease or condition, infectious disease or condition, musculoskeletal disease or condition, endocrine disease or condition, metabolic disease or condition, immune disease or condition, central nervous system (CNS) disease or condition, nervous system disease or condition, ophthalmic disease or condition, or cardiovascular disease or condition.

[0165] In one embodiment, the anticancer agent whose drug resistance leads to the signal of activating transcription factor covers biological therapeutic anticancer agent and chemotherapeutic agent.Exemplary biological therapeutic anticancer agent includes but is not limited to interferon, cytokine (for example, tumor necrosis factor, interferon alpha, interferon g), vaccine, hematopoietic growth factor, monoclonal serum therapy, immunostimulant and / or immunomodulator (for example, IL-1, 2, 4, 6 or 12), immune cell growth factor (for example, GM-CSF) and antibody (for example, HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)). Exemplary chemotherapeutic agents include, but are not limited to, antiestrogens (e.g., tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g., goserelin and leuprolide), antiandrogens (e.g., flutamide and bicalutamide), photodynamic therapy (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A (demethoxy- A) (2BA-2-DMHA), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU)), alkyl sulfonic acids (e.g., busulfan and treosulfan), triazines (e.g., dacarbazine, temozolomide), platinum-containing compounds (e.g., cisplatin, carboplatin, and oxaliplatin), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g.,Paclitaxel or paclitaxel equivalents such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamic acid-bound paclitaxel (PG-paclitaxel, paclitaxel polyglutamic acid, CT-2103, XYOTAX), tumor activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three paclitaxel molecules), paclitaxel-EC-1 (bound to erbB 2 recognition peptide EC-1) and glucose-coupled paclitaxel, such as 2'-paclitaxel methyl 2-pyranosyl glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), antimetabolites, DHFR inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., 5-fluorouracil (5-FU), floxuridine, deoxyfluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g., cytarabine), C), cytosine arabinoside and fludarabine), purine analogs (e.g., mercaptopurine and thioguanine), vitamin D3 analogs (e.g., EB 1089, CB 1093 and KH1060), prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., l-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g.,Bleomycin A2, Bleomycin B2, Peplomycin), anthracyclines (e.g., daunombicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zombicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca, 2+ ATPase inhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN TM , AZD2171), dasatinib ( BMS-354825), erlotinib Gefitinib Imatinib CGP57148B, STI-571), lapatinib Lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib Semaxanib, (semaxiib, SU5416), sunitinib ( SU11248), toceranib Vandetanib ( ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab Bevacizumab Rituximab Cetuximab Panitumumab Ranibizumab Nilotinib Sorafenib Everolimus Alemtuzumab Gemtuzumab ozogamicin Temsirolimus ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK TM ), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647 and / or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors ( For example, rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Novartis), XL765 (Sanofi Aventis), Aventis), PF-4691502 (Pfizer), GDC0980 (Genentech), SF1126 (Semafoe), and OSI-027 (OSI), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone one), dexamethasone, camptothecin, plicamycin, asparaginase, aminopterin, methotrexate, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethyl melamine.

[0166] In one embodiment, PGMs are used to treat "autoimmune diseases or conditions," which refers to diseases or conditions caused by an inappropriate immune response of the subject's body against substances and tissues normally present in the body. In other words, the immune system mistakes some parts of the body for pathogens and attacks its own cells. This dysfunction may be limited to certain organs (e.g., autoimmune thyroiditis) or involve specific tissues in different parts (e.g., Goodpasture's disease, which may affect the basement membranes of the lungs and kidneys). Treatment of autoimmune diseases often employs immunosuppression, such as drugs that reduce the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjögren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, cardiomyopathy.

[0167] In one embodiment, PGMs are used to treat "cancer," which refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl, ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, angiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); breast); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchial cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial cancer; ependymoma; endothelial sarcoma (e.g., Kaposi sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familial eosinophilia hypereosinophilia); gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancer (e.g., leukemias such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML) and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL));Lymphomas such as Hodgkin's lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin's lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia (aldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphocytic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome (Sezary syndrome syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemias / lymphomas as described above; and multiple myeloma (MM), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumor; immune cell amyloidosis; renal cancer (e.g., Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); myoma; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative diseases (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloid metaplasia (agnogenicmyeloid metaplasia) (AMM) also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma;Neurofibromas (e.g., neurofibromatosis (NF) type 1 or 2, schwannomas); neuroendocrine cancers (e.g., gastroenteropancreatic neuroendocrine tumors (GEP-NETs), carcinoid tumors); osteosarcomas (e.g., bone cancer); ovarian cancers (e.g., cystadenocarcinomas, ovarian embryonal carcinomas, ovarian adenocarcinomas); papillary adenocarcinomas; pancreatic cancers (e.g., pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), islet cell tumors); penile cancers (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumors (PNTs); plasma cell neoplasms; paraneoplastic syndromes syndromes); intraepithelial neoplasia; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small intestine cancer (e.g., appendix cancer); soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). ;

[0168] In one embodiment, PGMs are used to treat "genetic diseases or conditions," which refers to diseases or conditions caused by one or more abnormalities in the subject's genome, such as diseases that are present from birth. Genetic diseases or conditions may be hereditary and may be inherited from the genes of a parent. Genetic diseases or conditions may also be caused by mutations or changes in the subject's DNA and / or RNA. In this case, if the genetic disease or condition occurs in the germline, the genetic disease or condition will be heritable. Exemplary genetic diseases or disorders include, but are not limited to, Aarskog-Scott syndrome, Aase syndrome, achondroplasia, acroostosis, addiction, adrenoleukodystrophy, albinism, ablepharon-macrostomia syndrome, alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, Alport's syndrome, Alzheimer's disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten disease, Beckwith-Wiedemann syndrome, Best syndrome, disease, bipolar disorder, brachydactyly, breast cancer, Burkitt's lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn's disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob diseasedisease), cystic fibrosis, deafness, depression, diabetes, disabling dysplasia, DiGeorge syndrome, Down syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal dysplasia, Ehlers-Danlos syndrome, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Lriedreich's ataxia, Gaucher disease, glaucoma, glucose-galactose malabsorption, glutaric aciduria, gyrate atrophy atrophy, Goldberg Shprintzen syndrome (velocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary nonpolyposis colorectal cancer (HNPCC), Huntington's disease, immunodeficiency with hyper-IgM, juvenile onset diabetes mellitus, Klinefelter's syndrome, Kabuki syndrome, Leigh's disease, long QT syndrome, lung cancer, malignant melanoma, manic depression, Marfan syndrome syndrome, Menkes syndrome, miscarriage, mucopolysaccharidosisdisease), multiple endocrine tumors, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson's disease, aroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsum disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida bifida, spinal muscular atrophy, spinocerebellar atrophy, sudden adult death syndrome, Tangier disease, Tay-Sachs disease, thrombocytopenia absentradius syndrome, Townes-Brocks syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, vonHippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Wemer syndrome, Williams syndrome, Wilson's disease, xeroderma piginentosum, and Zellweger syndrome.

[0169] In one embodiment, PGM is used to treat "blood diseases or conditions", which include diseases or conditions that affect hematopoietic cells or tissues. Blood diseases or conditions include diseases or conditions associated with abnormal blood content and / or function. Examples of blood diseases or conditions include diseases caused by bone marrow radiation or chemotherapy for cancer, diseases such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections (such as malaria, trypanosomiasis, HIV, hepatitis viruses or other viruses), osteomyelitis anemia caused by bone marrow deficiency, renal failure resulting from anemia, and other diseases. anemia), anemia, polycythemia, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilms tumor, Ewing's sarcoma, retinoblastoma, hemophilia, diseases associated with an increased risk of thrombosis, herpes, thalassemia, antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical damage to red blood cells such as microangiopathic hemolytic anemia, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, parasitic infections such as Plasmodium, chemical injuries such as lead poisoning, and hypersplenism.

[0170] In one embodiment, PGM is used to treat "inflammatory diseases or disorders" and "inflammatory disorders", which are used interchangeably herein to refer to diseases or disorders or conditions that are initiated, caused or result in inflammation. Inflammatory diseases or disorders and conditions include diseases, disorders or conditions characterized by pain (pain caused by noxious substances and nerve stimulation), fever (fever caused by vasodilation), redness (redness caused by vasodilation and increased blood flow), swelling (tumors caused by excessive influx or restricted outflow of fluid), and / or loss of function (which may be partial or complete, temporary or permanent). Inflammation has various forms, including but not limited to acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrotic, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, occlusive, substantial, plastic, productive, proliferative, pseudomembranous, suppurative, sclerotic, serum plastic, serous, simple, specific, subacute, suppurative, toxic, traumatic and / or ulcerative inflammation. The term "inflammatory disease" can also refer to an inflammatory response that is dysregulated, which causes an overreaction of macrophages, granulocytes and / or T lymphocytes, thereby causing abnormal tissue damage and / or cell death. Inflammatory diseases can be acute or chronic inflammatory conditions, and can be caused by infection or non-infectious causes. Inflammatory diseases include, but are not limited to, atherosclerosis, arteriosclerosis, autoimmune diseases, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, osteoarthritis, rheumatoid arthritis, inflammatory arthritis, Sjögren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease), mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonia (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia (IIP),pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of vasculitis (temporal arteritis and polyarteritis nodosa), inflammatory skin diseases, hepatitis, delayed hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, reactions), asthma, hay fever, allergies, acute allergic reactions, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, Orchitis, osteitis, otitis media, pancreatitis, mumps, pericarditis, pharyngitis, pleurisy, phlebitis, non-infectious pneumonia (pneumonitis), proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis, tonsillitis, urethritis, cystitis (urocystitis), uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis and necrotizing enterocolitis. Inflammatory diseases of the eye include, but are not limited to, postoperative inflammation.

