Small drug-responsive domains for regulation of function / abundance and delivery of polypeptide payloads

Abstract

Provided herein is an engineered regulatable polypeptide comprising a payload and a drug responsive domain (DRD), wherein the DRD is operably linked to the payload, and the DRD is responsive to a ligand. The DRD is optionally either wild-type or a variant of FKBP13.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 269,582, filed March 18, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing A sequence listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference. The sequence listing has been submitted as an electronic document via the PatentCenter in ASCII format encoded as XML. The electronic document was created on March 14, 2023, is entitled "079445-000741US-1361439_ST26.xml", and is 114,195 bytes in size. [Background technology]

[0003] The administration of biological agents by providing genetically modified cells to subjects or using gene editing methods such as CRISPR / Cas9 methods shows great promise but presents substantial challenges. To optimize therapeutic benefits and reduce off-target effects, a means of regulating genetically modified cells or gene editing agents is needed. Regulation of payloads in genetically modified cells can be achieved by coupling the payload to a drug-responsive domain (DRD) that binds to a ligand, such that administration of the ligand dose-dependently regulates the activity of the payload. However, the size of the payload and other components in the nucleic acid construct that encodes the regulatable payload operably linked to the DRD may prevent the use of known DRDs due to limited capacity in the selected viral or non-viral vector. Thus, regulation of larger payloads such as gene editing agents, or transduction using smaller vectors such as adeno-associated virus (AAV) vectors present substantial challenges in regulation. Summary of the Invention

[0004] The present disclosure relates to compositions and systems that can use DRDs to deliver tunable payloads, as well as methods of making and using the compositions and systems. By way of example, gene editing function can be regulated by, among other things, modifying and / or modulating the abundance and / or activity of the payload. The abundance and / or activity of the gene editing payload can be regulated by operably linking the payload to a drug-responsive domain (DRD) that responds to a ligand, and providing a ligand to the DRD operably linked to the gene editing payload. In some cases, it is necessary to adjust the DRD so that the nucleic acid encoding the DRD can be accommodated in an expression system, such as a viral vector, together with other nucleic acids encoding additional elements that comprise the components of the gene editing system. Thus, small DRDs and nucleic acids encoding the same are provided herein.

[0005] The small DRD is optionally FKBP13 or its variants that respond to ligands such as FK506 (tacrolimus) and / or rapamycin (sirolimus). In the presence of ligand, the abundance or activity of payload increases. Thus, if the payload comprises a gene editing component, FKBP13 in the presence of ligand increases the abundance of gene editing component and / or increases gene editing activity. In the absence of ligand, the payload is downregulated or disabled, and gene editing is reduced or eliminated.

[0006] Provided herein is a nucleic acid that encodes a payload and a FKBP13 or its variant that binds to a ligand, and upon expression, the FKBP13 or its variant is operably linked to the payload. Optionally, the FKBP13 variant comprises one or more mutations in the ligand-binding site, and can further comprise one or more mutations in the non-ligand-binding region. For example, the FKBP13 can be a fragment of FKBP13 (e.g., a C-terminal fragment of less than 100 amino acids). The fragment optionally comprises one or more mutations in the ligand-binding site, and optionally further comprises one or more mutations outside the ligand-binding site. Optionally, the one or more mutations differentially affect the binding of FK5065 and rapamycin. For example, the encoded FKBP13 variant can be designed to modulate the biological activity of the payload more in response to FK506 than in response to rapamycin. Optionally, this differential effect between the response to rapamycin and the response to FK506 is achieved by mutations that reduce the binding affinity of the FKBP13 variant for rapamycin.

[0007] The nucleic acid encoding FKBP13 or a variant thereof and a payload may further comprise or encode additional components, such as one or more promoters or linkers.

[0008] The encoded payload can be one or more biological molecules with a desired biological activity. The desired biological activity can be, for example, gene editing. Thus, the payload optionally includes an RNA-guided endonuclease (e.g., Cas9 endonuclease). The payload optionally further includes at least one guide RNA. By way of example, a nucleic acid encoding a ligand-responsive FKBP13 or its variant is provided herein, which is configured to be packaged in an AAV vector as a single nucleotide sequence comprising a promoter sequence, a guide RNA, and a sequence encoding a Cas9 endonuclease payload, and the promoter sequence, the guide RNA, and the sequence encoding the Cas9 endonuclease comprise at least 3000 base pairs.

[0009] Also provided herein are genetically modified FKBP13, for example, FKBP13 fragments (e.g., C-terminal fragments) having one or more mutations in the ligand binding site and / or one or more mutations in the non-ligand binding site. The genetically modified FKBP13 fragments optionally have a lower rapamycin binding affinity and / or a higher FK506 binding affinity than a control FKBP13 fragment that does not have the mutations.

[0010] Provided herein is a recombinant polypeptide comprising a DRD and a payload, the payload being operably linked to FKBP13 or a variant thereof, and the activity of the payload in a biological system (e.g., mammalian blood) is regulatable by the concentration of a ligand contacting FKBP13. Optionally, the EC of FK506 for FKBP13 or a variant thereof in the biological system is 50 C of FK506 in biological systems max is about 2 to 20 times lower than that of

[0011] Further provided is a vector comprising the expressible nucleic acid described herein, and a cell comprising the vector or expressible nucleic acid.For example, the vector or cell can comprise a nucleotide sequence encoding an RNA-guided endonuclease, and one or more guide RNA sequences, comprising a first nucleotide sequence that hybridizes with a target DNA in the genome of the cell and a second nucleotide sequence that is configured to interact with the RNA-guided endonuclease.Optionally, the vector is a viral vector (e.g., an AAV vector).

[0012] Also provided is a method for producing recombinant cells by introducing the vector into target cells.Furthermore, provided is a method for modulating payload in cells by contacting the target cells containing the vector with a ligand.When the payload comprises a component for gene editing, the method or modulation results in the modulation of gene editing by contacting the target cells containing the vector with a ligand to modify the DNA expressed in the cells.

[0013] For example, a method for modifying a target DNA in a cell includes introducing into the cell a vector comprising (1) an RNA-guided endonuclease-encoding nucleic acid, the nucleic acid comprising a sequence encoding FKBP13 or a variant thereof responsive to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease, and (2) one or more guide RNAs, the nucleic acid comprising a nucleotide sequence that hybridizes to a target DNA in the genome of the cell and a nucleotide sequence configured to interact with the RNA-guided endonuclease. The method further includes contacting the vector-containing cell with an effective amount of a ligand to induce or increase the activity of the RNA-guided endonuclease, where the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically binds to and cleaves the target DNA in the cell. Also provided is a method of treating a disease or disorder responsive to genetic modification in a subject in need thereof, by introducing a vector into one or more cells of the subject and contacting one or more vector-containing cells with an effective amount of a ligand to induce or increase the activity of an RNA-guided endonuclease, where the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically binds to and cleaves the target DNA in one or more cells of the subject. Also provided is a method of treating a disease or disorder responsive to genetic modification in a subject in need thereof, by administering one or more cells containing a vector to the subject and administering an effective amount of a ligand to the subject to induce the activity of an RNA-guided endonuclease, where the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically modifies the genome of one or more cells.

[0014] Disclosed herein is a method for controlling the dose or duration of administration of a payload to a subject, comprising administering to the subject one or more of the nucleic acid constructs, vectors, or cells described herein. The method optionally further comprises controlling the activity level of the payload administered via the one or more nucleic acid constructs, vectors, or cells by administering to the subject a selected amount of a ligand to provide a selected activity of the payload to the subject.

[0015] Also disclosed herein is a method for regulating the expression of downstream target protein or peptide of gene editing process. Such a method may include engineering cells to express engineered regulatable oligomer or polypeptide, which includes a payload, such as Cas9 protein or transcription factor protein, involved in the regulation of gene editor, and this payload is operably linked to DRD. In such a method, the nucleic acid construct or vector described herein is administered to cells (in vivo or in vitro). Optionally, the cell containing the nucleic acid or vector is administered to a subject, and the expression of the target protein or peptide by the cell is regulated.

[0016] The specific embodiments are merely exemplary and therefore non-limiting. The details of one or more non-limiting embodiments of the invention are set forth in the accompanying drawings and the description below. Other embodiments of the invention will be apparent to those skilled in the art from consideration of this disclosure. [Brief description of the drawings]

