Allogeneic t-cells and methods for production thereof

Abstract

To provide methods for producing allogeneic T-cells.SOLUTION: An allogeneic T-cell line comprises a CRISPR-associated (Cas) nuclease under the control of a controllable promoter, incorporated into the genome of the T-cell line. The CRISPR-associated nuclease may be a Cas9 nuclease or a Cas12 nuclease. The controllable promoter may be selected from the group consisting of an inducible promoter and a derepressible promoter. By preparing T-cells using an inducible nuclease, large volumes of cells can be prepared, each of which has the ability to individually produce the desired nuclease.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention provides a method for producing allogeneic T cells, including the use of engineered nucleases under the control of a controllable promoter. By preparing T cells using inducible nucleases, large numbers of cells can be prepared, each capable of individually producing a desired nuclease. These cells can then be modified as needed through the introduction of a gene of interest, or undesirable genes can be knocked out. Allogeneic T cells for use in various therapeutic applications are also provided herein. [Background technology]

[0002] As the clinical adoption of advanced cell therapies accelerates, attention is focused on the manufacturing strategies underlying these therapies that can benefit patients worldwide. Successful results from immunotherapy trials using chimeric antigen receptor (CAR) T cells offer new hope for patients suffering from previously untreatable cancers. While cell therapies hold great clinical potential, high manufacturing costs relative to reimbursement present a significant barrier to commercialization.

[0003] One of the challenges facing CAR T-cell therapy is the generation of allogeneic cells that can be used in any patient. Scaling up allogeneic T-cell therapy can be prohibitively expensive and require long lead times due to the need for large amounts of viral vectors and / or recombinant endonucleases.

[0004] To overcome these challenges, what is needed is a method for producing T cell lines that does not require a large investment of time and money, yet still provides cell lines that can be used in multiple patient populations. The present invention meets these needs. Summary of the Invention

[0005] In some embodiments, provided herein are methods of producing a T cell line for use in allogeneic applications, comprising introducing a nucleic acid molecule encoding an engineered nuclease under the control of a regulatable promoter into a T cell line, integrating the nucleic acid molecule into the genome of the T cell line, and expanding the T cell line.

[0006] In a further embodiment, a method of producing a genetically modified T cell line is provided, comprising introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T cell line, integrating the nucleic acid molecule into the genome of the T cell line, expanding the T cell line, inducing expression of the CRISPR-associated nuclease by activating the controllable promoter, introducing a guide RNA and a gene of interest into the expanded T cell line, knocking out expression of a T cell receptor and introducing the gene of interest into the genome of the T cell line, and recovering the genetically modified T cell line.

[0007] Also provided herein is a method of producing a chimeric antigen receptor (CAR) T cell line, comprising introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T cell line, integrating the nucleic acid molecule into the genome of the T cell line, expanding the T cell line, inducing expression of the Cas9 nuclease by activating the controllable promoter, introducing a guide RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T cell line, knocking out expression of the T cell receptor and introducing a nucleic acid encoding the CAR into the genome of the T cell line, and recovering the CAR T cell line.

[0008] In additional embodiments, provided herein are allogeneic T cell lines comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line. [Brief explanation of the drawings]

[0009] [Figure 1]Schematic diagrams of both autologous and allogeneic approaches to T cell therapy are shown. [Figure 2] 1 illustrates steps in the generation of allogeneic T cells. [Figure 3] Three exemplary phases of allogeneic T cell generation are shown. [Figure 4A] 1 shows an exemplary derepressible promoter system used herein. [Figure 4B] 1 shows an exemplary derepressible promoter system used herein. [Figure 4C] 1 shows an inducible vector system for use in embodiments of the present invention. [Figure 4D] 1 shows an inducible vector system for use in embodiments of the present invention. [Figure 4E] 1 shows the TRE3G Tet-On system used in an embodiment of the present invention. [Figure 5A] 1 shows a therapeutic protocol for transduction of T cells with Cas9-inducible vectors. [Figure 5B] 1 shows a therapeutic protocol for transduction of T cells with Cas9-inducible vectors. [Figure 5C] 1 shows a therapeutic protocol for transduction of T cells with Cas9-inducible vectors. [Figure 6A] Figure 1 shows the results of transduction of T cells with Cas9-inducible vectors. Please refer to the legend for the symbols to follow the line graph. [Figure 6B] Figure 1 shows the results of transduction of T cells with Cas9-inducible vectors. Please refer to the legend for the symbols to follow the line graph. [Figure 7A] The number of surviving cells after selection and during cell expansion is shown. [Figure 7B] The number of surviving cells after selection and during cell expansion is shown. [Figure 8] Measurements of exhaustion markers, senescence markers, activation markers, and T cell markers for the three treatment protocols are shown. [Figure 9]1 shows the results of inducing Cas9 expression in T cells. [Figure 10A] Shows T cell proliferation after cryopreservation. [Figure 10B] Shows T cell proliferation after cryopreservation. [Figure 11] Three approaches for TRAC gene knockout are presented. [Figure 12A-1] TRAC knockout is shown 4 days after nucleofection. [Figure 12A-2] TRAC knockout is shown 4 days after nucleofection. [Figure 12A-3] TRAC knockout is shown 4 days after nucleofection. [Figure 12A-4] TRAC knockout is shown 4 days after nucleofection. [Figure 12A-5] TRAC knockout is shown 4 days after nucleofection. [Figure 12A-6] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-1] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-2] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-3] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-4] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-5] TRAC knockout is shown 4 days after nucleofection. [Figure 12B-6] TRAC knockout is shown 4 days after nucleofection. [Figure 12C] TRAC knockout is shown 4 days after nucleofection. [Figure 13A-1] TRAC knockout is shown 7 days after nucleofection. [Figure 13A-2] TRAC knockout is shown 7 days after nucleofection. [Figure 13A-3] TRAC knockout is shown 7 days after nucleofection. [Figure 13A-4] TRAC knockout is shown 7 days after nucleofection. [Figure 13A-5] TRAC knockout is shown 7 days after nucleofection. [Figure 13A-6] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-1] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-2] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-3] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-4] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-5] TRAC knockout is shown 7 days after nucleofection. [Figure 13B-6] TRAC knockout is shown 7 days after nucleofection. [Figure 13C] TRAC knockout is shown 7 days after nucleofection. [Figure 14A-1] TRAC knockout is shown 14 days after nucleofection. [Figure 14A-2] TRAC knockout is shown 14 days after nucleofection. [Figure 14A-3] TRAC knockout is shown 14 days after nucleofection. [Figure 14A-4] TRAC knockout is shown 14 days after nucleofection. [Figure 14A-5] TRAC knockout is shown 14 days after nucleofection. [Figure 14A-6] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-1] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-2] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-3] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-4] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-5] TRAC knockout is shown 14 days after nucleofection. [Figure 14B-6] TRAC knockout is shown 14 days after nucleofection. [Figure 14C] TRAC knockout is shown 14 days after nucleofection. [Figure 15A] A summary of the knockout experiments is shown. [Figure 15B] A summary of the knockout experiments is shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] The use of the word "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0011] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value. Typically, the term is meant to encompass a variation of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0012] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0013] As used in the specification and claims, the terms "comprising" (and any form of "comprising," e.g., comprise and comprise), "having" (and any form of having, e.g., have and has), "including" (and any form of "including," e.g., includes and include), or "containing" (and any form of containing, e.g., contains and contain) are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host T cell, expression vector, and / or composition of the invention. Furthermore, the compositions, systems, cells, and / or nucleic acids of the invention can be used to achieve any of the methods described herein.