[0171] Additional exemplary inflammatory conditions include, but are not limited to, inflammation associated with acne; acne-associated inflammation, anemia (e.g., aplastic anemia, autoimmune hemolytic anemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis, and Reiter's arthritis), ankylosing spondylitis, amyloidosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, disease), chronic obstructive pulmonary disease, dermatomyositis, diverticulitis, diabetes (e.g., type I diabetes, type II diabetes), skin diseases (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infections, ischemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity reactions, headaches (e.g., migraines, tension headaches), intestinal obstruction (e.g., postoperative ileus and intestinal obstruction during sepsis), diopathic thrombocytopenic purpura (diopathic thrombocytopenic purpura), thrombocytopenicpurpura), interstitial cystitis (bladder pain syndrome), gastrointestinal disorders (e.g., selected from peptic ulcer, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastroenteropathy (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome (Behcet's syndrome, indeterminate colitis, and inflammatory bowel syndrome (IBS), lupus, multiple sclerosis, morphea, myasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious anemia, peptic ulcer, polymyositis, primary biliary cholangitis, neuroinflammation associated with brain disorders (e.g.,In some embodiments, the inflammatory condition is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis, and prostatitis. In some embodiments, the inflammatory condition is an acute inflammatory condition (e.g., inflammation caused by infection). In some embodiments, the inflammatory condition is a chronic inflammatory condition (e.g., a condition caused by asthma, arthritis, and inflammatory bowel disease).

[0172] In one embodiment, PGM is used to treat a "liver disease or disorder" or "hepatic disease," which refers to damage or disease of the liver.Non-limiting examples of liver diseases or disorders include intrahepatic cholestasis (e.g., alagille syndrome, biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye's syndrome), hepatic vein thrombosis, hepatolenticular degeneration (i.e., Wilson's disease), hepatomegaly, liver abscess (e.g., amebic liver abscess), liver cirrhosis (e.g., cirrhosis (e.g., alcoholic, biliary, and experimental cirrhosis), alcoholic liver disease (e.g., fatty liver, hepatitis, cirrhosis), parasitic liver disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal hypertension, hepatomegaly enlargement), ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis, chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C, hepatitis D, drug-induced chronic hepatitis), toxic hepatitis, viral human hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary sclerosing cholangitis, hepatic encephalopathy, varicose veins, primary biliary cholangitis, primary sclerosing cholangitis, hepatocellular adenoma, hemangioma, gallstones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastasis, cystic liver metastasis, fibrolamellar liver carcinoma, hepatic adenoma, hepatoma, liver cyst (e.g., simple cyst, polycystic liver disease, hepatobiliary adenoma, choledochal cyst), mesenchymal tumors (mesenchymal tumors) (mesenchymal hamartoma, infantile hemangioendothelioma, hemangioma, hemangiopurpura hepatis, lipoma, inflammatory pseudotumor), epithelial tumors (e.g., biliary hamartoma, biliary adenoma), focal nodular hyperplasia, nodular regenerative hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma, cystadenocarcinoma, vascular tumors, angiosarcoma, Kaposi's sarcoma, hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, primary lymphoma, hepatic purpura, erythrohepatic porphyria, hepatic porphyria (e.g., acute intermittent porphyria, porphyria cutanea tarda), and Zellweger's syndrome.

[0173] In one embodiment, the PGM is used to treat a "pulmonary disease or condition" or a "lung-related disease or condition," which refers to an injury or disease of the lungs. Examples of pulmonary diseases or disorders include, but are not limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema, cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis, aspergilloma, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia, bronchopneumonia, interstitial pneumonia), pulmonary fibrosis, tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., non-small cell lung cancer (e.g., adenocarcinoma, squamous cell lung cancer, large cell lung cancer)), small cell lung cancer.

[0174] In one embodiment, PGM is used to treat "neurological diseases or conditions", which refers to any disease or condition of the nervous system, including diseases or conditions involving the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves) and the autonomic nervous system (parts of which are located in the central and peripheral nervous systems). Neurodegenerative diseases or conditions refer to a class of nervous system diseases or conditions characterized by nerve cell loss, including but not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, tauopathy (including frontotemporal dementia) and Huntington's disease. Examples of nervous system diseases or conditions include but are not limited to headaches, stupor and coma, dementia, epileptic seizures, sleep disorders, trauma, infection, tumors, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord diseases, and peripheral nerve, muscle and neuromuscular junction diseases. Addiction and mental illness include but are not limited to bipolar disorder and schizophrenia, which are also included in the definition of nervous system diseases. Other examples of neurological disorders include acquired epileptic aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; corpus callosum dysgenesis; agnosia; Acardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Alzheimer's disease; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; hypoxia; aphasia; apraxia; arachnoid cyst; arachnoiditis; Amold-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia-telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten's disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal blepharospasm; focal; muscular atrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch-Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumor (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome; central pontine myelinolysis;Cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; brain atrophy; cerebral gigantism; cerebral palsy; progressive nephrotic muscular atrophy; chemotherapy-induced neuropathy and neuropathic pain; cerebellar tonsillar herniation malformation; chorea; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; Coffin-Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorder; Cushing's syndrome; cytomegalovirus infection; dancing eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; Demosier syndrome Morsier's syndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonia; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephalocele; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; syncope; familial spastic palsy; febrile seizures; Fisher syndrome; Friedreich's ataxia; frontotemporal dementia and other "tauopathies", Gaucher's disease; Gerstmann's syndrome. syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTFV-1-associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia;Heredopathiaatactica polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (also a neurologic manifestation of AIDS); holoprosencephaly; Huntington's disease and other glutamine duplication disorders; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile; phytic acid storage disease; infantile Refsum disease; infantile spasms; inflammatory myopathy; intracranial cysts; intracranial hypertension; Jobert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome syndrome; Klippel-Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Fafora disease; Fambert-Eaton myasthenic syndrome; Fandau-Kleffner syndrome; Wallenberg syndrome; learning disabilities; Leigh's disease; Fennox-Gastaut syndrome; Fesch-Nyhan syndrome; leukodystrophy; dementia with Lewy bodies; lissencephaly; locked-in syndrome; Lou Gehrig's disease disease) (also known as motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease-neurological sequelae; Machado-Joseph disease; brain malformation (macrencephaly); megalencephaly;Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-stroke; mitochondrial myopathy; Mobius syndrome; monosomic muscular atrophy; motor neuron disease; moyamoya disease; mucopolysaccharidosis; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating diseases; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis sclerosis; myoclonic encephalopathy of infancy; myoclonus; myopathy; myotonia congenita; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurologic manifestations of AIDS; neurologic sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; spondylodysplasia recessive sequence; Ohtahara syndrome; oligopontine cerebellar atrophy; opsoclonusmyoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesias; Parkinson's disease; paramyotonia congenita congenita; paraneoplastic diseases; paroxysmal seizures; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorder; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary tumor;Polymyositis; foramen encephalopathy; post-polio syndrome; postherpetic neuralgia (PHN); postinfectious encephalomyelitis; orthostatic hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion disease; progressive; hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliomyelitis; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types I and II); asmussen's encephalitis; reflex sympathetic dystrophy syndrome; Refsum's disease; repetitive movement disorder; repetitive stress injury; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus dance dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; herpes zoster; Shy-Drager syndrome; Sjögren's syndrome; sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumor; spinal muscular atrophy; stiff-person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease disease; temporal arteritis; tethered cord syndrome; Thomson's disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathy; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraplegia; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis, including temporal arteritis; von Hippel-Lindau disease (VHL); Wallenberg's syndrome; Werdnig-Hoffman disease;West syndrome; whiplash; Williams syndrome; Wilson disease; and Zellweger syndrome.

[0175] In one embodiment, PGM is used to treat "neurodegenerative diseases or conditions", which refers to a class of neurological diseases or conditions characterized by loss of nerve cells, including but not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, tauopathy (including frontotemporal dementia) and Huntington's disease. In some embodiments, the neurodegenerative disease or condition is Alzheimer's disease. The cause of Alzheimer's disease is poorly understood, but it is believed to be genetically related in most cases. The disease is characterized by the loss of neurons and synapses in the cerebral cortex, resulting in atrophy of the affected area. From a biochemical point of view, Alzheimer's disease is a protein misfolding disease caused by the accumulation of plaques of abnormally folded amyloid beta protein and tau protein in the brain. Symptoms of Alzheimer's disease include but are not limited to difficulty remembering recent events, language problems, disorientation, mood swings, loss of motivation, self-neglect and behavioral problems. Ultimately, physical function is gradually lost, and Alzheimer's disease eventually leads to death. Current treatments focus on treating cognitive problems caused by the disease (e.g., using acetylcholinesterase inhibitors or NMDA receptor antagonists), psychosocial interventions (e.g., behaviorally or cognitively oriented approaches), and general care. There is currently no treatment that can completely stop or reverse the progression of the disease.