[0017] [Figure 1A]FIG. 1 is a schematic diagram showing the general organization of an exemplary nucleic acid construct, including (i) a nucleic acid sequence encoding a potential FKBP DRD sequence, (ii) a nucleic acid sequence encoding Aequorea coerulescens GFP (AcGFP), (iii) a nucleic acid sequence encoding an exemplary self-cleaving peptide (Porcine Teschovirus-1 2A (P2A)), and (iv) a nucleic acid sequence encoding mCherry. [Figure 1B] We provide fluorescence-activated cell sorting (FACS) data for a tacrolimus dose-response study in transiently transfected HEK293 cells. Several FKBP13 mutations (D90G, M54A, and M96V) were effective in destabilizing AcGFP and reducing AcGFP fluorescence. [Diagram 2] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-039, FKBP-040, FKBP-041, FKBP-042, FKBP-043, FKBP-044, and FKBP-045 stably transfected into Jurkat cells. [Diagram 3] Superposition of rapamycin and tacrolimus represented as sticks. [Figure 4] Graph showing fluorescence-activated cell sorting (FACS) data of tacrolimus dose response for Jurkat cells stably transduced with FKBP13 constructs designated FKBP-044, FKBP-052, FKBP-053, FKBP-054, FKBP-055, FKBP-056, FKBP-057, FKBP-058, FKBP-059, FKBP-060, FKBP-061, FKBP-062, FKBP-063, or FKBP-064. [Diagram 5] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-044, FKBP-066, FKBP-067, FKBP-068, FKBP-069, FKBP-070, FKBP-071, and FKBP-072 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 6] FIG. 1 is a graph showing tacrolimus dose-response curves for FKBP13 constructs designated FKBP-044, FKBP-073, FKBP-074, FKBP-075, FKBP-076, and FKBP-077 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 7] FIG. 1 is a graph showing tacrolimus dose-response curves for FKBP13 constructs designated FKBP-044, FKBP-083, FKBP-084, FKBP-085, and FKBP-086 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 8] FIG. 1 is a graph showing tacrolimus dose-response curves for FKBP13 constructs designated FKBP-044, FKBP-087, FKBP-088, FKBP-089, FKBP-090, and FKBP-091 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 9] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-044, FKBP-092, FKBP-093, FKBP-094, FKBP-095, FKBP-096, and FKBP-097 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 10] FIG. 1 is a graph showing tacrolimus dose response FACS data (GFP mean fluorescence intensity (MFI)) for Jurkat cells stably transduced with FKBP13 constructs designated FKBP-044, FKBP-059, FKBP-068, FKBP-077, FKBP-084, FKBP-085, FKBP-091, FKBP-094, FKBP-095, and FKBP-097. [Figure 11]FIG. 1 is a graph showing tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-098, FKBP-099, FKBP-100, and FKBP-101 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 12] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-102, FKBP-103, FKBP-104, FKBP-105, and FKBP-106 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 13] FIG. 1 is a graph showing tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-107, FKBP-108, FKBP-109, FKBP-110, and FKBP-111 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 14] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-112, FKBP-113, FKBP-114, FKBP-115, and FKBP-116 transiently transfected into HEK293 cells, as well as the EC50 value for each construct. [Figure 15A] FIG. 1 is a graph showing FACS data of tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-117, FKBP-118, FKBP-119, FKBP-120, FKBP-121, FKBP-122, and FKBP-123 in stably transduced Jurkat cells measured using FACS and MFI of GFP. [Figure 15B]Another representation of tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-117, FKBP-118, FKBP-119, FKBP-120, FKBP-121, FKBP-122, and FKBP-123, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 15C] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-124, FKBP-125, FKBP-126, FKBP-127, FKBP-128, FKBP-129, and FKBP-130, measured using FACS and MFI of GFP. [Figure 15D] Another representation of tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-124, FKBP-125, FKBP-126, FKBP-127, FKBP-128, FKBP-129, and FKBP-130, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 15E] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-131, FKBP-132, FKBP-133, FKBP-134, FKBP-135, FKBP-136, and FKBP-137, measured using FACS-based GFP MFI. [Figure 15F] Another representation of tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-131, FKBP-132, FKBP-133, FKBP-134, FKBP-135, FKBP-136, and FKBP-137, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 15G]FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-138, FKBP-139, FKBP-140, FKBP-141, FKBP-142, FKBP-143, and FKBP-144, measured using FACS-based GFP MFI. [Figure 15H] Another representation of tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-138, FKBP-139, FKBP-140, FKBP-141, FKBP-142, FKBP-143, and FKBP-144, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 15I] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-091, FKBP-145, FKBP-146, FKBP-147, FKBP-148, FKBP-149, and FKBP-150, as measured using FACS-based GFP MFI. [Figure 15J] Another representation of tacrolimus dose-response curves for FKBP13 constructs designated FKBP-091, FKBP-145, FKBP-146, FKBP-147, FKBP-148, FKBP-149, and FKBP-150, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 16A] Graph showing tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-151, FKBP-152, and FKBP-153, as measured using FACS-based GFP MFI. [Figure 16B] Another representation of tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-151, FKBP-152, and FKBP-153, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 17A]Graph showing tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-153, FKBP-155, and FKBP-157, as measured using FACS-based GFP MFI. [Figure 17B] Another representation of tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-153, FKBP-155, and FKBP-157, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 18A] Graph showing tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-154, FKBP-155, and FKBP-156, as measured using FACS-based GFP MFI. [Figure 18B] Another representation of tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-154, FKBP-155, and FKBP-156, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 19A] Graph showing tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-157, FKBP-158, and FKBP-159, as measured using FACS-based GFP MFI. [Figure 19B] Another representation of tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-157, FKBP-158, and FKBP-159, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 20A] FIG. 13 is a graph showing tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-044 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 20B]Another representation of tacrolimus (T) vs. rapamycin (S) response dose curves for FKBP13 constructs designated FKBP-044 and FKBP-117, with data expressed as the ratio of GFP MFI to mCherry MFI. [Figure 21] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-044, FKBP-059, FKBP-091, and AcGFP-001, as measured using FACS-based GFP MFI. [Figure 22] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-191, FKBP-199, FKBP-197, FKBP-200, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 23] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-201, FKBP-243, FKBP-244, FKBP-251, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 24] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-195, FKBP-198, FKBP-209, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Diagram 25] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-214, FKBP-215, FKBP-220, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 26] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-223, FKBP-226, FKBP-240, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 27]FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-192, FKBP-203, FKBP-228, FKBP-229, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 28] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-230, FKBP-233, FKBP-255, FKBP-91 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 29] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-256, FKBP-257, FKBP-258, FKBP-259, and FKBP-195, as measured using FACS-based GFP MFI. [Diagram 30] FIG. 13 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-230, FKBP-233, FKBP-255, FKBP-91, and FKBP-117, with data expressed as the ratio of GFP MFI to mCherry MFI. [Diagram 31] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-260, FKBP-262, FKBP-263, FKBP-265, FKBP-268 and FKBP-195, as measured using FACS-based GFP MFI. [Diagram 32] FIG. 13 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-260, FKBP-262, FKBP-263, FKBP-265, FKBP-268, and FKBP-195, with data expressed as the ratio of GFP MFI to mCherry MFI. [Diagram 33]FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-270, FKBP-271, FKBP-273, FKBP-274, FKBP-275 and FKBP-195, as measured using FACS-based GFP MFI. [Diagram 34] FIG. 13 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-270, FKBP-271, FKBP-273, FKBP-274, FKBP-275, and FKBP-195, with data expressed as the ratio of GFP MFI to mCherry MFI. [Diagram 35] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-276, FKBP-277, FKBP-278, FKBP-279, FKBP-117, FKBP-195 and FKBP-291, as measured using FACS-based GFP MFI. [Diagram 36] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-280, FKBP-281, FKBP-282, FKBP-283, FKBP-117, FKBP-195 and FKBP-291, as measured using FACS-based GFP MFI. [Figure 37] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-284, FKBP-285, FKBP-286, FKBP-287, FKBP-117, FKBP-195 and FKBP-251, as measured using FACS-based GFP MFI. [Figure 38] FIG. 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-288, FKBP-289, FKBP-290, FKBP-291, FKBP-292, FKBP-195, FKBP-251 and FKBP-117, as measured using FACS-based GFP MFI. [Figure 39]FIG. 13 is a graph showing drug dose response curves for FKBP13 constructs designated FKBP-272 and FKBP-195, as measured using FACS-based GFP MFI. [Diagram 40] Graph showing drug dose response curves for FKBP13 constructs designated FKBP-258, FKBP-251, and FKBP-259, as measured using FACS-based GFP MFI. [Diagram 41] Graph showing drug dose response curves for FKBP13 constructs designated FKBP-256, FKBP-257, and FKBP-228, as measured using FACS-based GFP MFI. [Diagram 42] 1 is a graph showing a tacrolimus dose response curve for the FKBP13 construct designated FKBP-228, as measured using FACS-based MFI of GFP. BR1 and BR2 refer to replicate 1 and replicate 2. [Diagram 43] 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-256 and FKBP-257, as measured using FACS-based GFP MFI. BR1 and BR2 refer to replicate 1 and replicate 2. [Diagram 44] FIG. 13 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-167 and FKBP-168, as measured using FACS-based GFP MFI. [Diagram 45] 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-265 and FKBP-264, as measured using FACS-based GFP MFI. BR1 and BR2 refer to replicate 1 and replicate 2. [Figure 46] FIG. 13 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-197 and FKBP-251, as measured using FACS-based GFP MFI. [Figure 47]1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-258 and FKBP-259, as measured using FACS-based GFP MFI. BR1 and BR2 refer to replicate 1 and replicate 2. [Figure 48] 1 is a graph showing a tacrolimus dose response curve for the FKBP13 construct designated FKBP-215, as measured using FACS-based MFI of GFP. BR1 and BR2 refer to replicate 1 and replicate 2. [Figure 49] 1 is a graph showing tacrolimus dose response curves for FKBP13 constructs designated FKBP-262 and FKBP-263, as measured using FACS-based GFP MFI. BR1 and BR2 refer to replicate 1 and replicate 2. [Figure 50] none. [Figure 51] FIG. 13 is a graph showing a tacrolimus dose response curve for an FKBP13 construct called FKBP-195 at various serum concentrations as measured using FACS-based MFI of GFP. [Figure 52] FIG. 13 is a graph showing a tacrolimus dose response curve for an FKBP13 construct called FKBP-251 at various serum concentrations as measured using FACS-based MFI of GFP. [Figure 53] FIG. 13 is a graph showing a tacrolimus dose response curve for an FKBP13 construct called FKBP-195 at various serum concentrations as measured using FACS-based MFI of GFP. [Figure 54] FIG. 13 is a graph showing a tacrolimus dose response curve for an FKBP13 construct called FKBP-251 at various serum concentrations as measured using FACS-based MFI of GFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure provides compositions and systems for regulatory control of payloads. The abundance and / or activity of the payload can be regulated by operably linking the payload to a drug-responsive domain (DRD) that responds to a ligand, and providing a ligand to the DRD operably linked to the payload, such that the abundance and / or activity of the payload increases in a dose-dependent manner in the presence of the ligand. The DRD is optionally FKBP13 or a variant thereof that responds to a ligand such as FK506 (tacrolimus) and / or rapamycin (sirolimus). By way of example, if the payload comprises a gene editing component, FKBP13 in the presence of the ligand increases the abundance of the gene editing component and / or increases the gene editing activity. In the absence of the ligand, the payload abundance is downregulated, resulting in reduced or absent gene editing. Accordingly, provided herein are regulatable polypeptides comprising a payload, a DRD, and optionally one or more additional components (e.g., linkers, hinge and transmembrane domains, tails, tags); nucleic acids encoding the regulatable polypeptides; vectors comprising nucleic acids encoding the regulatable polypeptides; cells comprising the nucleic acids or vectors described herein; and methods of making and using the regulatable polypeptides (e.g., methods of gene editing and methods of treating a subject with a regulated payload).

[0019] composition Engineered Regulatable Polypeptides Provided herein is an engineered regulatable polypeptide having at least one payload and at least one FKBP13 drug response domain (DRD), wherein the FKBP13 DRD is operably linked to the payload, and the FKBP13 DRD is responsive to a ligand. The activity level of the payload ranges from a basal activity level in the absence of the ligand to a maximum activity level in the presence of a saturating amount of the ligand. A saturating amount of the ligand refers to an amount of the ligand equal to or greater than the amount that results in the maximum abundance and / or activity of the payload. For example, the activity level of at least one payload ranges from a basal activity level in the absence of the ligand to a maximum activity level in the presence of a saturating amount of the ligand.

[0020] Without intending to be limited by theory, it is believed that DRDs are unstable polypeptides that degrade in the absence of a corresponding stabilizing ligand (also referred to as counter-ligand or ligand), but whose stability is rescued by binding to a stabilizing ligand. Because the binding of the ligand to the DRD is reversible, subsequent removal of the ligand may result in the DRD unfolding, becoming unstable, and eventually being tagged for degradation by the ubiquitin-proteasome system ("UPS"). Thus, it is believed that when a DRD, such as the FKBP13 DRD, is operably linked to a payload, the entire construct (i.e., the DRD and payload) becomes unstable and is degraded by the UPS. However, in the presence of the counter-ligand, the construct is stabilized and the payload remains usable. Furthermore, it is believed that the conditional nature of DRD stability allows for a rapid and unperturbed switch from a stable polypeptide to an unstable UPS substrate, which may facilitate regulation or modulation of the activity level of the payload.

[0021] Since the abundance and availability of the payload are related to the activity of the payload, for the purposes of this disclosure, the terms abundance, availability, activity, and abundance and / or activity (and similarly, abundance level, availability level, activity level, and abundance and / or activity level) are used interchangeably throughout this disclosure and generally referred to as activity, unless expressly stated otherwise or the context otherwise requires. Furthermore, abundance or availability measurements are used as proxies for activity levels and may be used herein to reflect activity levels. Thus, the change in abundance or availability of the payload in the presence of an effective amount of the ligand compared to the absence of the ligand optionally serves as a proxy for measuring the change in activity level.

[0022] Exemplary components of the regulatable polypeptides described herein are referenced throughout this disclosure and are provided below. Any of the polypeptides described herein may include at least a payload, such as a gene editing payload, and a DRD. Optionally, the polypeptide may include one or more additional components, such as a linker, a hinge (e.g., a shedding hinge and a non-shedding hinge), a tail (e.g., a cytoplasmic tail), and a transmembrane domain. As described below in the section related to the method of production, a person skilled in the art can select from various components using the guidelines provided below to achieve a desired outcome (e.g., the location of the payload or DRD relative to the cell in which it is expressed, and the desired activity of the payload).