[0014] Chimeric antigen receptor T cells Chimeric antigen receptor T cells, or "CAR T cells," are T cells (also referred to herein as T cells) that have a chimeric antigen receptor (CAR) modified to more specifically target cancer cells. Generally, CARs contain three parts: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain is the region of the receptor exposed to extracellular fluid and contains three parts: a signal peptide, an antigen recognition region, and a spacer. The signal peptide directs the nascent protein to the endoplasmic reticulum. In CARs, the signal peptide is a single-chain variable fragment (scFv). The scFv contains variable fragments of light chains linked by a short linker peptide. In some embodiments, the linker contains glycine and serine. In some embodiments, the linker contains glutamic acid and lysine.

[0015] The transmembrane domain of the CAR is a membrane-spanning hydrophobic α-helix. In some embodiments, the transmembrane domain of the CAR is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain results in a highly expressed CAR. In some embodiments, the transmembrane domain of the CAR is a CD3-ζ transmembrane domain. In some embodiments, the CD3-ζ transmembrane domain results in a CAR integrated into a natural T cell receptor.

[0016] The endodomain of a CAR is generally considered the "functional" end of the receptor. After antigen recognition by the antigen recognition region of the ectodomain, the CAR clusters and a signal is transmitted to the cell. In some embodiments, the endodomain is a CD3-ζ endodomain, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs). In this case, the ITAMs transmit activation signals to T cells after antigen binding, inducing a T cell immune response. Additional CAR designs known in the art can also be utilized in the implementation of the methods described herein.

[0017] During the production of CAR-T cells, T cells are removed from a human subject, genetically modified, and then reintroduced into the patient to attack cancer cells. CAR-T cells can be derived from either the patient's own blood (autologous) or another healthy donor (allogeneic). Generally, CAR-T cells are developed to be specific for antigens that are overexpressed in tumors compared to healthy cells.

[0018] Methods for producing T cells for use in allogeneic applications While autologous T cells represent a significant opportunity for the treatment of various diseases, particularly cancer, the use of allogeneic cells, particularly methods for producing cell lines that can be modified as needed for each individual patient, represents a breakthrough in T cell-based therapy. Producing T cell lines that can be used for any patient not only dramatically increases the supply of potential cells, but also the cost of producing such cells.

[0019] Conventional methods for producing allogeneic T cells after removing cells from a patient donor include expanding the cells before introducing the desired CAR for the required therapy. However, in such methods, the T cell receptor, which is required for expansion, must be removed before introduction into the patient, so all CAR introduction must occur after cell expansion. This requires not only a significant amount of virus and the desired CAR, but also a significant amount of any engineered nucleases (which may be required for gene editing). This significantly increases cost and complexity.

[0020] However, the methods described herein allow for the production of T cells that contain an engineered nuclease under the control of a controllable promoter, but still retain the native T cell receptor, allowing for proliferation. Once the cells are expanded, the desired CAR can then be inserted, the T cell receptor removed, and the cells further processed and suitable for injection into a patient.

[0021] Figure 1 shows a schematic diagram of both autologous and allogeneic approaches to T cell therapy. In autologous applications, T cells are isolated from a patient, a CAR construct is virally transduced into the patient's T cells, and the CAR T cells are then transferred back into the same patient. In allogeneic approaches, T cells are isolated from a healthy donor and the cells are virally transduced with the desired CAR construct. At the same time, the T cell receptor is knocked out to prevent graft-versus-host disease (GvHD), a prerequisite for universal CAR T therapy (additional genes, including B2M and PD1, which help prevent GvHD, can also be knocked out). Allogeneic T cells containing the desired CAR can then be transferred into any patient. As described herein, producing a source of T cells that can be expanded prior to introducing the CAR construct allows for a significant increase in scale-up and also reduces the resources and costs required.

[0022] In embodiments, then, provided herein are methods for producing T cell lines for use in allogeneic applications. As used herein, "T cell line" refers to lymphocyte cells that originate in the thymus and contain T cell receptors on their surface. T cells include immortalized T cells. As used herein, "allogeneic" or "allogeneic applications" refers to the use of cells from one or more donor sources (often healthy donors) in therapeutic applications to one or more patients (which may be independent of the donor source).

[0023] In embodiments, the methods described herein include introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T cell line. Methods for introducing nucleic acid molecules into T cell lines include the use of various transduction or transfection systems, including various viral systems; for example, the nucleic acid molecule can be introduced into a T cell line using a lentiviral vector. Additional transduction or transfection systems include nucleofection, the use of an exosome system, the use of a liposome system, the use of a polymer-based system, etc.

[0024] As used herein, "nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymeric compound containing covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or miRNA. Nucleic acids also include RNA, which is introduced into a cell, then reverse-transcribed into DNA, which is then integrated into the cell's genome.

[0025] As used herein, "gene" refers to an assembly of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can function as a regulatory sequence before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. In some embodiments, the gene is integrated in multiple copies. In some embodiments, the gene is integrated in a predetermined number of copies.

[0026] As used herein, "transfection" refers to the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. A "transfected" cell contains an exogenous nucleic acid molecule within the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change within the cell. The transfected nucleic acid molecule may be introduced into the cell as RNA, reverse transcribed into DNA by the cell, and then integrated into the genomic DNA of the host T cell and / or maintained extrachromosomally by the cell for a transient or long-term period. A host T cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transformed," or "transgenic" organism. Many transfection techniques are commonly known in the art. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Preferably, transfection of T cells with one or more of the vectors described herein utilizes a transfection agent such as polyethyleneimine (PEI) or other suitable agent, including various lipids and polymers, to integrate the nucleic acid into the genomic DNA of the host T cell. In some embodiments, transfection comprises viral infection (also referred to as "transduction"), transposon, mRNA transfection, electroporation, or a combination thereof. In some embodiments, transfection comprises electroporation. In additional embodiments, transfection comprises viral transduction. The vector can be, for example, a viral vector such as a lentiviral vector, a gammaretroviral vector, an adeno-associated viral vector, or an adenoviral vector.In embodiments, transfection comprises introducing a viral vector into activated T cells in cell culture. In additional embodiments, the vector is delivered as a viral particle.

[0027] As shown in Figure 2, T cells are suitably contacted with the nucleic acid molecule contained within the viral particle, allowing the nucleic acid to be integrated into the genome of the T cell line. The nucleic acid is introduced as RNA, reverse transcribed by the cell into DNA, and then integrated into the genome of the cell. This provides a T cell line, each containing an engineered nuclease integrated into the genome under the control of a controllable promoter. These T cells can then be expanded as described herein to produce T cell lines for use in allogeneic applications.