[0176] In one embodiment, PGM is used to treat "proliferative diseases or conditions", which refers to diseases or conditions that occur due to abnormal growth or extension caused by cell proliferation (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, England, 1990). Proliferative diseases or conditions may be associated with: 1) pathological proliferation of normal quiescent cells; 2) pathological migration of cells from their normal locations (e.g., metastasis of tumor cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) pathological angiogenesis, such as proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancer (i.e., "malignancies"), benign tumors, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0177] Target Gene

[0178] The target gene used in the present disclosure is not particularly limited, as long as it is a gene that produces and expresses RNA (mRNA, IncRNA, miRNA, etc.) in vitro or in a cell (preferably, wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell). In one embodiment, the target gene encodes a protein. In one embodiment, the target gene encodes a microRNA. In one embodiment, the target gene encodes a long non-coding RNA. The target gene can be selected from any gene whose expression increase or decrease is beneficial to the treatment of a selected disease, disorder, cell stress or injury and other physiological or pathological states, examples of which are described below.

[0179] In one embodiment, there is an endogenous TFBS in the target gene, where the TF can be translocated from the PGM to the endogenous binding site due to proximity. In one embodiment, the TF bound to the PGM can remain bound to the PGM regardless of whether there is a binding site in the gene. Since the effect of the TF may depend on their overall proximity to the transcription start site or the chromatin associated with the gene, co-linearity of the bound DNA with the gene is not required.

[0180] In one embodiment, the target gene is a gene having a binding site for a TF introduced by a PGM. In this case, it is known that the TF can regulate the expression of the gene. In some embodiments, the presence of a TFBS in a target gene of a TF in a PGM is first identified using the methods of the present disclosure.

[0181] In another embodiment, the target gene has no known binding site for the TF introduced by the PGM. Instead, the PGM introduces the TF near the promoter of the target gene through the DNA binding module, which is sufficient for the TF to enhance or reduce the expression of the target gene. In other words, through the PGM, the target gene can be controlled by the TF, otherwise the TF does not regulate the expression of the target gene in the absence of the PGM. Therefore, the PROTEGE platform is extended to regulate the expression of any desired / undesirable target gene by the PGM, because the PGM is designed to specifically bind to the promoter of the target gene through the sgRNA / dCas part, and then brings the TF to the target gene, which is activated by the TFBS of the PGM via a signal associated with the condition of interest (e.g., HIF-1α activated by hypoxia).

[0182] Therefore, the present disclosure provides a method for strongly specifically regulating the expression of a target gene in a cell-specific manner, because only cells exposed to a signal that activates a TF will bring the TF into close proximity with the target gene through the PGM. In the absence of a signal, the PGM may bind to the promoter region of the target gene, but since there is no TF in the PGM, nothing will happen. The TFBS is empty until a signal activates the TF, which then binds to the PGM and activates or reduces the expression of the target gene.

[0183] In one embodiment, the target gene is selected from the following categories: Fc receptor, IgG-Fc control, cytokine, interleukin, growth factor, kinase, nuclease, protease, enzyme, stem cell protein, epigenetic protein, cancer protein, immunotherapy protein, CD molecule protein, receptor protein (e.g., cytokine, growth factor, B cell, monocyte, granulocyte, NK cell, stem cell, T cell and dendritic cell receptor), TNF superfamily, B7 family, TGFβ family, cell therapy protein, immune checkpoint protein.

[0184] In one embodiment, the target gene is selected from pro-inflammatory genes and anti-inflammatory genes. In one embodiment, such genes are selected from cytokines (GM-CSF, IFNα, IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A (CTLA-8) and TNFα; chemokines (IP-10 (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4); and / or cell adhesion and inflammatory response genes (ICAM-1, CD62E (E- In one embodiment, the target gene encodes an anti-inflammatory cytokine. In one embodiment, the cytokine may be selected from IL-1β, IL-4, IL-6, IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13 and TGFβ, and its expression needs to be upregulated with PGM. In one embodiment, the target gene encodes a pro-inflammatory cytokine and its expression needs to be downregulated with PGM. In one embodiment, the pro-inflammatory cytokine is IL-1β, IL-6 and TNF-α.

[0185] In one embodiment, the target gene is selected from a receptor associated with innate immunity. Table 3. Innate immune receptors that recognize pathogens also play an important role in signaling responsible for the induction of local inflammation, recruitment of new effector cells, containment of local infection, and initiation of adaptive immune responses. In one embodiment, the target gene is a co-stimulatory immune checkpoint target or a co-inhibitory immune checkpoint target, which can be used to treat cancer and respond to various cellular and extracellular signals. Table 4 and Table 5.

[0186] Table 4: Exemplary target genes related to immunity

[0187]

[0188]

[0189]

[0190] In one embodiment, the expression of the target gene contributes to the treatment of cancer. In one embodiment, the target gene is selected from those listed in Table 5.

[0191] Table 5: Exemplary target genes whose expression is useful for cancer treatment and their therapeutic effects

[0192]

[0193] In one embodiment, the target gene is a cytokine that plays a role in asthma. Asthma is different from other chronic inflammatory diseases (such as rheumatoid arthritis, Crohn's disease and psoriasis) in that asthma exhibits a characteristic cytokine response dominated by Th2 cytokines, most of which are encoded in small clusters on chromosome 5q32-34. It is believed that this coordinated regulation of immune responses that favors Th2 cytokines (including interleukin (IL) -3, IL-4, IL-5, IL-6, IL-9, IL-13 and granulocyte-macrophage colony stimulating factor (GM-CSF)) is the result of the weakening of the inhibitory effects of Th1 cytokines (especially IL-18, IL-12 and interferon-γ), so by default it will lead to Th2 polarization of the immune response. This imbalance between Th1 and Th2 immunity will be manifested early in life, even prenatally, in those who are destined to become atopic allergies.

[0194] In one embodiment, the target gene is a gene associated with rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by persistent strong immune activity, local destruction of bone and cartilage, and multiple systemic manifestations. CD4 T cells play a central role in the initiation and continuation of the chronic autoimmune response characteristic of rheumatoid inflammation. In one embodiment, the target gene is IL-4, IFN-γ, IL-10, or a Th1 / Th2 cytokine.

[0195] In one embodiment, the target gene is associated with sepsis. In one embodiment, the target gene is selected from IL-1 family members, IL-1 receptor family members, TNF family members, TNF receptor family members, interferons, IFN receptors, members of the IL-6, IL-10, IL-6 receptor and IL-10 receptor families, members of the TGFβ or TGFβ receptor family, chemokines and chemokine receptors.

[0196] In one embodiment, the target gene is a tumor suppressor gene and the expression of the gene is favorable, in which case PGM is designed to enhance its expression. In one embodiment, the target gene is a mutated tumor suppressor gene, and its expression is unfavorable, in which case PGM is designed to inhibit its expression. The human genome encodes more than 2000 different TFs, many of which are expressed in a cell type-specific manner to coordinate gene expression programs in a large number of cell processes (see, e.g., Lee TI, Young RA. Transcriptional regulation and its misregulation in disease. Cell. 2013; 152: 1237–1251). In one embodiment, the target gene is a pro-apoptotic gene, and although the expression of some other apoptotic genes is triggered by extracellular signals (e.g., glucocorticoids), it is expected that additional pro-apoptotic genes are expressed in cells in response to the signal. In this case, PGM is designed to bind to a TF that responds to glucocorticoids, and PGM TF is closely approached to the desired pro-apoptotic gene by sgCNA. In one embodiment, glucocorticoids usually trigger the expression of pro-apoptotic BIM genes (BCL2-interacting cell death mediators) in cancer cells, but PGMs bring glucocorticoid-responsive TFs into close proximity with one or more other pro-apoptotic target genes, and their expression is also beneficial, but will not be activated in the absence of PGMs. Examples of tumor suppressor genes include TP53 and MYC. Examples of pro-apoptotic genes (i.e., proteins) include caspases, amyloid-B peptides, some members of the Bcl-2 protein family, p53 genes, BAX, BAK, BCLX, BAD, BID BIK, HRK, and heat shock proteins. Examples of anti-apoptotic genes include BCL-2, BCL-XL, BCL-W, BFL-M, BRAG-1, MCL-1, and A1 / BFL-1.

[0197] In some embodiments, the target gene is an enzyme. In some embodiments, the enzyme is selected from the enzymes having one or more functions described in Table 5.

[0198] Table 5: Enzyme target genes and their functional classification

[0199]

[0200]

[0201]

[0202]

[0203] Exemplary PGMs and Their Applications Wound Healing

[0204] In one embodiment, the PGM is designed to correct the imbalance between TGF-β1 and TGF-β3 in wounds, which can slow wound healing and lead to scarring. Figure 4A and 4B In one embodiment, the transcription factor FOXO1 or SMAD can be activated by inflammatory signals and then brought to the promoter of the TGF-β3 gene by the PROTEGE platform to promote its expression and accelerate wound healing and reduce scar formation. In one embodiment, the PGM is locally delivered to fibroblasts.

[0205] Radiation exposure

[0206] In one embodiment, PGM is designed to reduce the side effects of radiation exposure. In one embodiment, PGM targets the GCSF gene, and its expression promotes the mobilization and hematopoiesis of hematopoietic stem cells. In one embodiment, PGM has TFBS for NF-kB or Nrf-2 transcription factors. They can be activated by free radicals generated during radiation exposure and contact the promoter region of the GCSF gene, promoting its expression by PGM, thereby reducing the side effects of radiation exposure. In one embodiment, PGM is delivered intravenously to bone marrow adipocytes.