[0023] DRD The DRD interacts with a ligand such that when the DRD is operably linked to a payload, it results in ligand-dependent reversible modulation of a payload characteristic (e.g., activity). Although referred to as a drug-responsive domain, the ligand to which the DRD responds does not have to be a drug. Suitable FKBP13 DRDs (and their paired ligands), sometimes referred to as destabilizing domains or ligand-binding domains, are provided elsewhere herein.

[0024] The FKBP13 DRD can be, for example, an amino acid sequence comprising a wild-type FKBP13 DRD or a variant (e.g., genetically modified) FKBP13 DRD that responds to a ligand. Optionally, the FKBP13 DRD is a full-length wild-type FKBP13 polypeptide (SEQ ID NO: 1) or a fragment thereof (i.e., a DRD functional fragment that binds to a ligand). For example, the FKBP13 DRD can be a fragment of FKBP13, for example, a C-terminal fragment of SEQ ID NO: 1, such as SEQ ID NO: 2. In some embodiments, the C-terminal fragment comprises less than 100 amino acids. In some embodiments, the fragment comprises SEQ ID NO: 2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2.

[0025] The genetically modified FKBP13 optionally comprises one or more mutations compared to wild-type FKBP13 DRD (SEQ ID NO: 1). Optionally, the genetically modified FKBP13 is a C-terminal fragment of SEQ ID NO: 1 of less than 100 amino acids, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-109, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-109. One or more genetic modifications, such as mutations (including truncations, substitutions and deletions) in the amino acid sequence of FKBP13 may be advantageous, as outlined in the Examples.

[0026] The term identity or substantial identity, as used in the context of the polypeptide or polynucleotide sequences described herein, refers to a sequence having at least 80% sequence identity with a reference sequence. Alternatively, the percent identity can be any integer between 80% and 100%. Exemplary embodiments include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by comparison with a reference sequence using programs known to those of skill in the art, such as BLAST, using standard parameters, as described below.

[0027] In sequence comparison, typically, one sequence is used as a reference sequence, and test sequences are compared with it. When using sequence comparison algorithm, test sequences and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. Then, sequence comparison algorithm calculates the percent sequence identity of test sequence relative to reference sequence based on program parameters.

[0028] A comparison window as used herein includes reference to a segment of any one of the following numbers of consecutive positions selected from the group consisting of 20-600, about 20-50, about 20-100, about 50-200, or about 100-150, within which a sequence may be compared to a reference sequence of the same number of consecutive positions after optimally aligning the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be accomplished by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0029] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available via the National Center for Biotechnology Information (NCBI) website. This algorithm requires first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction is stopped if: the cumulative alignment score deviates from its maximum performed value by an amount X; if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or if the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.

[0030] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One of the similarity measures provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0031] In some embodiments, genetically modified FKBP13 comprises one or more mutations in the binding site of FK506.In some embodiments, genetically modified FKBP13 fragment has lower rapamycin binding affinity than control FKBP13 fragment, for example, control FKBP13 fragment lacking specific mutations in or outside the binding site, which change the three-dimensional structure of the binding site.In some embodiments, genetically modified FKBP13 fragment has higher FK506 binding affinity than control FKBP13 fragment that does not have mutation in the ligand binding site.In some embodiments, genetically modified FKBP13 fragment has higher FK506 binding affinity than control FKBP13 fragment, and optionally has lower rapamycin binding affinity than control FKBP13 fragment.

[0032] Although numerous FKBP13 DRDs are described herein, those skilled in the art can identify additional FKBP13 DRDs suitable for use in regulatable compositions according to the present disclosure. As an example, FKBP13 DRDs can be identified by using library screening and structure-guided engineering to select optimal FKBP13 DRD variants that have sufficient instability in the absence of ligand and sufficient stability in the presence of ligand. Random mutagenesis screening by transducing mutant FKBP13 DRD candidates into cells (e.g., Jurkat cells) can be used to generate variant libraries. To create enriched libraries, cells with desired characteristics (low basal activity / expression and high dynamic range) are selected by testing the abundance of the polypeptide at various ligand concentrations. Single cell clones are then produced and characterized to identify candidate FKBP13 DRDs. The FKBP13 DRDs described herein respond to counter-ligands (also referred to as stabilizing ligands or simply ligands).

[0033] Optionally, the EC of FK506 against FKBP13 or a variant thereof in a biological system. 50 C of FK506 in biological systems max The biological system may be about 2-fold to about 20-fold lower than. Biological systems include, but are not limited to, tissues, cells, or blood (e.g., mammalian blood).

[0034] payload For example, the payload can be any polypeptide(s) having a desired biological function. Such payload can be a modified polypeptide, such as a glycosylated polypeptide or a lipopeptide, or any active portion thereof. For example, the payload can be an RNA-guided endonuclease, such as a Cas9 polypeptide, or an active portion thereof.

[0035] In some embodiments, the payload of the present disclosure can be one or more components of a gene editing system. In such embodiments, the engineered regulatable polypeptide regulates the activity of the gene editing system, thereby regulating the expression of a downstream target protein. As used herein, a target protein refers to a protein selected for gene editing, including, for example, a protein that has a genetic mutation that causes a deleterious effect in a subject or cell. Any of the polypeptides described herein, including one or more components of a gene editing system, can be used to modify the genome of a cell in vitro or in vitro to change the expression and / or activity of a target protein in the cell.

[0036] When the payload comprises an RNA-guided endonuclease, any one of several endonucleases can be selected. For example, the selected RNA-guided endonuclease can be a Cas protein (CRISPR-associated protein), such as Cas9 and Cas12. CRISPR / Cas system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. Wild-type type II CRISPR / Cas system utilizes an RNA-mediated nuclease, such as Cas9, complexed with guide and activator RNA to recognize and cleave foreign nucleic acids. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual-activity guide RNAs are referred to as single guide RNAs (sgRNAs).

[0037] As used herein, the term Cas9 refers to an RNA-mediated endonuclease (e.g., derived from bacteria or archaea, or derived / modified therefrom). Exemplary RNA-mediated nucleases in the payload include Cas9 proteins and their homologs. Other RNA-mediated nucleases include Cpf1 (Cas12a) (see, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015) and its homologs. Of course, in any of the embodiments described herein, the Cas9 nuclease can be replaced with a Cpf1 nuclease or any other guided nuclease.

[0038] Cas9 homologs are found in a wide variety of eubacteria, including but not limited to bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologs are described, for example, in Chylinksi et al., RNA Biol. 2013 May 1; 10(5): 726-737, Makarova et al., Nat. Rev. Microbiol. 2011 June; 9(6): 467-477, Hou et al., Proc Natl Acad Sci USA. 2013 Sep 24; 110(39): 15644-9, Sampson et al., Nature. 2013 May 9; 497(7448): 254-7, and Jinek et al., Science. 2012 Aug 17; 337(6096): 816-21. Any variant of the Cas9 nuclease provided herein can be optimized for efficient activity or improved stability in a host cell. Thus, engineered Cas9 nucleases are also contemplated. See, e.g., Slaymaker et al., Science 351(6268):84-88(2016)).

[0039] In some cases, Cas9 protein is a nickase, and thus when it binds to target nucleic acid as part of a complex with guide RNA, it introduces a single-strand break or nick into the target nucleic acid.By targeting a pair of Cas9 nickases, each of which is bound to a structurally different guide RNA, to two proximal sites of the target genomic region, a pair of proximal single-strand breaks can be introduced into the target genomic region.Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations (see, for example, Ran et al., Cell 154(6):1380-1389(2013)).

[0040] The RNA-guided endonuclease, e.g., Cas9 endonuclease, may be an inactive Cas9, e.g., dCas9. As used herein, a dCas9 polypeptide is a non-activated or nuclease-inactive Cas9 modified to inactivate Cas9 nuclease activity. Modifications include, but are not limited to, changing one or more amino acids to inactivate nuclease activity or nuclease domain. Other modifications include removing all or part of the nuclease domain of Cas9, such that no sequence exhibiting nuclease activity is present in Cas9. Thus, dCas9 may include a polypeptide sequence modified to inactivate nuclease activity, or removal of a polypeptide sequence(s) to inactivate nuclease activity. dCas9 retains the ability to bind to DNA even when nuclease activity is inactivated. Thus, dCas9 contains the polypeptide sequence(s) necessary for DNA binding, but contains modified nuclease sequences or lacks the nuclease sequences involved in nuclease activity. Of course, similar modifications can be made to inactivate nuclease activity in other site-directed nucleases, such as Cpf1.

[0041] Optionally, a polypeptide comprising an RNA-guided endonuclease, such as Cas9 endonuclease, linked to the FKBP13 DRD described herein is complexed with one or more guide RNAs in vitro, and the resulting complex is introduced into a cell. Optionally, the complex may comprise a nucleotide sequence, such as a DNA template, for insertion into a specific site in the genome sequence of the cell. As used herein, the phrase "introduce" in the context of introducing a nucleic acid, polypeptide, or complex described herein refers to the translocation of a nucleic acid sequence, polypeptide, or complex from outside the cell to inside the cell. In some cases, introducing refers to the translocation of a nucleic acid, polypeptide, or complex from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-penetrating peptides, liposome-mediated translocation, etc.

[0042] As used herein, the phrase modifying in the context of modifying the genome of a cell refers to inducing a structural change in the sequence of the genome at a target genomic region. For example, modifying can take the form of inserting a nucleotide sequence into the genome of a cell. For example, a nucleotide sequence encoding one or more polypeptides can be inserted into the genomic sequence of a cell at a specific locus. Such modification can be achieved, for example, by inducing a double-stranded break in the target genomic region, or a pair of single-stranded nicks on opposite strands that flank the target genomic region. Methods for inducing a single-stranded or double-stranded break in or within a target genomic region include the use of a Cas9 nuclease domain or a derivative thereof, and a guide RNA or a pair of guide RNAs that are directed to the target genomic region.

[0043] Other gene editing payloads include nucleases (e.g., zinc finger nucleases, TALENs (transcription activator-like effector-based nucleases), or meganucleases) and / or recombinases such as Cre recombinase. As used herein, the term "TALEN" refers to a protein that includes a transcription activator-like (TAL) effector binding domain and a nuclease domain, including monomeric TALENs that are functional alone and others that require dimerization with another monomeric TALEN. Dimerization can result in homodimeric TALENs when both monomeric TALENs are identical, or heterodimeric TALENs when the monomeric TALENs are different. Although TALENs are often used in pairs, monomeric TALENs are known. Genetic modifications performed by TALENs or other tools can be selected from the list consisting of, for example, insertions, deletions, insertions of exogenous nucleic acid fragments, and substitutions. Generally, a target DNA site is identified and a TALEN pair is created that specifically binds to the site. TALEN is provided to cell or embryo, for example, as protein, mRNA, or by the vector that codes for TALEN.TALEN cuts DNA to generate double-strand breaks, which are subsequently repaired, often resulting in the creation of indels, or the sequence or polymorphism contained in the accompanying exogenous nucleic acid is integrated, which is either inserted into chromosome or serves as a template for repairing breaks with modified sequence.

[0044] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by fusing zinc finger DNA binding domains to DNA cleavage domains. Zinc finger domains can be engineered to target desired DNA sequences, allowing zinc finger nucleases to target unique sequences in complex genomes. These reagents can be used to alter the genomes of higher organisms by exploiting endogenous DNA repair mechanisms. ZFNs can be used in methods to inactivate genes. Materials and methods for using zinc fingers and zinc finger nucleases to generate genetically modified animals are disclosed, for example, in US8,106,255, US2012 / 0192298, US2011 / 0023159, and US2011 / 0281306.