[0028] As used herein, the term "engineered nuclease" refers to a nuclease that has been isolated, modified, mutated, and / or altered from its natural state as a nuclease. A "nuclease" refers to an enzyme that can cleave DNA and / or RNA molecules. By engineering a nuclease, the specific location of cleavage can be designed and tailored to the cell type and / or gene of interest.

[0029] Exemplary engineered nucleases that can be inserted into T cells include, for example, meganucleases, methyltransferases, zinc finger nucleases, transcription activator-like effector-based nucleases (TALENs), FokI nucleases, and CRISPR-associated nucleases. Generally, engineered nucleases use DNA-binding proteins that have both the desired catalytic activity and the ability to bind to a desired target sequence through protein-nucleic acid interactions in a manner similar to restriction enzymes. Examples include meganucleases, which are naturally occurring or engineered rare sequence cleavage enzymes; zinc finger nucleases (ZFNs), which contain FokI catalytic nuclease subunits linked to modified DNA binding domains and can each cleave a specific sequence; or transcription activator-like nucleases (TALENs). In ZFNs, the binding domain consists of a chain of amino acids that folds into a customized zinc finger domain. In TALENs, a 34-amino acid repeat derived from a transcription factor folds into a large DNA-binding domain. In the case of gene targeting, these enzymes can cleave genomic DNA to form double-strand breaks (DSBs) or generate nicks, which can be repaired by one of two repair pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The NHEJ pathway can potentially result in specific mutations, deletions, insertions, or substitutions. The HR pathway results in the replacement of the target sequence with the supplied donor sequence. An exemplary FokI and methyltransferase-based system is described in U.S. Patent No. 10,220,052 (the disclosure of which is incorporated herein by reference in its entirety).

[0030] In a preferred embodiment, the CRISPR-associated nuclease is a Cas9 nuclease or can be another Cas nuclease (e.g., a Cas12 nuclease, a Cas13 nuclease, a Cas14 nuclease). In an embodiment, the Cas9 nuclease is a Cas9 nuclease with reduced immunogenicity as disclosed in U.S. Published Patent Application No. 2018-0319850 (the disclosure of which is incorporated herein by reference in its entirety).

[0031] Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (CRISPR-associated nucleases, or Cas proteins), including the CRISPR-Cas system, were first identified in select bacterial species and form part of the prokaryotic adaptive immune system. See Sorek, et al., “CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea,” Nat. Rev. Microbiol. 6(3)181-6(2008), incorporated herein by reference in its entirety. CRISPR-Cas systems have been classified into three main types: Type I, Type II, and Type III. The main defining features of the different types are the various cas genes used and the respective proteins they encode. The cas1 and cas2 genes appear to be universal across the three major types, while cas3, cas9, and cas10 are thought to be specific to Type I, Type II, and Type III systems, respectively. See, e.g., Barrangou, R. and Marraffini, LA, “CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity,” Mol. Cell. 54(2):234-44 (2014), incorporated herein by reference in its entirety.

[0032] In general, CRISPR-Cas systems function by capturing short regions of invading viral or plasmid DNA and integrating the captured DNA into the host genome, spaced apart by repeats within the CRISPR locus, to form so-called CRISPR arrays. This DNA capture into the CRISPR array is followed by transcription and RNA processing.

[0033] Depending on the bacterial species, CRISPR RNA processing proceeds differently. For example, in the Type II system first described in Streptococcus pyogenes, the transcribed RNA is paired with a transactivating RNA (tracrRNA) and then cleaved by RNase III to form individual CRISPR-RNAs (crRNAs). The crRNA is further processed to generate mature crRNAs after binding of the Cas9 nuclease. The crRNA / Cas9 complex then binds to DNA containing a sequence complementary to the capture region (called a protospacer). The Cas9 protein then cleaves both strands of DNA in a site-specific manner, forming double-strand breaks (DSBs). This provides DNA-based memory and leads to rapid degradation of viral or plasmid DNA upon repeated exposure and / or infection. Natural CRISPR systems have been comprehensively reviewed (see, e.g., Barrangou, R. and Marraffini, L.A., 2014).

[0034] Since its initial discovery, multiple groups have conducted extensive research on the potential applications of CRISPR systems in genetic engineering, including gene editing (Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337(6096):816-21 (2012); Cong et al., “Multiplex genome engineering using CRISPR / Cas systems,” Science 339(6121):819-23 (2013); and Mali et al., “RNA-guided human genome engineering via Cas9,” Science 339(6121):823-26, each of which is incorporated herein by reference in its entirety). One major development has been the use of chimeric RNAs targeting the Cas9 protein, designed around individual units from a CRISPR array fused to tracrRNA. This generates a single RNA species called a small guide RNA (gRNA), and sequence modifications in the protospacer region can site-specifically target the Cas9 protein. Considerable research has been conducted to understand the nature of the base-pairing interaction between the chimeric RNA and the target site and its tolerance to mismatches, which is highly relevant for predicting and assessing off-target effects (see, e.g., Fu et al., "Improving CRISPR-Cas nucleases using truncated guide RNAs," Nature Biotechnology 32(3):279-84 (2014) and supporting materials, which are incorporated herein by reference in their entirety).

[0035] The CRISPR-Cas9 gene editing system has been successfully used in a wide range of organisms and cell lines to induce the formation of double-strand breaks using wild-type Cas9 proteins or to nick single DNA strands using a mutant protein called Cas9n / Cas9 D10A (see, e.g., Mali et al., (2013) and Sander and Joung, “CRISPR-Cas systems for editing, regulating, and targeting genomes,” Nature Biotechnology 32(4):347-55 (2014) (each of which is incorporated herein by reference in its entirety). Formation of double-strand breaks (DSBs) results in the generation of small insertions and deletions (indels) that can disrupt gene function, while the Cas9n / Cas9 D10A nickase avoids the generation of indels (as a result of repair by non-homologous end joining) while stimulating endogenous homologous recombination machinery. Thus, Cas9n / Cas9 D10A nickase can be used to insert regions of DNA into the genome with high fidelity.

[0036] As described herein, the CRISPR-associated nuclease that is inserted into the genome of T cell is preferably Cas9 nuclease.By placing Cas9 nuclease under the control of a controllable promoter, nuclease can be kept dormant or silent before its desired use as a gene editing tool.As used herein, " controllable promoter " refers to a promoter that can be turned on or off depending on the desired control of the gene that is under the control of the promoter.

[0037] In addition to Cas9 nuclease, Cas12, Cas13, and Cas14 nucleases can also be utilized in the methods described herein. Cas12 nuclease generates staggered cuts in dsDNA (5-nucleotide 5' overhand dsDNA cuts). Cas12 processes its own guide RNA, resulting in increased multiplexing capacity. Cas13t targets RNA, not DNA. When activated by an ssRNA sequence complementary to the crRNA spacer, nonspecific RNase activity is released, destroying all nearby RNAs regardless of their sequence. See, e.g., Yan et al., "CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR Cas9," Cell Biology and Toxicology, pages 1-4 (August 29, 2019), the disclosure of which is incorporated herein by reference in its entirety.