[0207] Viral infection

[0208] In one embodiment, PGM is designed to treat viral infection. In one embodiment, PGM targets IFN gene, whose expression inhibits viral replication. In one embodiment, PGM has TFBS for NF-kB, which can be activated in the presence of viral RNA, and then contacted with the promoter of IFN gene by PGM to promote its expression, thereby treating viral infection. In one embodiment, PGM is delivered to the respiratory endothelium by intranasal / inhalation. In one embodiment, PGM is delivered intravenously.

[0209] Diabetic nephropathy

[0210] In one embodiment, the PGM is designed to treat diabetic nephropathy. In one embodiment, the PGM targets the Klotho gene, whose expression inhibits renal fibrosis. In one embodiment, the PGM has a TFBS for USF2, which can be activated by high glucose levels, and then the PGM contacts the promoter of the Klotho gene to promote its expression, thereby inhibiting renal fibrosis. In one embodiment, the PGM is delivered intravenously to the glomerular endothelium and / or glomerular mesangial cells.

[0211] Atherosclerosis

[0212] In one embodiment, PGM is designed to treat atherosclerosis. In one embodiment, PGM targets FGF-21 gene and / or Klotho gene, whose expression reduces inflammation and oxidative stress. In one embodiment, PGM has one or more TFBS for NFAT, EGR1, STAT3, SREBP and / or Nrf2, which can be activated in the presence of oxidized phospholipids, and then PGM contacts the promoter of FGF-21 gene and / or Klotho gene to promote its expression, thereby reducing inflammation and oxidative stress. In one embodiment, PGM is delivered intravenously to coronary artery endothelial cells.

[0213] Cystic fibrosis

[0214] In one embodiment, the PGM is designed to treat cystic fibrosis. In one embodiment, the PGM targets HNF-3β and / or CaCC genes, whose expression reduces mucin levels (HNF-3β), balances Cl - / Na + In one embodiment, the PGM has a TFBS for the NF-kB gene, which can be activated by mucosal accumulation and then contacted with the promoter of the HNF-3β and / or CaCC gene by the PGM to promote their expression, thereby reducing mucin levels (HNF-3β), balancing Cl - / Na + Levels and water accumulation (CaCC). In one embodiment, the PGM is delivered to the airway mucosal epithelium via intranasal / inhalation.

[0215] Alzheimer's disease

[0216] In one embodiment, PGM is designed to treat Alzheimer's disease. In one embodiment, PGM targets NDBF and / or NGF genes, and its expression promotes neurite survival. In one embodiment, PGM has TFBS for NFAT, which can be activated by the presence of high Ca2+ levels, and then contacted with the promoter of NDBF and / or NGF genes by PGM to promote its expression, thereby promoting neurite survival. In one embodiment, PGM is delivered to entorhinal neurons via intrathecal administration.

[0217] Oxidative stress and inflammation

[0218] In one embodiment, PGM is designed to treat oxidative stress and / or inflammation. In one embodiment, PGM targets the klotho gene, which encodes a membrane-bound and circulating protein that inhibits oxidative stress and inflammation. In one embodiment, the PGM comprises a TFBS for Nrf2, which can be activated by oxidative stress and / or inflammatory signals (which can be simulated by adding tert-butylhydroquinone (tBHQ)), and then the PGM is used to contact the promoter of the klotho gene to promote its expression, thereby inhibiting oxidative stress and inflammation. In one embodiment, PGM is delivered to the airway mucosal epithelium by intranasal / inhalation. In one embodiment, PGM is delivered intravenously to the coronary artery endothelium. In one embodiment, PGM is delivered to the respiratory endothelium by intranasal / inhalation. In one embodiment, PGM is delivered intravenously.

[0219] cancer

[0220] In one embodiment, the PGM is designed to treat cancer. In one embodiment, the PGM targets a BH3-only gene that encodes a protein that promotes apoptosis of tumor cells. In one embodiment, the PGM comprises a TFBS for one or more of HIF-1, p73, Sp1, or Fox03a, which can be activated by hypoxia, low pH, and / or high levels of lactic acid in the tumor microenvironment and contacted with the promoter of the BH3-only gene by the PGM, thereby inducing apoptosis of tumor cells.

[0221] In one embodiment, the PGM targets one or more genes encoding glycolytic enzymes such as hexokinase or phosphoglycerate kinase, which stimulate glucose uptake by regulating the glucose transporters GLUT1 and GLUT3. In one embodiment, the PGM comprises a TFBS of a TF that responds to glucose levels.

[0222] In one embodiment, the expression of the target gene is increased by at least 1.5 fold, at least 2.0 fold, at least 2.5 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, at least 10.0 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 21 fold, at least 22 fold, at least 23 fold, at least 24 fold, at least 25 fold, at least 26 fold, at least 27 fold, at least 28 fold, at least 29 fold, at least 30 fold, at least 31 fold, at least 32 fold, at least 33 fold, at least 34 fold, at least 35 fold, at least 36 fold, at least 37 fold, at least 38 fold, at least 39 fold, at least 40 fold, at least 41 fold, at least 42 fold, at least 43 fold, at least 44 fold 32 times, at least 33 times, at least 34 times, at least 35 times, at least 36 times, at least 37 times, at least 38 times, at least 39 times, at least 40 times, at least 41 times, at least 42 times, at least 43 times, at least 44 times, at least 45 times, at least 46 times, at least 47 times, at least 48 times, at least 49 times, at least 50 times, at least 51 times, at least 52 times, at least 53 times, at least 54 times, at least 55 times, at least 56 times, at least 57 times, at least 58 times, at least 59 times, at least 60 times, at least 61 times, at least 62 times, at least 63 times, at least 64 times, at least 65 times, at least 66 times, at least 67 times, at least 68 times, at least 57 times, at least 58 times, at least 59 times, at least 60 times, at least 61 times, at least 62 times, at least 63 times, at least 64 times, at least 65 times, at least 66 times, at least 67 times, at least 68 times, at least 69 times, at least 70 times, at least 71 times, at least 72 times, at least 73 times, at least 74 times, at least 75 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times.

[0223] In one embodiment, the expression of the target gene is reduced by at least 1.5 fold, at least 2.0 fold, at least 2.5 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, at least 10.0 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 21 fold, at least 22 fold, at least 23 fold, at least 24 fold, at least 25 fold, at least 26 fold, at least 27 fold, at least 28 fold, at least 29 fold, at least 30 fold, at least 31 fold, at least 32 fold, at least 33 fold, at least 34 fold, at least 35 fold, at least 36 fold, at least 37 fold, at least 38 fold, at least 39 fold, at least 40 fold, at least 41 fold, at least 42 fold, at least 43 fold, at least 44 fold 32 times, at least 33 times, at least 34 times, at least 35 times, at least 36 times, at least 37 times, at least 38 times, at least 39 times, at least 40 times, at least 41 times, at least 42 times, at least 43 times, at least 44 times, at least 45 times, at least 46 times, at least 47 times, at least 48 times, at least 49 times, at least 50 times, at least 51 times, at least 52 times, at least 53 times, at least 54 times, at least 55 times, at least 56 times, at least 57 times, at least 58 times, at least 59 times, at least 60 times, at least 61 times, at least 62 times, at least 63 times, at least 64 times, at least 65 times, at least 66 times, at least 67 times, at least 68 times, at least 57 times, at least 58 times, at least 59 times, at least 60 times, at least 61 times, at least 62 times, at least 63 times, at least 64 times, at least 65 times, at least 66 times, at least 67 times, at least 68 times, at least 69 times, at least 70 times, at least 71 times, at least 72 times, at least 73 times, at least 74 times, at least 75 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times.

[0224] Nucleic acids and vectors

[0225] In one embodiment, the disclosure provides a nucleic acid comprising one or more components of a PGM disclosed herein. In one embodiment, the nucleic acid comprises one or more of the following: a cRNA and / or a cRNA module, a tracrRNA and / or a tracrRNA module, and a sgCNA, and / or a nucleic acid having a transcription factor binding site or a transcription factor binding site module.

[0226] In some embodiments, the transcription factor binding site comprises one or more modifications relative to the native sequence. In some embodiments, one or more modifications include one or more transitions, one or more transversions, one or more insertions, one or more deletions, one or more inversions, or any combination thereof. In one embodiment, one or more transitions are selected from the following groups: (a) T to C; (b) A to G; (c) C to T; and (d) G to A. In one embodiment, one or more transversions are selected from the following groups: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T. In one embodiment, the crRNA carries one or more modifications relative to the crRNA that is completely hybridized to the target gene. In one embodiment, the one or more modifications comprise altering a native DNA gene sequence encoding a DNA binding protein (e.g., dCas) so as to introduce at least one of the following changes: (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair. In one embodiment, the one or more modifications comprise insertions or deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, optionally wherein one or more edits comprise insertions or deletions of 1-15 nucleotides. In some embodiments, the transcription factor binding site has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or homology relative to the native sequence. In one embodiment, the nucleic acid contains one or more chemically modified or non-natural nucleotides. In some embodiments, inclusion of chemically modified or non-natural nucleotides increases the functional lifespan of the PGM in the cell.