[0045] Linker Many linker sequences (linkers) are known in the art. Linkers include, for example, GS linkers, GSG linkers, and GGSG linkers. These linkers are one or more repeats of subunits. Thus, GS linkers are GSn linkers, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Similarly, GSG linkers are GSGn linkers, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. GGSG linkers are GGSGn linkers, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0046] Hinge and transmembrane domains Hinge sequences are short amino acid sequences that lend themselves to flexibility between the connected components. Hinge sequences can be any suitable sequence derived or obtained from any suitable molecule. Hinge sequences can be derived from all or part of the immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of the immunoglobulin (e.g., IgG4 Fc hinge), or the extracellular region of type 1 membrane proteins, such as CD8α CD4, CD28, and CD7, which can be wild-type sequences or derivatives thereof. Some hinge regions include the immunoglobulin CH3 domain or both the CH3 and CH2 domains. In some embodiments, the hinge is derived from a transmembrane domain.

[0047] Transmembrane domains useful in the engineered regulatable polypeptide constructs of the present disclosure can include, for example, an MHC1 transmembrane domain, a CD8α transmembrane domain, a B7-1 transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 Fc region.

[0048] Intracellular / cytoplasmic or transmembrane tails Optionally, the polypeptide construct provided herein comprises an intracellular / cytoplasmic or transmembrane tail.Optionally, the intracellular / cytoplasmic or transmembrane tail is CD8, CD40L, LIGHT, NKG2C or B7.1 intracellular tail.The absence of a transmembrane region in the construct can be designed, for example, for secreted payloads or payloads with intracellular or nuclear activity.

[0049] tag Optionally, the polypeptide described herein comprises a tag. Such tag allows the polypeptide to be isolated or detected, or the payload to be isolated or detected. Such tag optionally comprises a fluorescent protein (e.g., green fluorescent protein), His tag, HA tag, Myc tag, FLAG tag, mCherry, CD20, CD34, nerve growth factor receptor (NGFR), truncated NGFR (tNGRR), epidermal growth factor (EGFR), or truncated EGFR (tEGFR).

[0050] Ligand The ligand can be any agent that binds to the FKBP13 DRD of an engineered regulatable polypeptide described herein, an effective amount of which results in a measurable change in the characteristics (e.g., abundance, availability, activity) of a payload operably linked to the FKBP13 DRD. In some embodiments, the ligand can be a synthetic molecule. In some embodiments, the stabilized ligand of the present disclosure can be a small molecule compound. The stabilized ligand is optionally a small molecule therapeutic agent that has been previously approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA). Optionally, the FKBP13 DRD responds to a counter ligand that is a small molecule drug, such as an FDA-approved small molecule, e.g., tacrolimus (FK506) and / or sirolimus (rapamycin). As described herein, the DRD can be modified to affect the binding affinity of the ligand to the DRD. Optionally, the DRD can be modified to differentially affect the binding of a first ligand (e.g., tacrolimus) compared to a second ligand (e.g., sirolimus). For example, the modified DRD may exhibit increased binding affinity to a first ligand and decreased binding affinity to a second ligand compared to a control unmodified DRD.

[0051] nucleic acid Provided herein is an expressible nucleic acid construct encoding one or more engineered regulatable polypeptides described herein. For example, provided herein is a nucleic acid encoding a ligand-responsive FKBP13 or variant thereof (e.g., genetically modified FKBP13) and a biologically active payload, where upon expression of the nucleic acid, FKBP13 is operably linked to the payload. Upon expression of the nucleic acid, FKBP13 can interact with an effective amount of the ligand to modulate the biological activity of the payload. In some embodiments, the nucleic acid construct encodes a payload (e.g., a gene editing payload) and a DRD, and optionally encodes additional components such as hinges, linkers, transmembrane domains, tags, and intracellular / cytoplasmic and transmembrane tails as described herein. The nucleic acid construct also optionally encodes additional components such as signal sequences and cleavage sites including shedding domains. The construct optionally further comprises a promoter sequence and other regulatory elements, such as enhancers, translational control elements (e.g., IRES), and elements that control half-life.

[0052] The term nucleic acid, as used throughout, refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. It will be understood that when a DNA sequence is described, its corresponding RNA is also described, in which case thymidine is represented as uridine. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence specified, but also modified variants, alleles, orthologs, SNPs, and complementary sequences thereof.

[0053] Optionally, the nucleic acid encodes a ligand-responsive FKBP13 fragment. Optionally, the FKBP13 fragment, e.g., a C-terminal fragment, is less than 100 amino acids. Optionally, the encoded FKBP13 fragment is less than 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 amino acids. Optionally, the encoded FKBP13 fragment is 90-99 amino acids.

[0054] In some embodiments, the nucleic acid encodes SEQ ID NO:1 (i.e., wild-type FKBP13) or a fragment thereof. Optionally, the nucleic acid encodes a variant of SEQ ID NO:1 or a fragment thereof. The encoded FKBP13 fragment is optionally SEQ ID NO:2. In some embodiments, the encoded FKBP13 is a variant of SEQ ID NO:2 that includes one or more mutations relative to SEQ ID NO:2, such as an FKPB13 that includes an amino acid sequence that has at least 80%, 85%, 90%, 95%, 99% or 100% identity to any one of SEQ ID NOs:3-109. In some embodiments, the encoded FKBP13 variant includes one or more mutations in the ligand binding site. Residues of FKBP13 that are within 4 angstroms of tacrolimus have been found to be part of the binding site of FKBP13. The binding site for FKBP13 includes residues Y56, F66, D67, F76, Q84, V85, I86, W89, Y112, R115, K120, I121 and F129.

[0055] The FKBP13 ligand is optionally FK506 (tacrolimus), rapamycin (sirolimus), or both FK506 (tacrolimus) and rapamycin (sirolimus), whereby the encoded FKBP13 binds to one or both ligands. In some embodiments, the encoded FKBP13 variant modulates the biological activity of the payload 5-10 fold, e.g., about 7 fold, greater in response to FK506 than in response to rapamycin.

[0056] Some nucleic acid sequences described herein encode a payload that includes an RNA-guided endonuclease, for example, a CRISPR / Cas-associated endonuclease. Optionally, the CRISPR / Cas-associated endonuclease is a Cas9 endonuclease or a Cas12 endonuclease. Optionally, the Cas9 endonuclease is a Staphylococcus aureus Cas9 (SaCas9) or a Staphylococcus lugdunensis (SluCas9). Optionally, the nucleic acid further encodes at least one guide RNA sequence. As used throughout, a guide RNA (gRNA) is a sequence that interacts with a site-specific or targeted nuclease (e.g., an RNA-guided endonuclease) and specifically binds or hybridizes to a target nucleic acid in the genome of a cell, such that the gRNA and the targeted nuclease are co-localized to the target nucleic acid in the genome of the cell. Each gRNA comprises a DNA targeting sequence or protospacer sequence of about 10-50 nucleotides in length that specifically binds or hybridizes to a target DNA sequence in a genome. For example, the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

[0057] In some embodiments, the RNA-guided endonuclease, e.g., the Cas9 protein, may be in an active endonuclease form such that upon binding to a target nucleic acid as part of a complex with a guide RNA, a double-stranded break is introduced into the target nucleic acid.

[0058] Vectors and cells Also provided herein is a vector for expressing one or more of the nucleic acids. Such vectors can be selected from viral and non-viral vectors, plasmids, cosmids, and artificial chromosomes. By way of example, the vector can be a viral vector, such as an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, or an adenovirus vector. The vector optionally comprises a nucleic acid sequence encoding a transposase and / or a nuclease. Optionally, the vector comprises one or more inverted terminal repeats (ITRs).

[0059] In some cases, the vector comprises any of the nucleic acid sequences described herein, wherein the encoded payload is an RNA-guided endonuclease, and the vector further comprises one or more guide RNA sequences comprising a first nucleotide sequence that hybridizes to a target DNA in the genome of the cell and a second nucleotide sequence configured to interact with the RNA-guided endonuclease.

[0060] In some embodiments, the nucleic acid is configured to be packaged in a vector, e.g., a viral vector, as a single nucleotide sequence comprising a promoter sequence, a guide RNA, and a sequence encoding a Cas9 endonuclease payload. Optionally, the nucleic acid is configured to be packaged in an AAV vector as a single nucleotide sequence comprising a promoter sequence, a guide RNA, and a sequence encoding a Cas9 endonuclease payload, and the promoter sequence, the guide RNA, and the sequence encoding Cas9 comprise at least 3000 base pairs. Such vector constructs require a small DRD, taking into account the limited capacity of the vector and the size of the payload. Nucleic acids encoding the FKBP13 DRD or variants thereof, such as fragments of FKBP13 with 100 amino acids or less, are particularly well suited for such vector constructs.

[0061] Cells are provided that include one or more nucleic acid constructs or vectors described herein. Optionally, the cells are immune cells, stem cells, muscle cells, etc. Optionally, the immune cells are primary human T cells, such as human peripheral blood mononuclear cells (PBMCs), PBMCs collected after stimulation with G-CSF, bone marrow, or T cells derived from umbilical cord blood. In some embodiments, the immune cells are tumor infiltrating lymphocytes (TILs), for example, collected from tumors. The immune effector cells may be NK cells, αβ T cells, iNKT cells, γδ T cells, macrophages, B cells, dendritic cells, bone marrow-derived progenitor cells, eosinophils, basophils, neutrophils, or Tregs. Optionally, the stem cells are hematopoietic stem cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs). The cells provided herein are optionally mammalian cells, or more specifically, human cells.

[0062] As used herein, the phrase hematopoietic stem cells refers to a type of stem cell that can give rise to blood cells. Hematopoietic stem cells can give rise to cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are found primarily in bone marrow, but can be isolated from peripheral blood. Various cell surface markers can be used to identify, select, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells can be expressed by the c-kit + Katsu - In some cases, human hematopoietic stem cells are identified as CD34 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells are identified as CD34 - , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells are identified as CD133 + , CD59 +, Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, hematopoietic stem cells are identified as CD150 + CD48 - CD244 - It is.

[0063] As used herein, the phrase hematopoietic cells refers to cells derived from hematopoietic stem cells. Hematopoietic cells may be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood or a fraction thereof). Alternatively, hematopoietic cells may be obtained or provided by isolating hematopoietic stem cells and differentiating the stem cells. Hematopoietic cells include cells with limited ability to differentiate into additional cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include lymphoid and myeloid cells, such as lymphocytes, erythrocytes, granulocytes, monocytes, and platelets. In some embodiments, the hematopoietic cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells. In some embodiments, the cells are innate immune cells.

[0064] As used herein, the term T cells refers to lymphoid cells that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells are CD4 + , CD8 + , or CD4 + and CD8 + T cells can also be both CD4 - , CD8 - , or CD4- and CD8 - T cells can be both helper cells, e.g., T H Type 1, T H Type 2, T H Type 3, T H Type 9, T H 17 type, or T FH The T cells can be cytotoxic T cells. The regulatory T cells can be FOXP3 + or FOXP3 - Optionally, the T cells may be CD4 + CD25 hi CD127 lo In some cases, the T cells are type 1 regulatory (Tr1), T H 3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or subpopulation thereof. + T cells. In some cases, the T cells are CD4 + CD25 lo CD127 hi Optionally, the T cell is a CD4 + CD25 lo CD127 hi CD45RA hi CD45RO - The T cells are naive T cells. The T cells may be genetically engineered recombinant T cells.

[0065] As used herein, the term primary in the context of primary cells refers to cells that are not transformed or immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from organisms, systems, organs, or tissues, optionally selected, and directly utilized without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contacting with (e.g., culturing in the presence of) CD3, CD28 agonist, IL-2, IFN-γ, or a combination thereof.