[0038] In further embodiments, an inactivated Cas9 enzyme (dCas9) can be linked to an active endonuclease and utilized in the methods described herein (e.g., including dCas9-Fok1 fusions).

[0039] As used herein, "under control" refers to a gene being regulated by a "promoter," "promoter sequence," or "promoter region," which refers to a DNA regulatory region / sequence capable of binding RNA polymerase and initiating transcription of a downstream coding or non-coding gene sequence. In other words, the promoter and gene are in operable combination or operably linked. As referred to herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linkage of nucleic acid sequences in such a manner as to produce a promoter capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid sequences in such a manner as to produce a functional protein.

[0040] In some examples of the present disclosure, the promoter sequence includes the transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. In some embodiments, the promoter sequence includes the transcription initiation site and a protein binding domain involved in the binding of RNA polymerase. Eukaryotic promoters often, but do not necessarily, include "TATA" boxes and "CAT" boxes.

[0041] Various promoters, including inducible promoters, can be used to drive gene expression, for example, in host T cells or vectors of the present disclosure. In some embodiments, the promoter is not a leaky promoter, i.e., the promoter does not constitutively express any of the gene products described herein. In other embodiments described herein, the promoter is a constitutive promoter that initiates mRNA synthesis independently of external regulatory influences. In exemplary embodiments, the promoter used to control the engineered nuclease is an inducible promoter. "Inducible promoter" refers to a group of promoters that can enhance expression of exogenous genes under the stimulation of specific physical, chemical, or pathogen signals. In embodiments herein, exemplary inducible promoters that can be used to control the engineered nuclease include, but are not limited to, a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, a TET-on system, or a glutamate-inducible promoter.

[0042] Preferably, the promoter used to control the engineered nuclease is a derepressible promoter. As used herein, a "derepressible promoter" refers to a structure comprising a functional promoter and additional elements or sequences that can bind to a repression element to cause repression of the functional promoter. "Repression" refers to reducing or inhibiting the initiation of transcription of a downstream coding or non-coding gene sequence by the promoter. A "repression element" refers to a protein or polypeptide that can bind to a promoter (or near a promoter) to reduce or inhibit the promoter's activity. The repression element can interact with the substrate or binding partner of the repression element such that the repression element undergoes a conformational change. This conformational change of the repression element eliminates the ability of the repression element to reduce or inhibit the promoter, resulting in "derepression" of the promoter, thereby allowing the promoter to proceed with transcription initiation. A "functional promoter" refers to a promoter that is capable of transcription initiation in the absence of the action of a repression element. A variety of functional promoters that can be used in the practice of the present invention are known in the art, including, for example, promoters of PCMV, PH1, P19, P5, P40, and adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).

[0043] Exemplary repression elements and their corresponding binding partners that can be used as derepressible promoters are known in the art, and include the cumate gene switch system (CuO operator, CymR repressor, and cumate binding partner) (see, e.g., Mullick et al., "The cumate gene-switch: a system for regulated expression in mammalian cells," BMC Biotechnology 6:43(1-18) (2006), the disclosure of which is incorporated herein by reference in its entirety, including the disclosure of the derepressible promoter system described therein) and the TetO / TetR system described herein (see, e.g., Yao et al., "Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells," Human Gene Therapy 9:1939-1950 (1998), the disclosure of which is incorporated herein by reference in its entirety. In exemplary embodiments, the derepressible promoter comprises a functional promoter and one of two tetracycline operator sequences (TetO or TetO2). In such embodiments, the nucleic acid introduced into the T cell further comprises a tetracycline repressor protein for controlling the TetO derepression system.

[0044] In an exemplary embodiment, as shown in Figure 3, in phase 1, T cells can be transfected with inducible Cas9 (iCas9) (or Cas9 under the control of a derepressible promoter) via a viral system such as lentivirus, resulting in T cells containing Cas9 (or other engineered nuclease) integrated into their genome.

[0045] In Phase 2, as shown in Figure 3, iCas9 T cells (or T cells containing another engineered nuclease) can be suitably expanded using a variety of methods for cell growth. The T cell expansion methods described herein can utilize any suitable reactor, including, but not limited to, stirred tank bioreactors, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or entrained bed bioreactors. As used herein, "reactor" can include a fermentor or fermentation unit, or any other reaction vessel, and the term "reactor" is used interchangeably with "fermentor." The terms fermentor or fermentation refer to both microbial and mammalian cultures. For example, in some aspects, an exemplary bioreactor unit can perform one or more, or all, of the following: feeding nutrients and / or carbon source, injecting a suitable gas (e.g., oxygen), flowing fermentation or cell culture medium inlet and outlet, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, can include multiple reactors within the unit; for example, a unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility can include multiple units with single or multiple reactors within the facility. In various embodiments, bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor may have a volume of from about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. 1, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Furthermore, suitable reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0046] In embodiments, the proliferation preferably includes activation of T cells. As described herein, the T cell receptor has not been removed from the engineered T cell line, and the cells can still be activated. In vivo, antigen-presenting cells (APCs), such as dendritic cells, act as a stimulus for T cell activation through the interaction of the T cell receptor (TCR) with the APC major histocompatibility complex (MHC). The TCR associates with CD3, a T cell co-receptor that serves to activate both cytotoxic T cells (e.g., CD8+ naive T cells) and T helper cells (e.g., CD4+ naive T cells). Generally, T cell activation follows a two-signal model, requiring stimulation of the TCR / CD3 complex as well as a co-stimulatory receptor.

[0047] Non-limiting examples of costimulatory molecules for T cells include CD28, a receptor for CD80 and CD86 on the membrane of APCs, and CD278 or ICOS (inducible T cell costimulator), a CD28 superfamily molecule expressed on activated T cells that interacts with ICOS-L. Thus, in some embodiments, the costimulatory molecule is CD28. In other embodiments, the costimulatory molecule is ICOS. In vivo, costimulatory signals can be provided by B7 molecules on APCs that bind to the CD28 receptor on T cells. B7 is a peripheral transmembrane protein found on activated APCs and can interact with CD28 or CD152 surface proteins on T cells to generate costimulatory signals. Thus, in some embodiments, the costimulatory molecule is B7.

[0048] Various activation methods have been utilized in vitro to simulate T cell activation. In embodiments, a culture of T cells is activated with an activation reagent. In further embodiments, the activation reagent is an antigen-presenting cell (APC). In yet further embodiments, the activation reagent is a dendritic cell. Dendritic cells are APCs that process antigens and present them on their cell surface to T cells. In some embodiments, the activation reagent is co-cultured with the T cell culture. Co-culture may require separate purification and culture of a second cell type, which can increase labor requirements and sources of variability. Therefore, in some embodiments, an alternative activation method is used.