[0227] In some embodiments, the disclosure provides a vector comprising one or more nucleic acids of the disclosure. In some embodiments, the vector comprises a nucleic acid encoding a DNA binding protein (dCas) of the disclosure. In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0228] Composition

[0229] In one embodiment, the present disclosure provides a composition comprising one or more nucleic acids and / or proteins of the present disclosure or a composition consisting of one or more nucleic acids and / or proteins of the present disclosure. In one embodiment, the present disclosure provides a composition comprising one or more cells of the present disclosure (preferably wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell). In some embodiments, the composition is a pharmacological composition. In some embodiments, the composition comprises one or more components of PGM as described herein or is composed of one or more components of PGM as described herein, and the composition can be given to cells, tissues or organisms by any suitable means, and the means are such as by gene therapy, mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP) delivery and combinations thereof, as described above.

[0230] In one embodiment, the present disclosure provides a composition for delivering the nucleic acid of the present disclosure to a cell. In one embodiment, the composition comprises RNA, DNA and / or protein components of the PGM of the present disclosure or is composed of it. In one embodiment, the composition comprises a complete PGM of the present disclosure or is composed of a complete PGM of the present disclosure. In one embodiment, the composition comprises cRNA and / or cRNA modules, tracrRNA and / or tracrRNA modules, Cas / DNA binding proteins, sgCNA and / or nucleic acids with transcription factor binding sites or transcription factor binding site modules. More compositions are described in the delivery method of the gene PGM system above.

[0231] In one embodiment, the composition comprises one or more cells comprising cRNA and / or cRNA modules, tracrRNA and / or tracrRNA modules, Cas / DNA binding proteins, sgCNA, nucleic acids with transcription factor binding sites or transcription factor binding site modules, and / or PGMs. In some embodiments, the composition further comprises a chemical substance that acts as an activation / upregulation / inhibition / downregulation signal for PGM-mediated gene expression. In some embodiments, the chemical is a drug.

[0232] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any composition disclosed herein. In some embodiments, the pharmaceutical composition comprises any composition disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises or consists of any polynucleotide and / or protein disclosed herein and a pharmaceutically acceptable carrier. Even if not explicitly disclosed or listed herein, any reference to a composition of the present disclosure "comprising" something also means the same composition "consisting of" the something, and also means the same composition "consisting essentially of (the something)".

[0233] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose or sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, cellulose acetate, etc.; (4) powdered trifoliate; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, etc. , olive oil, corn oil and soybean oil; (10) diols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as tromethamine, magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; (22) C2-C12 alcohols such as ethanol; (23) other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation.Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" are used interchangeably herein.

[0234] Reagent test kit

[0235] In one embodiment, the present disclosure provides a kit. In one embodiment, the kit comprises one or more nucleic acids and / or vectors of the present disclosure. In one embodiment, the kit further comprises a DNA binding protein (e.g., dCas). In one embodiment, the kit comprises instructions for use. In one embodiment, the kit comprises components for preparing a pharmaceutical composition having nucleic acids and / or cells of the present disclosure.

[0236] Alternative PGM

[0237] The present disclosure also provides modifications to the above-mentioned embodiments, wherein the DNA binding molecules do not cover Cas proteins. Therefore, in one embodiment, any part with sufficient DNA binding specificity to address a single site in a target genome (e.g., the human genome) can be used as a DNA binding module for PGM. In one embodiment, the DNA binding molecules are arranged in a tandem array of about six zinc finger motif units, which bind to a unique site selected in the human genome. In one embodiment, such zinc finger arrays can be pre-coupled with DNA modification molecules (such as nucleases and transcription factors) to target those DNA modification activities to the DNA close to the zinc finger recognition site. In one embodiment, PGM comprises a coupling of a nucleic acid to a peptide (such as a zinc finger array), and such methods can be used to attach a transcription factor binding module comprising a nucleic acid to a zinc finger array DNA binding module to produce a PGM. As an example, synthesizing a zinc finger array by solid phase peptide synthesis allows dibenzocyclooctyne (DBCO) groups to be incorporated into the amino termini of zinc finger peptides by standard peptide coupling procedures. Transcription factor binding modules comprising nucleic acids carrying an azide group attached to their 3' or 5' termini can be coupled to the terminal groups by a well-known strain-promoted azide-alkyne cycloaddition reaction. Nucleic acids carrying azide groups can be readily prepared by reacting linkers carrying azide groups (such as azidobutyrate NHS ester) with amino linkers on oligonucleotides synthesized by solid phase phosphoramidite chemistry.

[0238] In one embodiment, the DNA binding module of the PGM comprises a TAL (transcription activator-like) effector protein. The correspondence between the polypeptide sequence of the TAL effector and its DNA recognition sequence enables embodiments in which the protein binds to a desired unique DNA sequence. Any method known to those skilled in the art for coupling proteins to nucleic acids can be used to connect the transcription factor binding module comprising nucleic acids to the TAL effector DNA binding module to form a PGM. Such methods include, but are not limited to, using any of a variety of cross-linking agents to connect a dibenzocyclooctyne (DBCO) group to a protein, followed by coupling the nucleic acid module with an azide group by an azide-alkyne cycloaddition, or coupling the maleimide group attached to the nucleic acid module to the polypeptide by Michael addition through cysteine. In one embodiment, the TAL effector protein can be engineered to contain two different DNA binding domains, one that binds to the target DNA sequence of the PGM DNA binding module and the other that binds to the duplex DNA component of the transcription factor binding module.

[0239] In addition to using DNA oligonucleotides folded to contain known duplex DNA binding sites of endogenous transcription factors, transcription factor binding modules can be derived from DNA or RNA aptamers that bind to desired transcription factors. Therefore, in one embodiment, methods known to those skilled in the art can be used to generate DNA and RNA aptamers to bind to various molecules including proteins (including transcription factors). In one embodiment, a PGM with a transcription factor binding module comprising a DNA aptamer can be connected to a genomic DNA binding module to produce a PGM using, for example, a DNA ligase by the same method and chemistry as a DNA hairpin. In this case, aptamers can be synthesized with complementary sequences near the 3' and 5' ends of DNA to promote the formation of duplex regions with overhangs, thereby allowing connection to the cr and tracr components of sgCNA. In the case of RNA aptamers, a complete guide nucleic acid can be produced by transcription of a DNA template.

[0240] All publications, patents, patent applications, and / or other references cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, patent application, and / or other reference cited in this application were individually indicated to be incorporated by reference for all purposes.

[0241] While various specific embodiments / aspects have been shown and described, it will be appreciated that various modifications can be made without departing from the spirit and scope of the disclosure.

[0242] Unless otherwise stated or apparent from the context, if one, more than one, or all members of a group are present in, employed in, or otherwise associated with a given product or process, an embodiment or description containing "or" is considered to be included between one or more members of the group. The present invention includes embodiments in which one member of the group is present in, employed in, or otherwise associated with a given product or process. The present invention includes embodiments in which more than one, or all members of the group are present in, employed in, or otherwise associated with a given product or process. In addition, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms in one or more of the listed claims are introduced into another claim. For example, any claim that is subordinate to another claim can be modified to include one or more limitations present in any other claim that is subordinate to the same base claim. When elements are presented in the form of a list, for example, in a Markush group format, each subgroup of the element is also disclosed, and any element can be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as including specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are composed of such elements and / or features or are substantially composed of such elements and / or features. For simplicity, these embodiments are not specifically described herein. It should also be noted that the terms "comprising" and "containing" are intended to be open-ended and allow the inclusion of additional elements or steps. A given range includes endpoints. In addition, unless otherwise specified or apparent from the context and understanding of a person of ordinary skill in the art, the value represented by a range can adopt any specific value or sub-range within the range described in different embodiments of the present invention, up to one-tenth of the lower range unit, unless otherwise clearly stated in the context.

[0243] This application relates to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, the specification shall prevail. In addition, any specific embodiment of the present invention that belongs to the prior art can be explicitly excluded from any one or more embodiments. Since these embodiments are considered to be known to those of ordinary skill in the art, they can be excluded even if they are not explicitly stated here. Any specific embodiment of the present invention can be excluded from any embodiment for any reason, whether or not it is related to the existence of the prior art. Those skilled in the art should understand or be able to determine that many equivalent forms of the specific embodiments described herein can be obtained using no more than routine experiments. The scope of the embodiments described herein is not limited to the above description, but as described in the attached embodiments. It will be understood by those of ordinary skill in the art that various changes and modifications can be made to this description without departing from the spirit or scope of the present invention as defined in the embodiments clearly disclosed above, below and in the claims.

[0244] Example

[0245] Example 1

[0246] Preparation of chimeric guide nucleic acids

[0247] Cr module, tracr module and transcription factor binding module oligonucleotides were commercially obtained from suppliers and were fully deprotected and gel purified.

[0248] Cr module: 5'CGGAGGCAGUCCCGGCUCGCGUUUUAGAGCUAdAdCdCdCTdGdAdCTTdGdAdCdGT3'

[0249] (SEQ ID NO:64);

[0250] tracr module: 5'dAdAdGTdCdAdGdGdGTUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA CUUGAAAAGUGGCACCGAGUCCGGUGCUUUU3' (SEQ ID NO: 65);

[0251] Transcription factor binding module: 5'dATdGdAdCTdCdAdGdCdAdCdAdATdGdGdCdGdAdAdAdGdCdCdATTdGTdGd CTdGdAdGTdCdATdAdCdGTdC3' (SEQ ID NO: 66);

[0252] The Cr module targets the sequence 5'CGGAGGCAGUCCCGGCUCGC3' (SEQ ID NO: 67) in the promoter region of the klotho gene. This site was identified and selected using the CRISPRa function using the target site selection tool CRISPick (https: / / portals.broadinstitute.org / gppx / crispick / public).