[0066] The disclosure also includes any of the engineered or genetically modified cells, and populations of engineered or genetically modified cells produced by any of the methods described herein.

[0067] Method of preparation The present disclosure provides methods for making nucleic acid constructs and vectors encoding the polypeptides of the present disclosure, methods for making the polypeptides of the present disclosure by expression of the nucleic acid constructs, and methods for making cells comprising the nucleic acid constructs or vectors described herein. In general, a DRD / ligand pair is selected for modulation of a desired payload, e.g., FKBP13 / FK506. Optionally, the ligand is an FDA-approved small molecule drug. In some embodiments, the DRD / ligand pair is selected for clinical tractability. For example, the pair is selected such that the ligand can modulate the abundance of the payload or modulate the activity of the payload within the tolerance range of the ligand. Such range is optionally the FDA-approved dose range of the ligand. The nucleic acid or vector is then designed to encode the components / components of the regulatable polypeptides described herein, which, in the case of a polypeptide, include at least the payload and the FKBP13 DRD. The nucleic acid or vector can be further designed to encode additional components / components, such as a transmembrane domain, a linker, a hinge, a tag, or other components mentioned herein. Using this disclosure, one skilled in the art can select appropriate components and order them in a construct to achieve a desired outcome, for example, the order of components affects whether the DRD regulates the payload at the N-terminus or C-terminus.

[0068] As another example, the linker and linker length can affect the constitutive activity level (i.e., basal activity in the absence of ligand), and in certain embodiments, a particular linker and length is selected to maximize the on-state (e.g., maximum activity level in the presence of ligand) while maintaining a low basal activity level in the absence of ligand. As yet another example, a particular hinge can affect ligand responsiveness by allowing conformational changes, and thus the hinge is selected to provide sufficient dynamic range (i.e., acceptable payload activity range that corresponds to ligand variation from zero or minimal activity to maximum saturating activity) to obtain the desired range of payload abundance and biological activity.

[0069] In some cases, the nucleic acid construct is optionally configured to encode a payload associated with an intracellular or transmembrane domain, such that the payload is optionally anchored to the cell membrane.

[0070] The vehicle / vector is selected and designed to deliver the nucleic acid construct into the desired cell. For example, the vehicle can be selected from the above, including viral vectors (e.g., adeno-associated vectors, lentiviral vectors, retroviral vectors, etc.), plasmids, cosmids, and artificial chromosomes. Such vectors can be designed to encode transposases, nucleases, and elements that control translation (e.g., IRES). The choice of vector can also affect the selection of various building block components. For example, vectors that require smaller constructs may require the use of smaller DRDs. Appropriate components such as promoters, enhancers, multicistronic expression, translation control, and half-life control elements are selected to achieve the desired control of payload abundance or activity.

[0071] Additionally, the nucleic acid construct is designed for cistronic or multicistronic expression as required for the desired expression of various engineered components. In some embodiments, for multicistronic expression, two or more nucleic acids encoding two or more polypeptides, e.g., a fusion of FKBP13 and a payload and a second payload, may be separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, e.g., porcine teschovirus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot and mouth disease virus (F2A) peptide. The self-cleaving 2A peptide allows for the expression of multiple gene products from a single construct. (See, e.g., Chng et al., MAbs 7(2):403-412 (2015)). In some embodiments, the nucleic acid construct comprises two or more self-cleaving peptides. In some embodiments, the two or more self-cleaving peptides are all the same. In other embodiments, at least one of the two or more auto-cleaving peptides is different.

[0072] The cell that is supplied with the nucleic acid is selected at least in part based on the cell's ability to allow expression of the regulatable polypeptide disclosed herein and to allow payload activity in a sufficient dynamic range. Optionally, the cell expresses little or no payload in the absence of the nucleic acid or vector provided. In certain embodiments, the skilled artisan will select a cell that requires increased activity or abundance of the payload in a cell that naturally expresses the payload. In certain embodiments, the skilled artisan will select a cell that requires gene editing. In certain embodiments, the cell is selected as an effector cell, for example, an immune effector cell.

[0073] Those skilled in the art can apply knowledge from this disclosure to construct a variety of regulatable polypeptides, as well as nucleic acids and vectors encoding regulatable polypeptides, and cells engineered to express regulatable polypeptides, beyond those explicitly exemplified herein, within the scope of this disclosure.

[0074] How to use Disclosed herein is a method of modulating a payload, for example, modulating the abundance, availability, and / or activity of a gene editing payload. In some embodiments, the method of modulation is a method of modifying the activity of a payload by engineering a cell to express a payload operably linked to a FKBP13 DRD that responds to a ligand. The activity of the payload (e.g., corresponding to the abundance and / or availability of the payload) is reduced compared to the activity of the payload in a control cell, for example, a cell engineered to express the payload independently of the DRD.

[0075] For example, provided herein is a method of modulating a payload in a cell, the method comprising introducing into the cell a vector comprising a nucleic acid encoding a ligand-responsive FKBP13 or a variant thereof, the nucleic acid further comprising a promoter sequence and a sequence encoding a payload, the method further comprising contacting the cell with an effective amount of the ligand such that a desired level of payload activity is achieved.

[0076] Optionally, the payload has a biological activity level ranging from a basal activity level in the absence of the ligand to a maximum activity in the presence of a saturating amount of the ligand, and the method is a method of modulating the activity of the payload, comprising contacting a cell engineered to express a payload (e.g., a gene editing payload) operably linked to the FKBP13 DRD with an effective amount of the ligand such that the activity of the payload is increased relative to the basal activity level. In some embodiments, the method comprises contacting the cell with a selected amount of the ligand, the selected amount of the ligand resulting in a selected activity level of the payload. In certain embodiments, the method comprises alternatively contacting the cell with different selected amounts of the ligand to achieve different selected activity levels ranging from the basal level to the maximum level.

[0077] The contacting step can be performed in vivo or in vitro. The contacting step is optionally performed to achieve a sustained selected activity of the payload (i.e., to achieve a sustained on-state of the payload) or to achieve intermittent activity of the payload (i.e., to perform a pulsed supply of the payload between on-state and off-state). By off-state, it is meant that the payload activity is at a basal activity level. By on-state, it is meant that the selected activity level in the presence of an effective amount of the ligand is higher than the off-state. The sustained activity of the payload can be achieved by continuously contacting the FKBP13 DRD with an effective amount of the ligand or by performing a subsequent contacting step(s) of the FKBP DRD with the ligand, the subsequent contacting step(s) being performed before the activity level of the payload from the previous contacting step reaches the basal activity level. Each contacting step may be varied with respect to the amount of the ligand such that more payload activity occurs when more ligand is used and less payload activity occurs when less ligand is used within the dynamic range of the ligand / DRD pair. Thus, the amount of ligand can be varied in subsequent contacting steps to up- or down-regulate the amount and / or activity of the payload over time. Each contacting step can also be varied in frequency to achieve a desired pattern of activity levels.

[0078] Also provided is a method for modifying target DNA in a cell, by (a) introducing into the cell a vector comprising (1) an RNA-guided endonuclease-encoding nucleic acid, the nucleic acid comprising a sequence encoding FKBP13 or its variant that responds to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease, and (2) one or more guide RNAs, the nucleic acid comprising a nucleotide sequence that hybridizes to the target DNA in the genome of the cell and a nucleotide sequence configured to interact with the RNA-guided endonuclease, and (b) contacting the vector-containing cell with an effective amount of a ligand to induce the activity of the RNA-guided endonuclease, the RNA-guided endonuclease interacting with the guide RNA, and the RNA-guided endonuclease specifically binding to and cleaving the target DNA in the cell. Any of the methods for modifying cells described herein can be performed in vitro or in vivo. Any of the cells described herein can be genetically modified by any of the methods described herein.

[0079] Treatment Provided herein is a method of delivering a payload operably linked to a DRD to a subject by administering to the subject a nucleic acid or vector described herein or a cell comprising a nucleic acid or vector described herein. Thus, a method of delivering a payload to a subject in need thereof is provided. The payload can be delivered by administering to the subject a nucleic acid construct or vector described herein. The payload can be any biologically active payload, including, for example, a therapeutically effective gene editing payload. The method results in expression and gene editing in a target cell of the subject. Also provided is a method of delivering to a subject one or more cells comprising a nucleic acid or vector encoding a regulatable polypeptide. The one or more cells into which the vector is introduced can be derived from the same subject or a different subject. The one or more cells can be derived from the same or a different subject and then grown in culture before and / or after introduction of the vector.

[0080] By way of example, provided herein is a method of treating a disease or disorder responsive to genetic modification in a subject in need of genetic modification, by (a) introducing into one or more cells of the subject a vector comprising (1) an RNA-guided endonuclease-encoding nucleic acid comprising a sequence encoding FKBP13 or a variant thereof responsive to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease, and (2) at least one guide RNA comprising a nucleotide sequence that hybridizes to a target DNA in the genome of the cell and a nucleotide sequence configured to interact with the RNA-guided endonuclease; and (b) contacting the one or more vector-containing cells with an effective amount of a ligand to induce activity of the RNA-guided endonuclease, whereby the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically binds to and cleaves the target DNA in the one or more cells of the subject.

[0081] Also provided herein is a method of treating a disease or disorder responsive to genetic modification in a subject in need thereof, comprising: (a) administering to the subject one or more cells comprising a vector, the vector comprising (1) an RNA-guided endonuclease-encoding nucleic acid comprising a sequence encoding FKBP13 or a variant thereof responsive to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease, and (2) a guide RNA comprising a nucleotide sequence that hybridizes to a target DNA in the genome of the one or more cells and a nucleotide sequence configured to interact with the RNA-guided endonuclease; and (b) administering to the subject an effective amount of a ligand to induce activity of the RNA-guided endonuclease, wherein the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically modifies the genome of the one or more cells.

[0082] Any of the methods described herein in which one or more cells comprising a nucleic acid or vector encoding a regulatable polypeptide are provided to a subject may further include isolating cells from the subject, transducing the isolated cells with a nucleic acid or vector encoding a regulatable polypeptide described in this disclosure, propagating the cells in vitro, and providing the cells to the same or a different subject. The subject may have a genetic mutation. The cells may be isolated from the same subject that will receive the transduced cells (an autologous source), or the cells may be isolated from a different subject (e.g., an allogeneic source). In certain embodiments, the cells are administered at a concentration of about 1000 cells per injection to about 10 billion cells per injection, e.g., 1×10 cells per injection. 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×103 , 5×10 3 , or any range between any two of these numbers, inclusive. Optionally, 1×10 8 ~1×10 10 Cells are administered to the subject. Optionally, cells are administered once, twice, three times or four times as required. In some methods, one or more genetically modified cells as described herein or produced by the method described herein are administered to the subject.