[0049] In some embodiments, the activation reagent is an antibody. In some embodiments, the cell culture is activated with an antibody bound to a surface (including a polymer surface), including beads. In further embodiments, one or more antibodies are anti-CD3 and / or anti-CD28 antibodies. For example, the beads may be magnetic beads, such as DYNABEADS coated with anti-CD3 and anti-CD28. Anti-CD3 and anti-CD28 beads may suitably provide a stimulatory signal to support T cell activation. See, e.g., Riddell (1990), Trickett (2003).

[0050] In other embodiments, the cell culture is activated with a soluble antibody. In further embodiments, the soluble antibody is a soluble anti-CD3 antibody. OKT3 is a mouse monoclonal antibody of the immunoglobulin IgG2a isotype that targets CD3. Thus, in some embodiments, the soluble anti-CD3 antibody is OKT3. OKT3 is further described, for example, in Dudley (2003), Manger (1985), Ceuppens (1985), Van Wauwe (1980), and Norman (1995).

[0051] In some embodiments, the costimulatory signal for T cell activation is provided by accessory cells. The accessory cells may contain Fc receptors, for example, that allow cross-linking of CD3 antibodies with the TCR / CD3 complex on T cells. In some embodiments, the cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs). The PBMCs may contain accessory cells that can support T cell activation. For example, a CD28 costimulatory signal may be provided by B7 molecules present on monocytes in PBMCs. Thus, in some embodiments, the accessory cells comprise monocytes or monocyte-derived cells (e.g., dendritic cells). In additional embodiments, the accessory cells comprise B7, CD28, and / or ICOS. Accessory cells are further described in, for example, Wolf (1994), Chai (1997), Verwilghen (1991), Schwartz (1990), Ju (2003), Baroja (1989), Austin (1987), and Tax (1983).

[0052] As described herein, the activation reagent determines the phenotype of the CAR T cells produced and can promote a desired phenotype. In some embodiments, the activation reagent determines the ratio of T cell subsets (i.e., CD4+ helper T cells and CD8+ cytotoxic T cells). Cytotoxic CD8+ T cells typically play a role in killing cancer cells (i.e., anti-tumor response), infected (e.g., viral) cells, or otherwise damaged cells. CD4+ T cells typically produce cytokines, help regulate the immune response, and in some cases, can support T cell lysis. CD4+ cells activate APCs, which then prime naive CD8+ T cells for an anti-tumor response. Thus, in embodiments, the methods of the present disclosure further include producing CAR T cells of a predetermined phenotype (i.e., promoting cells of a desired phenotype). The predetermined phenotype can be, for example, a predetermined ratio of CD8+ cells to CD4+ cells. In some embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 1: 1, about 0.25: 1, or about 0.5: 1. In other embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 2: 1, about 3: 1, about 4: 1, or about 5: 1.

[0053] In some embodiments, the T cell culture is expanded to a predetermined culture size (i.e., number of cells). The predetermined culture size may contain a sufficient number of cells suitable for clinical use (i.e., transfusion to a patient, research and development work, etc.). In some embodiments, a clinical or therapeutic dose of T cells for administration to a patient is about 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, approximately 10 9 cells, or approximately 10 10In some embodiments, the methods produce at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 clinical doses of T cells (and thus ultimately CAR T cells). In embodiments, the number of T cells produced by the methods described herein is at least about 100 million (i.e., 100 x 10 6 ) cells, or at least about 1 billion (i.e., 1 × 10 9 ) cells, at least about 50 billion, at least about 100 billion, at least about 250 billion, at least about 500 billion, at least about 750 billion, or at least about 1 trillion (i.e., 1 x 10 12 ) cells, including at least about 2 trillion, at least about 3 trillion, at least about 4 trillion, at least about 5 trillion, or at least about 10 trillion T cells.

[0054] Following T cell expansion, the cells are suitably prepared for storage, including, for example, freezing the expanded T cell line after expansion. Methods for freezing expanded cells are known in the art and may involve the use of liquid nitrogen, dry ice, and various freeze-drying procedures. Preferably, the cells are frozen at a temperature of about -80°C to about 0°C and may involve the use of a cryoprotectant such as dimethyl sulfoxide (DMSO). The cells can be stored in a frozen state for weeks, months, and even years, until the desired time at which they can be thawed for further processing and / or genetic modification as described herein.

[0055] In a further embodiment, provided herein is a method for producing a genetically modified T cell line, comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T cell line; integrating the nucleic acid molecule into the genome of the T cell line; and propagating the T cell line. As described herein, the nucleic acid molecule encoding the CRISPR-associated nuclease is RNA, which is reverse transcribed into DNA and then integrated into the genome of the cell. As described herein, preferably, the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0056] After expansion as described herein, the T cells can be optionally frozen and stored, with the stored cells then suitably thawed prior to further processing.

[0057] As described herein, the method can further include inducing the expression of the CRISPR-associated nuclease by activating a controllable promoter. In the case of an inducible promoter (e.g., a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone-responsive element, or a glutamate-inducible promoter), the promoter is induced by adding, for example, 4-hydroxytamoxifen, rapamycin, a hormone, or glutamate, respectively. In the case of a derepressible promoter (e.g., a TetO sequence described herein linked to a CMV promoter), the addition of doxycycline releases repression, and the gene (engineered nuclease) is expressed via the CMV promoter. Preferably, the nucleic acid molecule encoding Cas9 also encodes a TetR repression element, preferably under the control of another promoter system, such as a constitutive promoter like the hPGK promoter. The controllable promoter can also be a Tet-on system, including the use of the TRE3G promoter sequence described herein.

[0058] Figures 4A-4B show an exemplary derepression system, the TetO system described herein. As shown in Figure 4A, two TetO sequences (along with a promoter sequence) are preferably oriented in front of the engineered nuclease (EN). When the tetracycline repressor protein (TetR, the repression element of the TetO sequence) binds to the TetO sequence, the promoter (e.g., CMV) is repressed; that is, little or no transcription occurs from these promoters. As shown in Figure 4B, when a binding partner (preferably doxycycline (Dox)) binds to TetR, the TetR protein changes conformation and is released from the TetO sequence, allowing the functional promoter (e.g., CMV) to begin its normal transcription process, as found in nature, resulting in the production of the engineered nuclease (EN).

[0059] Figure 4C shows an exemplary inducible vector (e.g., a lentiviral vector) that can be used to incorporate an inducible engineered nuclease (in this case, Cas9 nuclease under the control of the TET-on operating system (TRE3G)), allowing expression of Cas9 in cells upon induction with doxycycline. Figure 4D shows a more detailed vector map.

[0060] Figure 4E shows the operation of the TET-on operating system, TRE3G. As shown, in the absence of doxycycline, the Tet-On 3G transactivator protein does not bind to the TRE3G promoter sequence. In the presence of doxycycline, the Tet-On 3G transactivator protein binds to the TRE3G promoter sequence, which can activate transcription and cause expression of the Cas9 nuclease (or other nuclease, as appropriate).