[0253] The transcription factor binding module was designed to fold into a hairpin structure containing the Nrf2 response element: 5'-ATGACTCAGCA-3' (SEQ ID NO: 68).

[0254] Obtain both the Tracr module and the transcription factor binding module with 5' phosphate. The Tracr module (7 nanomoles) and the Cr module (8 nanomoles) were annealed in a total volume of 15 μL, heated to 65 ° C for 1 minute, and then cooled to room temperature within 60 minutes. The transcription factor binding module (8 nanomoles in 41 μL) was annealed by heating to 95 ° C for 1 minute and cooling to room temperature within 75 minutes. The annealed oligonucleotide modules were mixed and connected in an 80 μL reaction containing 1mM ATP, 1X ligase buffer and 16,000 units of T4 DNA ligase. After connecting overnight at room temperature, the ligase was inactivated by heating to 65 ° C for 15 minutes. The mixture was then extracted with phenol, and the aqueous layer was desalted by gel filtration and eluted with 400 μL of water. The nucleic acids were precipitated with ethanol and sodium acetate, dissolved in water, and desalted again by gel filtration.

[0255] Example 2

[0256] Preparation of programmable gene regulators

[0257] Chimeric guide nucleic acid (80 pmoles) in 10 μL of phosphate buffered saline was warmed to 37°C for 30 minutes and then cooled to room temperature over 30 minutes.

[0258] Chimeric guide nucleic acid sequence: 5'CGGAGGCAGUCCCGGCUCGCGUUUUAGAGCUAdAdCdCdCTdGdAdCTTdGd AdCdGTdATdGdAdCTdCdAdGdCdAdCdAdATdGdGdCdGdAdAdAdGdCdCdATTdGTdGdCTdGdAdGTdCdATdAdCdGTdCdAdAdGTdCdAdGdGdGTUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU3'(SEQ ID NO:69)

[0259] Klotho / Nrf2 sgRNA example sequence:

[0260] 5'-CGGAGGCAGTCCCGGCTCGCGUUUUAGAGCUAdAdCdCdCdTdGdA dCdTdTdGdAdCdGdTdAdTdGdAdCdTdCdAdGdCdAdCdAdAdTdGdGdCdGdAdAdAdGdCdCdAdTdTdGdTdGdCdTdGdAdGdTdCdAdTdAd CdGdTdCdAdAdGdTdCdAdGdGdGdTUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCCGAGUCGGUGCUUUU-3'(SEQ ID NO:4)

[0261] The annealed chimeric guide nucleic acid was mixed with 78 pmoles of recombinant dCas9 with nuclear localization sequences (NLS-dCas9-NLS, Novateinbio, PR-137213B) fused to the N- and C-termini.

[0262] Nuclear localization signal sequence-MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILSDILRVNTEITKAPLSASMIKRYDEHHQDLTLTLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY VTEGMRKPAFLSGEQKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI- Nuclear localization signal sequence (SEQ ID NO: 70)

[0263] The mixture was incubated at room temperature for 15 minutes to form the programmable gene regulator (PGM).

[0264] Example 3

[0265] In vitro assay for recruitment of transcription factors to target DNA

[0266] Preparation of test wells.

[0267] Biotinylated oligonucleotide duplexes containing a 20 bp target sequence from the human Klotho gene promoter or a control sequence containing a scrambled target sequence were immobilized on an 8-well strip coated with high-binding capacity streptavidin. The sense and antisense strands of the duplexes were purchased commercially, fully deprotected and gel purified.

[0268] Sense strand target DNA: 5'CCTCGGCGCCCCTGCCCCCGCCCCCAGTGCCAGGGCGGAGGCAGTCCCGG CTCGCAGGTAATTATTGCCAGCGGAGCCCGCCGGGGAGCG3' (SEQ ID NO: 71)

[0269] Antisense strand target DNA: 5'CGCTCCCCGGCGGGCTCCGCTGGCAATAATTACCTGCGAGCCGGGACTGC CTCCGCCCTGGCACTGGGGGCGGGGGCAGGGGCCGCCGAGG-Biotin 3' (SEQ ID NO: 72)

[0270] Scrambled sense strand target DNA: 5'CCTCGGCGCCCCTGCCCCCGCCCCCAGTGCCAGGGGGACGCGCGGGCACCGCTTCAGGTAATTATTGCCAGCGGAGCCCGCCGGGGAGCG3' (SEQ ID NO:73)

[0271] Scrambled antisense strand target DNA: 5'CGCTCCCCGGCGGGCTCCGCTGGCAATAATTACCTGAAGCGGTGCCCGCGCGTCCCCCTGGCACTGGGGGCGGGGGCAGGGGCGCCGAGG-biotin 3' (SEQ ID NO: 74)

[0272] The 3'-end of the antisense strand obtained was labeled with biotin. Sense and antisense oligonucleotides were combined in tris-buffered saline (TBS) at a concentration of 8 μM and annealed by heating to 95°C for 5 minutes and then cooling to room temperature within 60 minutes. The hybridized duplex was diluted twice with 5X concentrated TBS, and 100 μL of the resulting solution was added to each streptavidin-coated well and incubated at room temperature for 72 hours. Each DNA-coated well was washed with tris-buffered EDTA (TE) and then washed with TBS.

[0273] Preparation of nuclear extracts.

[0274] HEK293 cells were grown to 50-70% confluence in 10 cm culture dishes and treated with 50 μM freshly prepared tert-butylhydroquinone (tBHQ) in phosphate buffered saline (PBS) containing 30% DMSO for 24 hours to activate Nrf2. For the control, cells were treated with PBS / 30% DMSO. The cells in PBS were scraped from the culture dish and centrifuged at 3,200 rpm for 5 minutes at 4 ° C. The cells were washed once with PBS, and the pellet was gently resuspended in 100 μL of ice-cold hypotonic buffer solution (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 3 mM MgCl2). After incubation on ice for 15 minutes, 5 μL of 10% NP40 lysis buffer (Sigma Aldrich) was added, and the cells were vortexed vigorously for 10 seconds. The homogenate was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. The supernatant containing the cytoplasmic fraction was discarded. 50 μL Invitrogen cell extraction buffer (catalog number FNN0011, Invitrogen) supplemented with 1 mM PMSF and a mixture of protease inhibitors was added to the precipitate containing the nuclear fraction, and then incubated on ice for 30 minutes, vortexing every 10 minutes. The solution was centrifuged at 14,000 x g for 30 minutes at 4 ° C, and the supernatant containing the nuclear fraction was transferred to a separate tube. Total protein was quantified by Bradford assay using (Pierce Micro-BCA assay).

[0275] Nrf2 recruitment assay.

[0276] The binding of Nrf2 to PGM bound to target DNA was assessed using components in the Nrf2 transcription factor assay kit (Abcam, ab207223). Freshly prepared PGMs were added to the wells fixed with oligonucleotide duplexes as described above and incubated overnight at room temperature. Unbound PGMs were removed by washing with TE. Nuclear extracts (20 μg total protein) of HEK298 cells treated with tBHQ or vehicle alone were added to the wells and incubated for one hour at room temperature. Each well was washed 3 times with 200 μL of wash buffer provided by the assay kit. Rabbit anti-Nrf2 antibody (100 μL, 1:1000 dilution) provided by the kit was added, followed by incubation at room temperature for 1 hour and washing 3 times with 200 μL of wash buffer provided by the assay kit. Anti-rabbit HRP antibody (100 μL, 1:1000 dilution) was added, followed by incubation at room temperature for 1 hour and washing 4 times with 100 μL of wash buffer. Color development solution (100 μL) was added and the wells were incubated at room temperature for 10 minutes before adding stop solution (100 μL). Binding of Nrf2 to the wells was quantified by absorbance at 450 nm compared to control wells not developed with anti-Nrf2 antibody.

[0277] Example 4

[0278] Signal-dependent transcriptional regulation in cultured cells

[0279] HEK293 cells were cultured at 3 x 10 5 Cells / well were plated on 6-well plates containing 2mL complete growth medium (DMEM containing 10% FBS). When the cells reached 30-50% confluence, the cells were transfected with the above-mentioned PGM. PGM was freshly prepared as described above, and Opti-MEM medium (500 μL) was added to PGM, followed by 50 μL Cas9 Plus reagent (Invitrogen, CMAX00008). The resulting solution was added to a solution of 500 μL Opti-MEM and 30 μL CRISPRMAX transfection reagent (Invitrogen, CMAX00008). The mixture was briefly vortexed and incubated at room temperature for 10 minutes. In a control experiment lacking PGM, a blank control (mock) PGM solution was prepared by replacing the chimeric guide nucleic acid with water and replacing NLS-dCas9-NLS with Tris-HCl. PGM or blank control (mock) PGM solution (250 μL) was added to the cells and then incubated for 16 hours. Freshly prepared tBHQ solution or vehicle as described above was added to each well, and cells were incubated for an additional 24 h.