[0083] The method may further include controlling the dose or duration of administration of the payload to the subject by administering to the subject a selected amount of the paired ligand to provide a selected activity of the payload to the subject. The ligand may be administered to achieve sustained or intermittent payload activity in the subject. Sustained payload activity may be a substantially consistent activity level or the activity level may be modulated. Intermittent activity between an off state and an on state includes modulating activity between an off state and a substantially consistent on state or between an off state and a fluctuating on state activity level. If higher payload activity is desired, the ligand is administered at a higher dose or for a longer administration period, and if lower activity is desired, a reduced or absent ligand dose is selected. The dose and duration of ligand administration, as well as the resulting activity of the payload, may be selected to avoid unacceptable side effects or unwanted toxicity in the subject. The dosage of the ligand and the schedule for administering the dosage of the ligand may be empirically determined by one of skill in the art based on the resulting amount of payload, the activity of the payload, or based on one or more indications of the effect of the payload activity. The range for administration of the ligand can range from any amount above zero to a saturating dose, and the resulting payload activity can range from a basal level to a maximum level, optionally including sufficient dynamic range to allow for a desired dose response to the ligand and the associated activity range of the payload (e.g., for a given ligand and payload, the range of difference between the off-state and maximum payload activity will result from at least a 10-fold range of the ligand). This sufficient dynamic range allows for fine tuning and a dose-response curve that is not unacceptably steep. In embodiments, the dosage or frequency of administration of the ligand, and the resulting abundance and activity of the payload, are selected to avoid, reduce, or limit unacceptable or undesirable adverse side effects, and vary depending on the age, condition, and / or sex of the subject, as well as the type of condition being treated, the extent of the condition, or whether other therapeutic agents are included in the treatment regimen.Guidance on appropriate dosages for a given class of ligand can be found in the literature. Exemplary dosages, e.g., the clinically approved dosage of tacrolimus for various tissue / organ transplants, are 0.1-0.3 mg / kg body weight. Such dosages range from a C of 20-68 ng / ml. max values ​​can be achieved.

[0084] In the treatment methods described herein, the ligand administration regimen, including the selected amount of ligand to administer to the subject and the frequency of administration of the selected amount of ligand, is selected to modulate the payload and / or provide the desired outcome for the subject. The subject is optionally monitored for outcome. Thus, for example, in the treatment of a cancer in a subject, the number of malignant cells in a sample, the circulating tumor DNA in a sample, or the size of a solid tumor when imaged can be detected. When a desired endpoint is achieved (e.g., when successful treatment of a gene mutation is indicated), the ligand can be reduced or discontinued to reduce or eliminate the gene editing payload, e.g., to reduce the abundance, availability, and / or activity of the payload below a predefined threshold value to eliminate or reduce unwanted or undesired side effects. Similarly, the ligand can be reduced or discontinued if the subject develops unacceptable off-target effects or other adverse effects from the payload.

[0085] Also disclosed herein is a method for regulating expression of downstream target proteins of a gene editing process. In some embodiments, such methods include engineering cells to express an oligomer or engineered regulatable polypeptide that includes a payload, such as a CAS9 protein or a transcription factor protein, operably linked to a DRD. In certain embodiments, for a subject with a genetic mutation, a nucleic acid construct or vector according to the present disclosure is provided to the subject's cells to provide a payload that provides a nucleic acid editing polypeptide. The nucleic acid construct or vector is transduced into the subject's target cells, allowing the gene editing payload to modify the nucleic acid (e.g., genomic DNA or RNA) of the transduced cells. The activity level of the payload, and thus the expression of the target protein, can be regulated by administering a ligand to the subject.

[0086] Also provided are methods of treating a disease or disorder that include administering to a subject one or more cells that have been edited using any of the gene editing systems described herein, e.g., CRISPR / Cas9 editing, to edit the genome of the cell.

[0087] Any of the treatment methods described herein may be used to treat a disease or disorder associated with a genetic mutation, such as, but not limited to, Duchenne muscular dystrophy, myotonic dystrophy, cystic fibrosis, sickle cell, beta thalassemia, alpha-1 antitrypsin deficiency, APOL1-mediated kidney disease, or type 1 diabetes.

[0088] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0089] 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. For example, a reference to "a transcript" or "the transcript" may include a plurality of transcripts.

[0090] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise stated.

[0091] The terms "optional" and "optionally" mean that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes the occurrence or presence of the event, circumstance, or material, as well as its occurrence or absence.

[0092] Use of the terms "including," "comprising," or "having," and variations thereof herein are meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments described as "including," "comprising," or "having" particular elements are also contemplated as "consisting essentially of" and "consisting of" those particular elements.

[0093] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted in the alternative ("or").

[0094] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to include the materials or steps described and "those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ461,463 (CCPA 1976) (emphasis in original). See also MPEP §2111.03. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprising."

[0095] The terms "about" and "approximately," when used to refer to measurable values ​​such as an amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, location, length, etc., are meant to account for variations due to experimental error, which may encompass variations of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, location, length, etc. All measurements or numbers are implicitly understood to be modified by the word about, even if the measurement or number is not explicitly modified by the word about. When the terms "about" and "approximately" are used in reference to the location or position of a region within a reference polypeptide, these terms encompass variations of up to ±20 amino acid residues, up to ±15 amino acid residues, up to ±10 amino acid residues, up to ±5 amino acid residues, up to ±4 amino acid residues, up to ±3 amino acid residues, up to ±2 amino acid residues, or even ±1 amino acid residue.

[0096] Reference is made herein to "basal activity level." Basal level, as used herein, can be zero, near zero, or any amount in the absence of exogenous ligand. Basal activity can occur due to endogenous levels of the same or different ligand, or can occur due to resting levels of payload production in the absence of exogenous ligand.

[0097] Reference to "biological activity" is understood to mean under appropriate conditions, even if not so stated.

[0098] As used herein, "contacting" is understood to mean providing an agent (such as a ligand) to a target (such as a DRD) so that the agent and the target can contact each other. For example, contacting includes providing a ligand to a cell in vitro (e.g., when the DRD is extracellular or on the cell surface). As another example, contacting includes providing a ligand to a cell where the DRD is inside the cell, so that the ligand reaches the cytoplasm. Similarly, cells may be contacted in vivo by administering a ligand to a subject so that the ligand reaches the cell or the DRD.

[0099] "DRD" is understood to mean the ligand-responsive domain, even if not so specified.

[0100] The terms "ligand," "counter ligand," and "stabilizing ligand" are used interchangeably and mean the same thing when used in reference to a drug responsive domain ("DRD").

[0101] As used herein, "operably linked" means that in the presence of a counter ligand, the DRD is directly or indirectly linked to the payload to alter a measurable characteristic of the payload (e.g., altering the level of activity of the payload compared to the level of activity in the absence of the counter ligand). In some embodiments, the amount and / or measured level of activity of the payload is increased in the presence of an effective amount of the ligand compared to the measured level of expression or activity in the absence of the ligand. An effective amount of the ligand refers to the amount of the ligand required to see an increase in the amount or measure of activity of the payload. In some embodiments, the effective amount is not so large as to cause unacceptable toxicity or off-target effects. Optionally, the measurable characteristic is a therapeutic outcome, the amount of the payload in a sample, or the level of biological activity of the payload (for which measuring the amount of the payload can serve as a surrogate).

[0102] The term "payload" refers to an agent whose abundance, activity, availability, expression, function, or other characteristic is desired to be modulated by the DRD.

[0103] Whenever the words "connected" or "coupled" are used, they are understood to be accompanied by the words "directly or indirectly" unless expressly stated otherwise or otherwise indicated by context.

[0104] The details of one or more embodiments of the present disclosure are described in the present specification and the accompanying drawings. It is to be understood that other embodiments may be utilized and structural or process changes may be made without departing from the scope of the present disclosure. In other words, exemplary embodiments and aspects are described below. However, it will be understood that in developing such an actual embodiment, numerous implementation-specific decisions may be made to achieve the developer's particular goals, such as compliance with system-related and business-related constraints that may vary from implementation to implementation. Moreover, it will be understood that such a development effort may be complex and lengthy, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of the present disclosure.

[0105] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety.

[0106] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. EXAMPLES

[0107] Example 1 FKBP13 and its fragments were tested to identify DRDs that respond to FDA-approved drugs. For the purposes of these experiments, tacrolimus was used as an exemplary FDA-approved drug. The FKBP13 binding affinity of tacrolimus is 166 nM, which is substantially lower than the FKBP12 binding affinity of 0.2 nM. To increase the responsiveness of FKBP13 to tacrolimus, the binding site of FKBP13 was targeted with the goal of making the FKBP13 binding site more similar to that of FKBP12. To this end, the three-dimensional structures of FKBP12 and FKBP13 upon tacrolimus binding were compared. Based on visual inspection by experts, mutations that could increase the drug responsiveness of FKBP13 were designed. Library-based screening was also performed. DRDs corresponding to SEQ ID NOs: 3-109 were derived from library selection. This library was generated as a site saturation library using a binding site with the A117H variant (Q72R, Q84E, and A117H binding site mutations), where 117H is a residue found in the FKBP12 binding site. While the library was under selection, approximately five additional binding site mutations were designed, with the A117W mutation showing the highest EC 50It was shown that this likely improved binding site and resulted in a six-fold increase in binding affinity. When the library output was analyzed by sequencing, a DRD-genic mutation was generated in the A117W version of the binding site. As a control, five of the top FKBP13 mutants were also generated using the A117H sequence obtained from the library. FKBP-117, 119, 121, 123, and 125 were formatted with the improved binding site (A117W; identified in FKBP-091), whereas FKBP-118, 120, 122, 124, and 126 were as sequenced from the library and have the A117H mutation in the binding site. The remainder of the library output (127–150) were formatted with only the improved binding site mutations (i.e., Q72R, Q84E, and A117W).

[0108] The starting full length wild type FKBP13 sequence is as follows: MRLSWFRVLTVLSICLSAVATATGAEGKRKLQIGVKKRVDHCPIKSRKGDVLHMHYTGKLEDGTEFDSSLPQNQPFVFSLGTGQVIKGWDQGLLGMCEGEKRKLVIPSELGYGERGAPPKIPGGATLVFEVELLKIERRTEL (SEQ ID NO: 1).

[0109] Table 1 shows exemplary FKBP13 DRD sequences used in constructs described throughout the Examples. The wild-type FKBP13 fragment is referred to as SEQ ID NO: 2. SEQ ID NOs: 3-109 are examples of genetically modified FKBP13 polypeptides derived from the fragment corresponding to SEQ ID NO: 2. As used throughout, reference to the construct FKBP-039-FKBP- [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

[0110] Constructs containing the DRD sequences shown in Table 1 were made to determine the effect of each DRD on an exemplary payload polypeptide (i.e., Aequorea coerulescens GFP). These constructs are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0111] To test the effect of mutations that increase the responsiveness of FKBP13 to tacrolimus in the presence and absence of binding site mutations (i.e., DRD-producing mutations), constructs FKBP13-039, FKBP13-040, FKBP13-041, FKBP13-042, FKBP13-043, FKBP13-044, and FKBP13-045 were made. The sequences of these constructs are shown below. In constructs FKBP13-039, FKBP13-040, FKBP13-041, FKBP13-042, FKBP13-043, FKBP13-044, and FKBP13-045, the FKBP13 DRD sequence is in bold, the AcGFP amino acid sequence is underlined, and the mCherry sequence is in italics. It will be appreciated that any of the constructs described herein that include the FBKP13 DRD sequence can also include the AcGFP amino acid sequence (SEQ ID NO:117, underlined in SEQ ID NOs:110-116 below) and / or the mCherry amino acid sequence (SEQ ID NO:118, italicized in SEQ ID NOs:110-116 below), as shown in any one of constructs FKBP13-039, FKBP13-040, FKBP13-041, FKBP13-042, FKBP13-043, FKBP13-044 and FKBP13-045. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0112] FIG. 1A is a schematic diagram showing the general organization of an exemplary nucleic acid construct including (i) a nucleic acid sequence encoding a potential FKBP DRD sequence, (ii) a nucleic acid sequence encoding Aequorea coerulescens GFP (AcGFP), (iii) a nucleic acid sequence encoding an exemplary self-cleaving peptide (Porcine Teschovirus-1 2A (P2A)), and (iv) a nucleic acid sequence encoding mCherry.