[0061] As depicted in Figure 4D, the controllable systems described herein for introducing a nuclease (e.g., Cas9 or Cas12) also preferably include a selectable marker, e.g., an antibiotic resistance (e.g., ampicillin resistance) gene, allowing for the production of inducible nuclease-containing cells (including T cells) that can be easily selected and enriched. Other controllable systems described herein (other inducible systems as well as derepressible systems) can also be used in combination with a selectable marker to allow for the selection and then enrichment of nuclease-expressing cells to provide a pure population of cells with the desired nuclease (e.g., Cas9 or Cas12) integrated into their genome.

[0062] In addition to activating expression of the Cas9 nuclease, a guide RNA and a gene of interest are also introduced into the expanded T cell line, as shown in phase 3 of Figure 3. As described herein, this introduction of the guide RNA and gene of interest can be via a transfection mechanism, such as nucleofection.

[0063] As a result of this introduction of guide RNA and gene of interest, T cell receptor is suitably knocked out, and gene of interest is introduced into the genome of T cell line.After introducing gene of interest, T cell is suitably propagated, and then genetically modified T cell line is collected.The method for proliferation is known in the art and is described herein.The method for collecting desired cell includes various filtration methods, centrifugation, and cell isolation and washing.

[0064] Suitable knockout of the T cell receptor includes knocking out the TRAC gene (T cell receptor alpha subunit), which leads to ablation of the entire T cell receptor. As described herein, various guide RNA sequences can be used to knock out the TRAC gene, including those described in the Examples and other sequences readily determined by one of skill in the art.

[0065] As described herein, to produce chimeric antigen receptor T cells, the gene of interest preferably encodes a chimeric antigen receptor (CAR). As shown in Figure 2, gene editing with Cas9 nuclease (or Cas12 nuclease) results in knockout of the T cell receptor and expression of the desired CAR on the T cells. Such T cells can be administered to a desired patient population based on the desired CAR.

[0066] Integration of the desired CAR construct into the T cell preferably occurs at the site of a knockout of the TRAC gene, such that the CAR is under the control of the endogenous promoter of the TRAC gene.

[0067] As described herein, the ability to expand T cells to significant numbers before inducing Cas9 expression and then subsequently integrate a gene of interest is demonstrated in 10 9 ~10 12 This allows the production of as many as 10 T cells.

[0068] In further embodiments, rather than introducing a gene of interest, one or more genes can be knocked out in T cells to produce the desired modification. For example, genes such as programmed cell death protein 1 (PD1) and / or the B2M gene (b2 microglobulin) are responsible for encoding serum proteins found in association with major histocompatibility complex (MHC) class I heavy chains. Knockout of such genes can occur using a variety of methods, including antisense, siRNA, microRNA, and other approaches known in the art.

[0069] In additional embodiments, provided herein are methods of producing a chimeric antigen receptor (CAR) T cell line, comprising introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T cell line, integrating the nucleic acid molecule into the genome of the T cell line, expanding the T cell line, inducing expression of the Cas9 nuclease by activating the controllable promoter, introducing a guide RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T cell line, knocking out expression of the T cell receptor and introducing a nucleic acid encoding the CAR into the genome of the T cell line, and recovering the CAR T cell line.

[0070] Described herein are examples of various regulatable promoters, including inducible promoters and derepressible promoters, as well as methods for inducing expression of a Cas9 nuclease through the introduction of a molecule that induces expression or derepresses a derepressible promoter.

[0071] In additional embodiments, provided herein are allogeneic T cell lines comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T cell line. As described herein, the Cas nuclease is preferably a Cas9 nuclease.

[0072] As described herein, preferably, the allogeneic T cell line comprises a controllable promoter that is an inducible promoter (e.g., comprising a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, or a glutamate-inducible promoter). The controllable promoter may be a Tet-on system.

[0073] In further embodiments, the regulatable promoter can be a derepressible promoter, such as using one or more tetracycline operator sequences (TetO). In such embodiments, the T cell further comprises a nucleic acid molecule encoding a tetracycline repressor protein.

[0074] As described, allogeneic T cells prepared according to the described embodiments may be at least about 10 9 T cells, at least about 10 10 T cells, at least about 10 11 T cells, or in embodiments, at least about 10 12 This allows for the production of individual T cells.

[0075] Also provided herein are methods of treating a mammalian subject, preferably a human subject, comprising administering CAR T cells prepared using the allogeneic T cells described herein, as well as CAR T cells prepared using the methods described herein. Administration to a human subject can include, for example, inhalation, injection, or intravenous administration, as well as other methods of administration known in the art. [Example]

[0076] Example 1: Transduction of T cells with a Cas9-inducible vector Three treatment protocols were investigated for use in transducing T cells with inducible Cas9 vectors. The vectors contain a green fluorescent protein (GFP) tag to determine the level of transduction.

[0077] As shown in Figures 5A-5C, Treatment 1 (IL2) included 24-hour activation with 15 ng / mL IL-2 and CD3 / CD28 on the day of T cell isolation. After viral transduction, proliferation included 15 ng / mL IL-2 (IL2) alone. Treatment 2 (IL2 + CD3 / CD28) included 24-hour activation with 15 ng / mL IL-2 and CD3 / CD28 on the day of T cell isolation. After transduction, proliferation included treatment with 15 ng / mL IL-2 and CD3 / CD28 at each medium change. Treatment 3 (IL2 + IL) included 24-hour activation with 15 ng / mL IL-2 and CD3 / CD28 on the day of T cell isolation. After transduction, cells were treated with 15 ng / mL IL-2 and CD3 / CD28. Proliferation was performed in the presence of IL-2 and IL-7 alone.

[0078] Figures 6A and 6B provide the transduction results. As shown in Figure 6A, the percentage (%) of GFP-positive cells had the highest transduction efficiency using the combination of CD3 / CD28 + IL-2 treatment with both the control vector (#) and the vector containing the Cas9 nuclease gene (@). Treatment with IL-2 + IL-7 ($) also showed good transduction. Figure 6B shows the dilution rate of the GFP-positive population.

[0079] The balsticidin resistance gene in the Cas9-inducible vector was used to select cells containing the Cas9 vector correctly integrated into the genome. Figure 7A shows the number of surviving cells for the three treatments described in this example. As shown, treatment with IL-2 + IL-7 showed the highest number of surviving cells. After selection and expansion for 12 days, both cells treated with CD3 / CD28 + IL-2 and IL-2 + IL-7 showed a large number of surviving cells (Figure 7B).

[0080] Markers of fatigue, senescence, and activation were measured for the three treatment protocols, and the results are shown in FIG.

[0081] Induction of Cas9 expression was performed on day 11 after blasticidin selection. Cells were induced with 1 μg / mL doxycycline for 24 hours, and protein lysates were analyzed by Western blot. As shown in Figure 9, cells grown with Treatment 3 (IL-2 + IL-7) showed higher Cas9 expression upon induction, but cells treated with CD3 / CD28 + IL-2 also showed expression of Cas9 nuclease.