[0280] Total RNA was isolated using the Purelink RNA Mini Kit and quantified by spectrophotometry. Total RNA (250 ng) of each sample was reverse transcribed using a 3:1 (volume:volume) random hexamer mixture with an anchored oligo-dT primer in a 30 μL reaction using the Thermo Scientific Verso cDNA Synthesis Kit according to the manufacturer's protocol. The target gene Klotho and the endogenous reference gene GAPDH were assayed under each condition using Taqman Gene Expression Assays (ThermoFisher assays were Hs00934627_m1 and Hs02786624_g1, respectively) and Taqman Fast Advanced Master Mix (ThermoFisher). Hs02786624_g1 covers a 157 nt amplicon in GAPDH exon 7. Hs00934627_m1 covers a 108 nt amplicon spanning exons 2 and 3 in KL. Reactions in 20 μL qPCR reactions in 96-well plates contained 4 μL of two-fold diluted cDNA. Data were collected using the Bio-Rad CFX96 Touch Real-Time PCR Detection System and analyzed using the ΔΔCq method to calculate relative gene expression.

[0281] Example 5

[0282] Association of physiologically responsive transcription factors with target DNA sequences under the guidance of programmable gene regulators (PGMs)

[0283] As previously explained in FIG. 1 , the principle of the physiological responsive gene expression regulator of the present disclosure is as follows: transcription factors (TFs) are activated by physiological stimuli. Examples of physiological stimuli include oxidative stress or growth factor signaling. The responsive gene expression regulator is a ribonucleoprotein complex comprising a disabled CRISPR-associated protein, dCas9, and a chimeric guide nucleic acid. The chimeric guide nucleic acid comprises a DNA hairpin, which incorporates a binding site, a crRNA sequence, and a tracrRNA sequence for the activated TF. The complex binds to the genomic DNA sequence adjacent to the target gene, causing the target gene to respond to physiological signals. The binding site is programmed by the crRNA sequence in the guide nucleic acid. The association of the activated TF with the bound dCas9 complex brings the TF close to the target gene, thereby regulating target gene transcription.

[0284] Here, a gene regulator comprising dCas9 and a DNA response element for the transcription factor Nrf2 recruits activated Nrf2 to a DNA sequence targeted by a guide nucleic acid. Figure 5A. The target DNA sequence is a 20 base pair sequence (pink and gold) contained in a DNA duplex fixed in a well of a multiwell plate. The gene regulator comprises dCas9 (yellow circles) complexed with a single guide nucleic acid, which comprises a crRNA module (turquoise) complementary to the target sequence, a tracrRNA module (duck green), and a DNA module forming a hairpin structure, the stem of which contains an Nrf2 response element (red). After the gene regulator binds to the fixed target DNA, nuclear extracts of Hek293 cells treated with tert-butylhydroquinone (tBHQ) are added to stimulate the activation and nuclear localization of Nrf2. After washing the wells to remove unbound Nrf2, bound Nrf2 is detected with an anti-Nrf2 antibody and visualized by absorbance at 450 nm after treatment with an HRP-conjugated anti-rabbit secondary antibody and development with an HRP substrate. Figure 3 B.-3D. Each value is the average of three replicates in different wells. Error bars are standard deviations of the mean. Figure 5B. Dependence of Nrf2 binding on the presence of PGM. For the "-PGM" wells, PBS was added instead of the PGM solution. Figure 5C. Dependence of Nrf2 binding on the presence of a fixed target DNA sequence in the wells. In the "-target DNA sequence" wells, the target sequence was replaced by a fixed duplex containing a scrambled form of the target sequence (same sequence composition, different sequence). Figure 5D. Dependence of Nrf2 binding on Nrf2 activation. Nuclear extracts added to the "-Nrf2" wells were from cells that were not treated with tBHQ.

[0285] Tert-butylhydroquinone (tBHQ) was added to cultured cells to activate Nrf2. tBHQ is a well-known Nrf2 activator. It has been shown to react with Keap1, a protein that localizes Nrf2 to the cytoplasm. The reaction of tBHQ with Keap1 promotes the translocation of Nrf2 to the nucleusLi, W. and Kong, AN (2009). Molecular mechanisms of Nrf2-mediated antioxidant response. Mol. Carcinog. 48, 91–104.

[0286] PGM binds to the target DNA, and then adding nuclear extracts from cells in which Nrf2 has been activated results in the association of Nrf2 with the target DNA. No Nrf2 binding was detected in the absence of PGM. Nrf2 binding also depends on the correct sequence in the target DNA: Nrf2 does not bind to wells in which the DNA sequence targeted by the guide nucleic acid has been replaced by a scrambled sequence. The association of Nrf2 with the immobilized target DNA also depends on the biochemical activation of Nrf2. Adding nuclear extracts from cells that have not been treated with tBHQ to activate Nrf2 to the wells does not result in Nrf2 binding.

[0287] Example 6

[0288] Transcriptional regulation of the PGM target gene klotho in response to biochemical activation of endogenous transcription factors

[0289] Figures 6A and 6B Nrf2-dependent regulation of klotho transcription in cultured cells is shown. Human embryonic kidney cells were treated with PGMs targeting the klotho promoter and containing Nrf2 response elements. After 16 hours, Nrf2 was activated by tBHQ. Total RNA was isolated after an additional 24 hours, and klotho expression relative to GAPDH expression was measured by RT-qPCR. Figure 6A. The target sequence of PGM is a 20 base pair sequence upstream of the transcription start site of the klotho gene, a membrane-bound cyclic protein that inhibits oxidative stress and inflammation. The transcription factor binding module of PGM contains Nrf2 response elements. The addition of PGM alone did not significantly affect the transcription of klotho relative to the transcription of the housekeeping gene GAPDH, and treatment of cultured cells with tBHQ resulted in a small decrease in klotho transcription. However, treatment with PGM followed by activation of Nrf2 with tBHQ resulted in a two-fold increase in klotho transcription compared to treatment with tBHQ alone. Figure 6B.

Claims

1. An engineered non-naturally occurring system comprising a programmable gene regulator (PGM) for reversibly altering the expression of a target gene of interest in a cell in response to one or more intracellular or extracellular environmental signals, or a sgCNA subcomponent thereof, the system comprising the following subcomponents: an endonuclease-defective DNA-binding polypeptide (preferably, a dCas polypeptide); and a chimeric nucleic acid (sgCNA) comprising a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), and at least one nucleic acid segment comprising at least one transcription factor binding site; The crRNA comprises a sequence complementary to a nucleic acid sequence in the promoter region of a target gene of interest, and the transcription factor binding sites in the PGM are each capable of binding to at least one endogenous transcription factor, which is activated in response to one or more environmental signals in a cell containing the PGM, and then recognizes and binds to the transcription factor binding site of the PGM, which binds to the promoter of the gene of interest through the crRNA, thereby bringing the transcription factor close to the gene of interest and responding to one or more environmental signals to activate or inhibit the expression of the gene of interest.

2. The engineered non-naturally occurring system of claim 1, or a sgCNA subcomponent thereof, wherein (i) at least one transcription factor binding site in the PGM is also present in the target gene and / or (ii) at least one transcription factor binding site in the PGM is not an endogenous transcription factor binding site in the target gene.

3. The engineered non-naturally occurring system according to any one of claims 1 and 2, or a sgCNA subcomponent thereof, wherein when one or more endogenous transcription factors are activated in response to one or more environmental signals, the PGM recruits the one or more endogenous transcription factors to a gene of interest, thereby activating gene expression in response to one or more environmental signals in a cell-specific manner.

4. The engineered non-naturally occurring system according to any one of claims 1 to 3, or a sgCNA subcomponent thereof, wherein the one or more transcription factors are identified as transcription factors that (i) are known to be activated in response to the environmental signal and (ii) are known or unknown to activate / repress expression of a target gene of interest.

5. The engineered non-naturally occurring system of any one of claims 1 to 4, or a sgCNA subcomponent thereof, wherein the DNA binding polypeptide is a nuclease-deficient cas polypeptide.

6. The engineered non-naturally occurring system of any one of claims 1 to 5, or a sgCNA subcomponent thereof, wherein a gene of interest is identified as a gene whose expression (a) produces a beneficial cellular response in response to the one or more environmental signals, but whose expression is undetectable, or whose expression is increased by the PGM relative to the level of gene expression in the absence of the PGM; or (ii) produces a deleterious effect on the cell and whose expression is decreased in response to the one or more environmental signals relative to the level of gene expression in the absence of the PGM.

7. The engineered non-naturally occurring system according to any one of claims 1 to 6, or a sgCNA subcomponent thereof, wherein the gene of interest encodes a protein, a microRNA or a long non-coding RNA.

8. The engineered non-naturally occurring system of any one of claims 1 to 7, or a sgCNA subcomponent thereof, wherein the signal is any physical signal such as a light signal (e.g., UV light), ionizing radiation, heat / temperature, hypertonic or hypotonic conditions; a mechanical signal such as pressure (e.g., touch), acoustic motion, and / or blood pressure; and / or any chemical signal such as a growth factor, a cytokine, a chemokine, cyclic AMP, a hormone, a neurotransmitter, an extracellular matrix component, a bacterial antigen, a viral antigen, a lipid, a lipopolysaccharide, a gas level (e.g., oxygen level, nitric oxide level), an ion level (e.g., calcium level, sodium level), pH, reactive oxygen species, heavy metals, oxidized LDL and / or free radicals, a cell-cell signal (e.g., T-cell binding, cell-cell contact), or a combination thereof.