[0113] HEK-293T cells were cultured in standard medium (Dulbecco's Modified Eagle Medium (DMEM) (Fisher Scientific, Hampton, NH, model no. 11-960-044) containing 10% FBS (Fisher Scientific, model no. 10-082-147) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, model no. 15140122)) in 6-well plates and then transfected with Lipofectamine® 2000 (Fisher Scientific, FKBP13-039, FKBP13-040, FKBP13-041, FKBP13-042, FKBP13-043, FKBP13-044 or FKBP13-045 constructs were transiently transfected using a 100% chromatin (Thermo Fisher Scientific, model no. 11-668-027) transfected HEK-293T cells. After 24 hours, transfected HEK-293T cells were treated with either 0, 1 or 10 μM FK506 (tacrolimus). After 24 hours of FK506 treatment, cells were harvested by trypsinization and flow analysis was performed using an Attune NXT flow cytometer (Thermo Fisher Scientific).

[0114] The DRD in FKBP13-039 was wild-type (WT) FKBP13 (i.e., the 98 amino acid fragment of SEQ ID NO: 1). The DRDs of FKBP13-040, FKBP13-041, and FKBP13-042 were designed to contain the DRD-producing mutations D90G, M54A, and M96V, respectively, but did not contain any binding site mutations. The FKBP13-043, FKBP13-044, and FKBP13-045 constructs contained the DRD-producing mutations D90G, M54A, and M96V, respectively, and three binding site mutations (Q72R, Q84E, and A117H).

[0115] As shown in Figure 1B, all DRD-producing mutations were effective in destabilizing AcGFP and reducing AcGFP fluorescence. In the presence of binding site mutations (FKBP13-043, FKBP13-044 or FKBP13-045), AcGFP was stabilized more effectively and the fluorescence signal was increased. This is evidenced by the constructs without binding site mutations being less responsive to 1 μM FK506. Interestingly, WT FKBP13 (FKBP13-043) also had DRD activity.

[0116] EC20 values ​​for any of the FKBP13 DRDs are below plasma drug concentrations achievable in humans 50 To determine whether these findings have value, a tacrolimus dose response experiment was performed. The results showed that, overall, the response to tacrolimus was improved in the presence of the binding site mutations (Table 3). [Table 3]

[0117] We also tested the regulation of AcGFP from the FKBP13-AcGFP-mCherry construct in Jurkat cells. Jurkat clone E6-1 cells were cultured in standard media (RPMI + GlutaMAX Supplement (Life Technologies, Carlsbad, CA part number 61870127), fetal bovine serum (FBS) (Life Technologies part number 10-082-147)). Jurkat cells were transduced with lentivirus produced with each of the FKBP-039, FKBP-040, FKBP-041, FKBP-042, FKBP-043, FKBP-044, or FKBP-045 constructs. Jurkat cells were transduced until they were 10-25% positive, as determined 5 days post-transduction by using mCherry as a transduction marker. Five days after transduction, cells were treated with doses of tacrolimus or DMSO starting at a maximum concentration of 10 μM. After a 24-hour incubation period, GFP expression was measured in cells treated with tacrolimus or DMSO. The geometric mean fluorescence intensity (MFI) of GFP expression in mCherry+ cells was plotted and dose-response curves were fitted using Prism Software (San Diego, CA). The dose-response curves are shown in Figure 2. The FKBP-044 construct showed the best EC 50 and fold change characteristics, providing guidance for further FKBP13 DRD development.

[0118] In a separate experiment, several FKBP13 DRD constructs were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, Mass.). Figure 4 shows FACS data of tacrolimus dose response for Jurkat cells stably transduced with FKBP-044, FKBP-052, FKBP-053, FKBP-054, FKBP-055, FKBP-056, FKBP-057, FKBP-058, FKBP-059, FKBP-060, FKBP-061, FKBP-062, FKBP-063, and FKBP-064 constructs.

[0119] There are two different Protein Data Bank (PDB) codes for the crystal structure of FKBP13 (2PBC and 4NNR). Unlike the 98 amino acid sequence of 4NNR (SEQ ID NO: 2) corresponding to the truncated WT, the sequence with PDB ID 2PBC has two mutations: H41G and C42S. Since disulfides are often involved in protein stabilization, the role that this disulfide bond plays in the stabilization of FKBP13 was investigated. Constructs FKBP-052, FKBP-062, and FKBP-063 have the disulfide disabled. The FKBP-052 construct has the wild-type FKBP13 sequence, whereas the FKBP-062 and FKBP-063 constructs have the same DRD-producing mutations as the FKBP-044 construct, with the FKBP-062 construct having two cysteine ​​to serine mutations and the FKBP-063 construct having only one. Unexpectedly, abolishing the disulfide bond did not result in a significant difference in DRD function, as shown in Figure 4. [Table 4]

[0120] To test the effect of other substitutions for methionine at residue number 54 and leucine at position 93, FKBP13 DRD constructs were generated, transiently transfected into HEK293 cells as described above, and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves for FKBP-044 (M54A), FKBP-066 (M54K), FKBP-067 (M54H), FKBP-068 (M54S), FKBP-069 (M54G), FKBP-070 (M54D), and FKBP-071 (M54V), FKBP-072 (L93K) are shown in Figure 5. FKBP-068 performed closest to the original M54A substitution. Tacrolimus dose-response curves for FKBP-044, FKBP-073 (L93H), FKBP-074 (L93S), FKBP-075 (L93G), FKBP-076 (L93D), and FKBP-077 (L93V) are shown in Figure 6. FKBP-077 performed similarly to FKBP-044.

[0121] In a separate experiment, to explore the possibility of further truncating FKBP13, constructs (FKBP-044, FKBP-083, FKBP-084, FKBP-085, and FKBP-086) were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves are shown in Figure 7. Combination of DRD-producing M54A with truncation of the N-terminal histidine reduces basal (FKBP-083).

[0122] In a separate experiment, several constructs with alternative binding sites designed to improve sensitivity to tacrolimus (FKBP-044, FKBP-087, FKBP-088, FKBP-089, FKBP-090, and FKBP-091) were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves are shown in Figure 8. FKBP-091, which has the mutation A117W instead of A117H in FKBP-044 combined with the DRD-producing mutation M54A, has superior drug response.

[0123] In a separate experiment, several constructs designed to explore additional DRD-producing mutations (FKBP-044, FKBP-092, FKBP-093, FKBP-094, FKBP-095, FKBP-096, and FKBP-097) were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves are shown in Figure 9.

[0124] Figure 10 shows FACS data of tacrolimus dose response for Jurkat cells stably transduced with FKBP-044, FKBP-059, FKBP-068, FKBP-077, FKBP-084, FKBP-085, FKBP-091, FKBP-094, FKBP-095, and FKBP-097 constructs. These experiments included the second round of binding site design, as well as several previously identified mutants of interest, M54S (068), L93V (077), L93A (059), and M54A (044). In this experiment, the EC 50 was found to be 30 nM. See Table 5. [Table 5]

[0125] The other constructs (FKBP-091, FKBP-098, FKBP-099, FKBP-100, and FKBP-101) were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, Mass.). Tacrolimus dose-response curves are shown in FIG.

[0126] In another experiment, several constructs (FKBP-091, FKBP-102, FKBP-103, FKBP-104, FKBP-105 and FKBP-106) designed to combine the binding site mutations identified in FKBP-091 with novel DRD-producing mutations were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves are shown in Figure 12. All constructs have second generation binding sites (i.e., the binding sites in FKBP-091 (Q72R, Q84E, A117W)) except for the FKBP-106 construct, which has a different binding site (Q72R, Q84E, A117Y).

[0127] In a separate experiment, several other constructs (FKBP-091, FKBP-107, FKBP-108, FKBP-109, FKBP-110, and FKBP-111) designed to combine the binding site mutations identified in FKBP-091 with novel DRD-producing mutations were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, MA). Tacrolimus dose-response curves are shown in Figure 13. All constructs have second generation binding sites (i.e., the binding sites in FKBP-091 (Q72R, Q84E, A117W)).

[0128] In a separate experiment, several constructs (FKBP-091, FKBP-112, FKBP-113, FKBP-114, FKBP-115, and FKBP-116) designed to combine the binding site mutations identified in FKBP-091 with novel DRD-producing mutations were transiently transfected into HEK293 cells as described above and analyzed using a Celigo Image Cytometer (Nexcelom, Lawrence, Mass.). Tacrolimus dose-response curves are shown in FIG.

[0129] Example 2 Additional experiments were performed with constructs FKBP-117 to FKBP-150 to determine their sensitivity to tacrolimus. Constructs FKBP-117 to FKBP-150 were stably transduced into Jurkat cells as described above and their response to tacrolimus was analyzed using flow cytometry. FKBP-091 was used as a comparison construct. EC50 vs. AcGFP for each construct was 50 Values, EC relative to AcGFP / mCherry 50 Values ​​and fold changes are shown in Table 6. Tacrolimus dose response curves for constructs FKBP-117 through FKBP-150 are shown in Figures 15A-15J. [Table 6]

[0130] Constructs FKBP-151 to FKBP-159 were analyzed for differences in tacrolimus and sirolimus (rapamycin) sensitivity. These constructs have mutations around the binding site of FKBP13 designed to allow differential sensitivity to tacrolimus and sirolimus. The differences in shape and size of rapamycin and tacrolimus (Figure 3) were used to design these mutants. Constructs FKBP-151 to FKBP-159, along with controls FKBP-044 and FKBP-117, were stably transduced into Jurkat cells as described above and their response to tacrolimus or sirolimus was analyzed using flow cytometry. EC for each construct against tacrolimus and rapamycin 50 Value, Norm EC 50 The values ​​and fold changes are shown in Tables 7 and 8, respectively. Normalized EC 50 Values ​​were calculated from mCherry-normalized GFP MFI values, which took into account differences in mCherry intensity between samples. Tacrolimus vs. rapamycin dose response curves for constructs FKBP-151, FKBP-152, and FKBP-153 are shown in Figures 16A and 16B. Tacrolimus vs. rapamycin dose response curves for constructs FKBP-153, FKBP-155, and FKBP-157 are shown in Figures 17A and 17B. Tacrolimus vs. rapamycin dose response curves for constructs FKBP-154, FKBP-155, and FKBP-156 are shown in Figures 18A and 18B. Tacrolimus vs. rapamycin dose response curves for constructs FKBP-157, FKBP-158, and FKBP-159 are shown in Figures 19A and 19B. Dose response curves of tacrolimus versus rapamycin for comparative constructs FKBP-044 and FKBP-117 are shown in Figures 20A and 20B. Constructs 153, 155, and 157 show the greatest difference between rapamycin and tacrolimus, as shown in Table 9. [Table 7] [Table 8] [Table 9]

[0131] Example 3 Additional experiments were performed to compare the abundance of constitutively expressed AcGFP with the abundance levels of FKBP13 / tacrolimus-regulated AcGFP. FKBP13 was fused to the protein of interest (i.e., AcGFP) to examine the effect on the maximum abundance of AcGFP. Constructs FKBP-044, FKBP-059, and FKBP-091 as well as AcGFP-001 were stably transduced into Jurkat cells as described above, and dose-response experiments were performed. MFI was read by flow cytometry (Figure 21). Regulated FKBP13 reached approximately 70% of the MFI value of constitutive AcGFP.