[0082] The cells were then frozen and thawed to determine whether the insertion of the Cas9 vector had any effect on cell viability. As shown in Figure 10A, the viability was almost the same before and after cryopreservation for both treatments described. Figure 10B shows the cell survival days after thawing, indicating that for both treatments, the cells were able to proliferate successfully.

[0083] Example 2: Knockout of the TRAC gene Three sgRNA sequences were selected to investigate their ability to knock out the TRAC gene in T cells transduced with a Cas9 vector. Figure 11 shows the three sgRNA sequences examined (SEQ ID NOs: 1-3) and the regions they target in the translated TRAC gene (SEQ ID NO: 4). The sgRNA sequences were transfected into T cells using a nucleofection procedure. Briefly, 1 x 10 T cells in 20 µL were transfected using the AMAXA P2 Primary Cell 4D Nucleofector X Kit and the EO-115 program. 6 Cells were transduced with approximately 3.3 μg of sgRNA at room temperature.

[0084] Knockout experiments were performed with sgRNAs TRAC#1-3 (SEQ ID NOs: 1-3) and calibrated for CD-3. Cas9 T cells (treated with CD3 / CD28 and 15 ng / mL IL-2 and IL-7, then selected with 15 μg / mL blasticidin) were thawed on day 1. On day 2, Cas9 was induced with doxycycline (2 μg / mL, 24 hours). On day 3, cells were transduced with sgRNAs TRAC#1 (SEQ ID NO: 1), TRAC#2 (SEQ ID NO: 2), and TRAC#3 (SEQ ID NO: 3) via nucleofection and expanded. FACS analysis of CD-3 and TCRαβ expression levels was performed on days 4, 7, and 14.

[0085] Figures 12A-1 through 12B-6 show TRAC knockout 4 days after nucleofection using the TRAC#1 through #3 sgRNA sequences. Figure 12C shows a summary of the results. As shown, TRAC#1 through #3 each resulted in approximately 41-47% knockout of the TRAC gene, with the TRAC#2 sgRNA showing the highest amount of knockout (47%).

[0086] Figures 13A-1 through 13B-6 show TRAC knockout 7 days after nucleofection using the TRAC#1 through #3 sgRNA sequences. Figure 13C shows a summary of the results. As shown, TRAC#1 through TRAC#3 each resulted in approximately 66-74% knockout of the TRAC gene, with the TRAC#2 sgRNA showing the highest amount of knockout (74%).

[0087] Figures 14A-1 through 14B-6 show TRAC knockout 14 days after nucleofection using the TRAC#1 through #3 sgRNA sequences. Figure 14C shows a summary of the results. As shown, each of TRAC#1 through #3 resulted in approximately 84-89% knockout of the TRAC gene, with the TRAC#2 sgRNA showing the highest amount of knockout (89%).

[0088] Figures 15A-15B show a summary of the 14 day experiment, demonstrating effective knockout of the TRAC gene using the methods described herein.

[0089] Example 3: Knock-in of a CAR construct The following experiments are designed to demonstrate the ability to knock-in a desired constructed CAR into Cas9-containing T cells.

[0090] CAR knock-in experiments are performed following the same procedure as the knock-out experiments described above, with the following addition: during nucleofection of gRNA (targeting the TRAC gene), a DNA template is added. This DNA template is designed to integrate into the TRAC locus using homologous recombination mechanisms, following the double-strand break generated by the nuclease.

[0091] Three types of DNA templates are examined: single stranded DNA Mini Circle DNA Linear double-stranded DNA.

[0092] Two constructs are investigated: conventional anti-CD19 CAR (assessed using FACS or functional assays), Anti-CD19-CAR fused to a green fluorescent protein (GFP) molecule on the cytoplasmic side (which can be easily detected using FACS).

[0093] Further Exemplary Embodiments Embodiment 1 is a method of producing a T cell line for use in allogeneic applications, comprising introducing a nucleic acid molecule encoding an engineered nuclease under the control of a regulatable promoter into the T cell line, integrating the nucleic acid molecule into the genome of the T cell line, and expanding the T cell line.

[0094] Embodiment 2 includes the method of embodiment 1, wherein the engineered nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector-based nucleases, and CRISPR-associated nucleases.

[0095] Embodiment 3 includes the method of embodiment 2, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0096] Embodiment 4 includes the method of any one of embodiments 1 to 3, wherein the controllable promoter is an inducible promoter.

[0097] Embodiment 5 includes the method of embodiment 4, wherein the inducible promoter is a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, or a glutamate-inducible promoter.

[0098] Embodiment 6 includes the method of any one of embodiments 1 to 3, wherein the regulatable promoter is a derepressible promoter.

[0099] Embodiment 7 includes the method of embodiment 6, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0100] Embodiment 8 includes the method of embodiment 7, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0101] Embodiment 9 includes the method of any one of embodiments 1 to 3, wherein the regulatable promoter comprises a Tet-on system.

[0102] Embodiment 10 includes the method of any one of embodiments 1 to 9, further comprising freezing the expanded T cell line after expansion.

[0103] Embodiment 11 includes the method of any one of embodiments 1 to 10, wherein the nucleic acid molecule is introduced into the T cell line using a lentiviral vector.

[0104] Embodiment 12 is a method for producing a T cell line comprising the steps of: 9 12. The method of any one of embodiments 1 to 11, comprising administering to the subject a therapeutically effective amount of T cells.

[0105] Embodiment 13 is a method of producing a genetically modified T cell line, comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into the T cell line; integrating the nucleic acid molecule into the genome of the T cell line; expanding the T cell line; inducing expression of the CRISPR-associated nuclease by activating the controllable promoter; introducing a guide RNA and a gene of interest into the expanded T cell line; knocking out expression of a T cell receptor and introducing the gene of interest into the genome of the T cell line; and recovering the genetically modified T cell line.

[0106] Embodiment 14 includes the method of embodiment 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0107] Embodiment 15 includes the method of embodiment 13 or embodiment 14, wherein the controllable promoter is an inducible promoter.

[0108] Embodiment 16 includes the method of embodiment 15, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0109] Embodiment 17 includes the method of embodiment 13 or embodiment 14, wherein the regulatable promoter is a derepressible promoter.

[0110] Embodiment 18 includes the method of embodiment 17, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0111] Embodiment 19 includes the method of embodiment 18, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0112] Embodiment 20 includes the method of embodiment 18 or embodiment 19, wherein activating the derepressible promoter comprises adding doxycycline to the T cell line.

[0113] Embodiment 21 includes the method of embodiment 13 or 14, wherein the regulatable promoter comprises a Tet-on system.

[0114] Embodiment 22 includes the method of embodiment 21, wherein activating the Tet-on system includes doxycycline added to the T cell line.

[0115] Embodiment 23 includes the method of any one of embodiments 13 to 22, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

[0116] Embodiment 24 includes the method of any one of embodiments 13 to 23, further comprising freezing the T cell line after expansion in c and thawing it prior to derivation in d.