9. The engineered non-naturally occurring system according to any one of claims 1 to 8, or a sgCNA subcomponent thereof, wherein the transcription factor is selected from the group consisting of forkhead transcription factors, nuclear receptors, POU-domain proteins, SMADs, preferably Nrf2, FOX01, NF-kB, USF2, NFAT, EGR1, STAT3 and / or SREBP.

10. The engineered non-naturally occurring system, or a sgCNA subcomponent thereof, or a sgCNA subcomponent thereof, according to any one of claims 1 to 9, wherein the TF binding module comprises (i) at least one TF binding segment (TFBS), wherein the TF binding segment comprises DNA and / or RNA, or (ii) wherein the TF binding module comprises at least one TF binding segment (TFBS), wherein the TF binding segment comprises a DNA aptamer or an RNA aptamer selected for binding to an endogenous transcription factor.

11. The engineered non-naturally occurring system according to any one of claims 1 to 10, or a sgCNA subcomponent thereof, wherein the TF binding module comprises a sequence derived from a naturally occurring RNA.

12. The engineered non-naturally occurring system according to any one of claims 1 to 11, or a sgCNA subcomponent thereof, wherein the TF binding segment (TFBS) comprises a double-stranded segment of DNA containing at least one TF response element.

13. The engineered non-naturally occurring system according to any one of claims 1 to 12, or a sgCNA subcomponent thereof, wherein the strands of the DNA portion of the sgRNA form a duplex and are joined by a loop sequence of arbitrary length.

14. The engineered non-naturally occurring system of claim 13, or a sgCNA subcomponent thereof, wherein the loop comprises four nucleotides.

15. The engineered non-naturally occurring system according to any one of claims 13 and 14, or a sgCNA subcomponent thereof, wherein the sequence of the loop comprises 5'-guanosine-adenosine-adenosine-adenosine-3'.

16. The engineered non-naturally occurring system of any one of claims 1 to 15, or a sgCNA subcomponent thereof, wherein the crRNA comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleobases that are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementary to a target nucleic acid sequence of a target gene of interest.

17. The engineered non-naturally occurring system according to any one of claims 1 to 16, or a sgCNA subcomponent thereof, wherein the endonuclease-defective sequence-specific DNA binding protein (preferably a dCas polypeptide) is fused to at least one copy of a nuclear localization signal.

18. The engineered non-naturally occurring system of any one of claims 1 to 17, or a sgCNA subcomponent thereof, wherein the crRNA comprises any one of the following sequences: SEQ ID NO: 6 to SEQ ID NO:

34.

19. The engineered non-naturally occurring system of any one of claims 1 to 18, or a sgCNA subcomponent thereof, wherein the transcription factor binding sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 1, 2, 3, 5 and those in Table 2.

20. The engineered non-naturally occurring system of any one of claims 1 to 19, or a sgCNA subcomponent thereof, wherein the tracrRNA is capable of binding to dCas9.

21. The engineered non-naturally occurring system of any one of claims 1 to 20, or a sgCNA subcomponent thereof, wherein the crRNA, TFBS and tracrRNA of the PGM are assembled in any one of the following molecular arrangements: 5′-crRNA-TFBS-tracrRNA-3′; 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-3' (wherein the TFBS is integrated anywhere within the tracrRNA sequence, including as an extension of one or more hairpin structures thereof); 5′-crRNA-tracrRNA-TFBS-3′; 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-3’; 5'-crRNA-TFBS-tracrRNA-TFBS-3'; 5’-crRNA-tracrRNA’-TFBS-tracrRNA”-TFBS-3’; 5’-crRNA-TFBS-tracrRNA’-TFBS-tracrRNA”-TFBS-3’; 5'-crRNA-TFBS-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’; 5'-crRNA-TFBS-tracrRNA'- TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’; 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-3’; 5'-crRNA-tracrRNA'-TFBS-tracrRNA"-TFBS-tracrRNA"'-TFBS-tracrRNA""-TFBS-3'; 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3'; 5'-crRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-tracrRNA-TFBS-3'; 5'-crRNA-tracrRNA'-TFBS-tracrRNA”-TFBS-tracrRNA”’-3’; 5'-crRNA-tracrRNA'- TFBS-tracrRNA”-TFBS-tracrRNA”’-TFBS-tracrRNA””-3’; wherein tracrRNA', tracrRNA", tracrRNA"' and tracrRNA"' are consecutive segments of the complete tracrRNA sequence.

22. The engineered non-naturally occurring system of any one of claims 1 to 21, or a sgCNA subcomponent thereof, wherein the PGM comprises one or more different TFBSs including response elements for multiple different transcription factors.

23. The engineered non-naturally occurring system of any one of claims 1 to 22, or a sgCNA subcomponent thereof, wherein the PGM comprises a nucleic acid backbone with one or more different TFBSs, wherein the continuity of the nucleic acid backbone is disrupted at one or more positions and the complete nucleic acid sequence is assembled by base pairing of nucleotides from different chains.

24. The engineered non-naturally occurring system of claim 23, or a sgCNA subcomponent thereof, wherein the discontinuity in the nucleic acid backbone is located within one or more TFBSs.

25. The engineered non-naturally occurring system of any one of claims 1 to 24, or a sgCNA subcomponent thereof, wherein the TFBS is separated from the crRNA or tracrRNA by a linker having at least 1, 5, 10, 20 or 30 DNA, RNA or modified nucleotides.

26. An isolated nucleic acid comprising any one or more of the sgCNA, crRNA, tracrRNA, transcription factor binding site, or any other segment of the sgCNA of the PGM of any one of claims 1 to 25; or encoding a DNA binding protein of the PGM of any one of claims 1 to 25.

27. The isolated nucleic acid of claim 26, wherein the nucleic acid contains one or more modified or non-natural nucleotides.

28. The isolated nucleic acid of any one of claims 26 and 27, wherein the nucleic acid is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-200, 200-300, 300-400, or 400-500 bases in length.

29. A vector comprising the isolated nucleic acid of any one of claims 26 to 28 under the control of a heterologous promoter, preferably wherein the vector is an AAV vector or another vector.

30. A virus comprising the isolated nucleic acid of any one of claims 26 to 28, preferably wherein the virus is a lentivirus or an adenovirus.

31. A cell comprising the PGM or sgCNA subcomponent thereof according to any one of claims 1 to 25, and / or the nucleic acid according to any one of claims 26 to 28, and / or the vector according to claim 29, and / or the virus according to claim 30.

32. The cell according to claim 31, wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell.

33. A composition comprising the PGM or sgCNA subcomponent thereof of any one of claims 1 to 25, the nucleic acid of any one of claims 26 to 28, the vector of claim 29, the virus of claim 30, the cell of claim 31, or a combination thereof.

34. The composition according to claim 33, further comprising a cationic or ionizable lipid or a cationic or ionizable polymer, preferably in the form of nanoparticles.

35. The composition according to any one of claims 33 and 34, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

36. A method for reversibly altering the expression of a target gene of interest in a cell in response to one or more intracellular or extracellular environmental signals, comprising contacting the cell with a PGM or sgCNA subcomponent thereof as described in any one of claims 1 to 25, a nucleic acid as described in any one of claims 26 to 28, a vector as described in claim 29, a virus as described in claim 30, a composition as described in any one of claims 33 to 35, or a combination thereof.

37. The method of claim 36, wherein the cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell, a non-human primate cell or a human cell.

38. A method of treating a disease, disorder or injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the PGM or sgCNA subfraction thereof of any one of claims 1 to 25, the isolated nucleic acid of any one of claims 26 to 28, the vector of claim 29, the virus of claim 30, the cell of any one of claims 31 and 32, the composition of any one of claims 33 to 35, or a combination thereof.

39. The method of claim 38, wherein the disease, disorder or injury is selected from cellular stress, excision or incision wounds, radiation exposure, viral or bacterial infection, sepsis, diabetic nephropathy, atherosclerosis, cystic fibrosis, Alzheimer's disease, oxidative stress, ischemia-reperfusion injury, inflammation, cancer, anticancer agent resistance, a genetic disease or any other proliferative disease or disorder, an inflammatory disease or disorder, an autoimmune disease or disorder, a liver disease or disorder, a spleen disease or disorder, a lung disease or disorder, a blood disease or disorder, a nervous system disease or disorder, a gastrointestinal (GI) tract disease or disorder, a genitourinary disease or disorder, an infectious disease or disorder, a musculoskeletal disease or disorder, an endocrine disease or disorder, a metabolic disease or disorder, an immune disease or disorder, a central nervous system (CNS) disease or disorder, a nervous system disease or disorder, an ophthalmic disease or disorder, or a cardiovascular disease or disorder.

40. The method of claim 38, wherein the disease, disorder or injury is selected from excisional or incisional wounds, radiation exposure, viral or bacterial infection, sepsis, diabetic nephropathy, atherosclerosis, cystic fibrosis, Alzheimer's disease, oxidative stress, ischemia-reperfusion injury, inflammation and cancer.

41. A kit comprising the PGM or sgCNA subcomponent thereof of any one of claims 1 to 25, the nucleic acid of any one of claims 26 to 28, the vector of claim 29, the virus of claim 30, the composition of any one of claims 33 to 35, or a combination thereof, and a container and / or instructions for using the kit.

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