[0132] Example 4 Additional experiments were performed on constructs FKBP-160-292. These constructs are shown in Table 10, and the top 30 drug-responsive domains from the Cas9 screen are shown in Table 11. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7]

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

Table 10-23

Table 10-24

Table 10-25

Table 10-26

Table 10-27

Table 10-28

Table 10-29

Table 10-30

Table 10-31

Table 10-32

Table 10-33

Table 10-34

Table 10-35

Table 10-36

Table 10-37

Table 10-38

Table 10-39

Table 10-40

Table 10-41

Table 10-42

Table 10-43

Table 10-44

Table 10-45

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Table 11-7

Table 11-8

Table 11-9

Table 11-10

Table 11-11

Table 12-1

[0133] Figures 22-38 show the results of characterization of FKBP-160-292. The constructs were stably transfected into Jurkat cells and their response to tacrolimus was analyzed using flow cytometry. FKBP-091 and FKBP-117 were used as controls for FKBP-160-FKBP-255. FKBP-195 was used as a control for FKBP-256-FKBP-275. FKBP-117, FPBP-195, and FKBP-251 were used as controls for FKBP-276-FKBP-292. [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21]

[0134] Figures 39-50 show the results from further engineering of the top DRDs from the Cas9 screen, in particular providing a direct comparison of constructs mutated to other amino acids or with removal of the M54A mutation. [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33]

[0135] Figures 51-54 show the results of the tacrolimus serum experiment. Tacrolimus dilutions were made at each % FBS shown in Figures 51-54. Incubation with increasing FBS results in a 2-fold increase in Ec50 at 80% FBS. [Table 34] [Table 35] [Table 36] [Table 37]

[0136] To summarize the results of the examples, initial characterization of FKBP mutants discovered from pooled saturation mutagenesis library screening identified the V85R mutation (FKBP-117), which, when combined with the original binding site mutations (Q72R, Q84E, A117W), significantly reduced the FKBP / tacrolimus Ec50 to 5 nM. To further improve the properties obtained from construct FKBP-117, a series of combinatorial mutants (FKBP-160 to FKBP-255) were generated to identify FKBP constructs that conferred favorable DRD properties for protein regulation, with an Ec50 of less than 50 nM and a low basal off-state in the absence of tacrolimus. To assess the regulation of FKBP mutants by using AcGFP expression as a readout, FKBP mutants were cloned to generate fusion proteins with AcGFP. Constructs were stably transduced into Jurkat cells and their response to tacrolimus was analyzed using flow cytometry. FKBP mutants that had a >10-fold change in AcGFP expression in response to 10 uM tacrolimus dosing were then further screened in a tacrolimus dose response to determine the Ec50 of these constructs. FKBP-195, FKBP-228, and FKBP-251 were found to be the top FKBP constructs resulting in the lowest basal expression with Ec50 values ​​of 35 nM, 19 nM, and 30 nM, respectively. FKBP-215 showed the lowest basal AcGFP expression with an Ec50 of 90 nM. From these initial combinatorial mutants, further iterative FKBP mutants (FKBP-256 to FKBP-292) were constructed to see if the DRD properties of FKBP could be further improved. FKBP-272 was constructed from FKBP-195 by changing the K135Y mutation to K135L, which resulted in a similar Ec50 but reduced basal AcGFP expression. FKBP-263 is based on FKBP-215 with the M54A mutation removed, improving the Ec50 to 30 nM while retaining low basal AcGFP expression.

Claims

1. (a) FKBP13 or a variant thereof that responds to a ligand; (b) a biologically active payload; A nucleic acid encoding The nucleic acid, wherein upon expression of the nucleic acid, the FKBP13 is operably linked to the payload.

2. The nucleic acid of claim 1, wherein upon expression of the nucleic acid, the FKBP13 interacts with an effective amount of the ligand to modulate the biological activity of the payload.

3. 3. The nucleic acid of claim 1 or 2, wherein the encoded FKBP13 is a variant of SEQ ID NO:

1.

4. The nucleic acid according to any one of claims 1 to 3, wherein the encoded FKBP13 variant comprises one or more mutations in the ligand binding site.

5. The nucleic acid according to any one of claims 1 to 4, wherein the ligand is FK506, rapamycin, or both FK506 and rapamycin.

6. 6. The nucleic acid of claim 5, wherein the encoded FKBP13 variant modulates the biological activity of the payload to a greater extent in response to FK506 than in response to rapamycin.

7. The nucleic acid according to any one of claims 3 to 6, wherein the encoded FKBP13 variant is a fragment of FKBP13.

8. The nucleic acid of claim 7, wherein the encoded FKBP13 variant is a C-terminal fragment of FKBP13.

9. The nucleic acid according to any one of claims 1 to 8, further encoding a promoter sequence.

10. The nucleic acid of any one of claims 1 to 9, wherein the payload comprises an RNA-guided endonuclease.

11. 11. The nucleic acid of claim 10, wherein the nuclease is a Cas9 endonuclease.

12. 12. The nucleic acid of claim 11, wherein the Cas9 endonuclease is Staphylococcus aureus Cas9 (SaCas9) or Staphylococcus lugdunensis (SluCas9).

13. The nucleic acid according to any one of claims 10 to 12, further encoding at least one guide RNA.

14. 14. The nucleic acid of claim 13, wherein the nucleic acid is configured to be packaged in an AAV vector as a single nucleotide sequence comprising a promoter sequence, a guide RNA, and a sequence encoding a Cas9 endonuclease payload, and wherein the promoter sequence, the guide RNA, and the sequence encoding the Cas9 comprise at least 3000 base pairs.

15. The nucleic acid according to any one of claims 7 to 14, wherein the encoded FKBP13 fragment is less than 100 amino acids.

16. The nucleic acid of claim 15, wherein the encoded FKBP fragment is 90 to 99 amino acids.

17. The nucleic acid of claim 16, wherein the encoded FKBP13 fragment has an amino acid sequence having at least 85% identity to SEQ ID NO:

2.

18. The nucleic acid of claim 17, wherein the encoded FKBP13 comprises an amino acid sequence selected from SEQ ID NOs: 3-109.

19. A genetically modified FKBP13 comprising one or more mutations in the binding site of FK506.

20. The genetically modified FKBP13 of claim 19, wherein the FKBP13 is a C-terminal fragment of SEQ ID NO:

1.

21. The genetically modified FKBP13 of claim 20, comprising an amino acid sequence selected from SEQ ID NOs: 3 to 66.

22. The genetically modified FKBP13 of any one of claims 19 to 21, wherein the FKBP13 fragment has a lower rapamycin binding affinity compared to a control FKBP13 fragment not having a mutation in the ligand binding site.

23. The genetically modified FKBP13 according to any one of claims 19 to 22, wherein the FKBP13 has a higher FK506 binding affinity than a control FKBP13 fragment not having a mutation in the ligand binding site.

24. A recombinant polypeptide encoded by the nucleic acid of any one of claims 1 to 18, wherein the payload is operably linked to the FKBP13 or a variant thereof, and the activity of the payload in a biological system is regulatable by the concentration of the ligand that contacts the FKBP13.

25. The EC of FK506 against the FKBP13 or its variant in the biological system ５０ is the C of FK506 in the biological system. ｍａｘ The recombinant polypeptide of claim 24, wherein the polypeptide is about 2-fold to about 20-fold lower than the

26. 26. The recombinant polypeptide of claim 24 or 25, wherein the biological system is a tissue or a cell.

27. 27. The recombinant polypeptide of claim 26, wherein the biological system is mammalian blood.

28. A vector comprising the nucleic acid according to any one of claims 1 to 18.

29. The vector of claim 28 , wherein the vector is a viral vector.

30. 30. The vector of claim 28 or 29, wherein the encoded payload is an RNA-guided endonuclease, and the vector further comprises one or more guide RNA sequences comprising a first nucleotide sequence that hybridizes to a target DNA in the genome of a cell and a second nucleotide sequence configured to interact with the RNA-guided endonuclease.

31. 31. The vector of claim 30, wherein the vector further comprises one or more inverted terminal repeat sequences.

32. A cell comprising the vector according to any one of claims 28 to 31.

33. The cell of claim 32 , wherein the cell is a mammalian cell.

34. The cell of claim 33 , wherein the mammalian cell is a human cell.

35. The cell according to any one of claims 32 to 34, wherein the cell is a muscle cell, a stem cell, or a lymphocyte.

36. A method for producing a recombinant cell, comprising introducing the vector according to any one of claims 28 to 31 into a cell.

37. 37. The method of claim 36, wherein the cell is a mammalian cell.

38. 38. The method of claim 37, wherein the mammalian cell is a human cell.

39. The method of claims 36 to 38, wherein the cell is a muscle cell, a stem cell, or a lymphocyte.

40. A cell produced by the method of any one of claims 36 to 39.

41. 1. A method for modulating a payload in a cell, comprising: (a) introducing into a cell a vector comprising a nucleic acid encoding a ligand-responsive FKBP13 or a variant thereof, the nucleic acid further comprising a promoter sequence and a sequence encoding the payload; and (b) contacting said cell with an effective amount of said ligand.

42. 1. A method for modifying target DNA in a cell, comprising: (a) injecting the cells with (1) An RNA-guided endonuclease-encoding nucleic acid comprising a sequence encoding FKBP13 or a variant thereof that responds to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease; and (2) one or more guide RNAs comprising a nucleotide sequence that hybridizes to a target DNA in the genome of a cell and a nucleotide sequence configured to interact with the RNA-guided endonuclease; introducing a vector comprising: (b) contacting the vector-containing cell with an effective amount of a ligand to induce activity of the RNA-guided endonuclease; The method, wherein the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically binds to and cleaves the target DNA in the cell.

43. 43. The method of claim 41 or 42, wherein the vector is an AAV vector.

44. The method of any one of claims 41 to 43, wherein the cell is a mammalian cell.

45. 45. The method of claim 44, wherein the mammalian cell is a human cell.

46. The method of any one of claims 41 to 45, wherein the cell is a muscle cell, a stem cell, or a lymphocyte.

47. A genetically modified cell produced by the method of any one of claims 42 to 46.

48. 1. A method of treating a disease or disorder responsive to genetic modification in a subject in need thereof, comprising: (a) administering to one or more cells of said subject, (1) An RNA-guided endonuclease-encoding nucleic acid comprising a sequence encoding FKBP13 or a variant thereof that responds to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease; and (2) at least one guide RNA comprising a nucleotide sequence that hybridizes to a target DNA in the genome of the cell and is configured to interact with the RNA-guided endonuclease; introducing a vector comprising: (b) contacting one or more of said vector-containing cells with an effective amount of a ligand to induce activity of said RNA-guided endonuclease; The method, wherein the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically binds to and cleaves the target DNA in one or more cells of the subject.

49. 1. A method of treating a disease or disorder responsive to genetic modification in a subject in need thereof, comprising: (a) administering to the subject one or more cells comprising a vector, the vector comprising: (1) An RNA-guided endonuclease-encoding nucleic acid comprising a sequence encoding FKBP13 or a variant thereof that responds to a ligand, a promoter sequence, and a sequence encoding an RNA-guided endonuclease; and (2) A guide RNA comprising a nucleotide sequence that hybridizes to a target DNA in the genome of the one or more cells and a nucleotide sequence configured to interact with the RNA-guided endonuclease. said administering comprising: (b) administering to the subject an effective amount of a ligand to induce activity of the RNA-guided endonuclease; The method, wherein the RNA-guided endonuclease interacts with the guide RNA, and the RNA-guided endonuclease specifically modifies the genome of the one or more cells.

50. 48. A method of treating a disease or disorder in a subject, comprising administering to the subject one or more genetically modified cells of claim 47.

51. 51. The method of any one of claims 48 to 50, wherein the disease or disorder is Duchenne muscular dystrophy, myotonic dystrophy, cystic fibrosis, sickle cell, beta thalassemia, alpha-1 antitrypsin deficiency, APOL1 mediated kidney disease, or type 1 diabetes.

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