[0117] Embodiment 25 is a method for producing a genetically modified T cell line comprising administering to a subject a subject a genetically modified T cell line comprising at least about 10 9 The method of any one of embodiments 13 to 24 includes the method of any one of embodiments 13 to 24, wherein the method comprises administering to the patient a therapeutically effective amount of T cells.

[0118]

[0033] Embodiment 26 is a method of producing a chimeric antigen receptor (CAR) T cell line, comprising introducing a nucleic acid molecule encoding a Cas9 nuclease under control of a controllable promoter into a T cell line, integrating the nucleic acid molecule into the genome of the T cell line, expanding the T cell line, inducing expression of the Cas9 nuclease by activating the controllable promoter, introducing a guide RNA and a nucleic acid encoding the chimeric antigen receptor (CAR) into the expanded T cell line, knocking out expression of the T cell receptor and introducing a nucleic acid encoding the CAR into the genome of the T cell line, and recovering the CAR T cell line.

[0119] Embodiment 27 includes the method of embodiment 26, wherein the controllable promoter is an inducible promoter.

[0120] Embodiment 28 includes the method of embodiment 27, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0121] Embodiment 29 includes the method of embodiment 26, wherein the regulatable promoter is a derepressible promoter.

[0122] Embodiment 30 includes the method of embodiment 29, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0123] Embodiment 31 includes the method of embodiment 30, wherein the nucleic acid molecule further comprises a tetracycline repressor protein.

[0124] Embodiment 32 includes the method of embodiment 30 or embodiment 31, wherein activating the derepressible promoter includes adding doxycycline to the T cell line.

[0125] Embodiment 33 includes the method of embodiment 26, wherein the regulatable promoter comprises a Tet-on system.

[0126] Embodiment 34 includes the method of embodiment 33, wherein the Tet-on system includes adding doxycycline to the T cell line.

[0127] Embodiment 35 includes the method of any one of embodiments 26 to 34, further comprising freezing the T cell line after expansion in c and thawing it prior to derivation in d.

[0128] Embodiment 36 is a method for producing a CAR T cell line comprising administering to a subject a subject the method comprising administering to a subject a subject a CAR T cell line ... 9 The method of any one of embodiments 26 to 35 includes the method of any one of embodiments 26 to 35, wherein the method comprises administering to the patient a single T cell.

[0129] Embodiment 37 is an allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a regulatable promoter integrated into the genome of the T cell line.

[0130] Embodiment 38 comprises the allogeneic T cell line of embodiment 37, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

[0131] Embodiment 39 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the regulatable promoter is an inducible promoter.

[0132] Embodiment 40 comprises the allogeneic T cell line of embodiment 39, wherein the inducible promoter is a 4HT-inducible promoter or a glutamate-inducible promoter.

[0133] Embodiment 41 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the regulatable promoter is a derepressible promoter.

[0134] Embodiment 42 comprises the allogeneic T cell line of embodiment 41, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

[0135] Embodiment 43 includes the allogeneic T cell line of embodiment 42, wherein the T cells further comprise a nucleic acid encoding a tetracycline repressor protein.

[0136] Embodiment 44 comprises the allogeneic T cell line of embodiment 37 or embodiment 38, wherein the regulatable promoter comprises a Tet-on system.

[0137] Embodiment 45 is a method for treating a pulmonary artery disease comprising administering to a subject a subject a pulmonary artery disease having a pulmonary artery disease, ... or a pulmonary artery disease. 9 The allogeneic T cell line of any one of embodiments 37 to 44, comprising 10 T cells.

[0138] Embodiment 46 is a method for producing a medicament comprising administering at least about 10 10 46. The allogeneic T cell line of embodiment 45, comprising 46 T cells.

[0139] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0140] Although particular embodiments have been illustrated and described herein, it is to be understood that the claims should not be limited to the specific forms or arrangements of parts described and shown. Although exemplary embodiments are disclosed and specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0141] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0142] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 930,617, filed November 5, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0143] [Sequence table] This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is incorporated herein by reference in its entirety. The ASCII copy, created on December 15, 2020, is named 0132-0081WO1_SL.txt and is 1,511 bytes in size.

Claims

1. An allogeneic T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a regulatable promoter integrated into the genome of the T cell line.

2. 2. The allogeneic T cell line of claim 1, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

3. 3. The allogeneic T cell line of claim 1 or 2, wherein the controllable promoter is selected from the group consisting of an inducible promoter and a derepressible promoter.

4. The allogeneic T cell line of claim 3, wherein the controllable promoter is an inducible promoter.

5. 5. The allogeneic T cell line of claim 4, wherein the inducible promoter is selected from the group consisting of a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, a TET-on system, and a glutamate-inducible promoter.

6. 6. The allogeneic T cell line of claim 5, wherein the inducible promoter is the 4HT-inducible promoter or the glutamate-inducible promoter.

7. The allogeneic T cell line of claim 3 , wherein the regulatable promoter is a derepressible promoter.

8. The allogeneic T cell line of claim 7, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

9. The allogeneic T cell line of claim 8, wherein the T cells further comprise a nucleic acid encoding a tetracycline repressor protein.

10. The allogeneic T cell line of claim 1 or 2, wherein the controllable promoter comprises a Tet-on system.

11. The allogeneic T cell line of claim 10, wherein activating the Tet-on system comprises adding doxycycline to the expanded T cell line.

12. 2. The allogeneic T cell line of claim 1, wherein the CRISPR-associated nuclease is a Cas9 nuclease and the controllable promoter comprises a Tet-on system.

13. A chimeric antigen receptor (CAR) T cell line comprising a CRISPR-associated (Cas) nuclease under the control of a regulatable promoter integrated into the genome of the T cell line.

14. 14. The CAR T cell line of claim 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

15. 15. The CAR T cell line of claim 13 or 14, wherein the controllable promoter is selected from the group consisting of an inducible promoter and a derepressible promoter.

16. 16. The CAR T cell line of claim 15, wherein the controllable promoter is an inducible promoter.

17. 17. The CAR T cell line of claim 16, wherein the inducible promoter is selected from the group consisting of a 4HT-inducible promoter, a rapamycin-inducible promoter, a hormone response element, a TET-on system, and a glutamate-inducible promoter.

18. 18. The CAR T cell line of claim 17, wherein the inducible promoter is the 4HT-inducible promoter or the glutamate-inducible promoter.

19. 16. The CAR T cell line of claim 15, wherein said regulatable promoter is said derepressible promoter.

20. 20. The CAR T cell line of claim 19, wherein the derepressible promoter comprises one or more tetracycline operator sequences (TetO).

21. 21. The CAR T cell line of claim 20, wherein the T cells further comprise a nucleic acid encoding a tetracycline repressor protein.

22. 15. The CAR T cell line of claim 13 or 14, wherein the controllable promoter comprises a Tet-on system.

23. 23. The CAR T cell line of claim 22, wherein activating the Tet-on system comprises adding doxycycline to the expanded T cell line.

24. 14. The CAR T cell line of claim 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease and the controllable promoter comprises a Tet-on system.

Images