Methods for expanding regulatory T cells

Abstract

Provided herein are ex vivo methods for expanding regulatory T cells (Tregs), including engineered Tregs, while maintaining their stability and activity. In addition to improving the growth and expansion of Tregs, the methods can include the use of discontinuous stimulation of regulatory T cells (DSORT™), which generates Tregs with improved stability and activity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 152,787, filed February 23, 2021, which is incorporated by reference in its entirety.

[0002] Reference to electronically filed sequence listing The sequence listing, created on February 22, 2022 and submitted on February 23, 2022 as a text file named "4195_022PC01_Seqlisting_ST25" and 11,366 bytes in size, is incorporated herein by reference. [Background technology]

[0003] Field Provided herein are ex vivo methods for expanding regulatory T cells (Tregs), including engineered Tregs, while maintaining their stability and activity.

[0004] background Regulatory T cells (Tregs) are important for preventing autoimmunity. Tregs are CD4+ T cells that suppress inflammation and effector T cell (Teff) activity to maintain homeostasis. + T cells. Defects in the number and / or function of Tregs are frequently observed in patients with autoimmune diseases. The potential therapeutic use of Tregs has been recognized, and Tregs are being evaluated in clinical trials for the treatment of various autoimmune conditions and graft-versus-host disease (GvHD).

[0005] Although clinical studies using freshly isolated Tregs have been performed in the past, the widespread use of Treg adoptive cell therapy (ACT) requires ex vivo expansion of Tregs to generate sufficient numbers of cells, which must be done in a way that does not compromise the purity, stability, and intrinsic suppressive function of Tregs.

[0006] The inability to effectively expand stable and active populations of Tregs poses a significant challenge. Clinical manipulation of Tregs typically uses closed GMP-certified magnetic enrichment systems such as CliniMACS for the isolation of CD25-expressing cells, which results in a large amount of contamination with activated effector T cells (Teff). The expansion of such cells can be at least partially prevented by adding the mTOR protein kinase inhibitor rapamycin to the cultures. While Tregs are relatively resistant to mTOR inhibition, studies have found that rapamycin reduces the expansion of Tregs by about 35% over a 35-day period. Furthermore, rapamycin affects the phenotype of Tregs by altering, for example, the expression of tissue homing receptors such as CCR4 and CXCR3, as well as their function (e.g., CTLA-4, LAP) and lineage (Helios and Foxp3), which may obscure or alter the effects of gene editing.

[0007] Rapamycin is not the only compound that has been used to improve Treg purity or induce proliferation in vitro. TGF-β, together with the vitamin A derivative all-trans retinoic acid (ATRA), have been shown in preclinical studies to support Treg proliferation in vitro, but have profound effects on Treg phenotype, such as upregulation of gut-homing receptors such as CCR9 and integrin-α4β7, and CD161.

[0008] Another approach using rapamycin and ATRA involves the use of naive CD45RA +The first step is to start with a more homogenous population of naive Tregs. Such isolated products contain few effector T cells and are therefore more suitable for long-term Treg expansion in the absence of rapamycin. Naive Tregs expanded ex vivo have been shown to maintain a naive phenotype and express lymph node (LN) homing markers such as CD62L and CCR7, which have been shown to be necessary for the prevention of autoimmune disease in preclinical models. However, time is one of the most important factors when it comes to Treg purity and stability. Tregs become suppressive when activated in vitro and maintain a high level of suppressive capacity throughout the first 1-2 weeks in culture, during which time Tregs effectively prevent the proliferation of contaminating effector T cells. However, over time, the ability of Tregs to control the proliferation of other T cells declines, non-Tregs rapidly accumulate, and the pure Treg population is lost.

[0009] Thus, currently available protocols for the ex vivo expansion of Tregs result in insufficient expansion and / or stability, and therefore there is a need for efficient methods of expanding Tregs to generate pure, stable, and highly functional Treg populations. Summary of the Invention [Means for solving the problem]

[0010] Provided herein are methods for expanding regulatory T cells (Tregs) that maximize Treg proliferation during the first 1-2 weeks of culture and take advantage of the enhanced suppressive activity of Tregs early after activation. As demonstrated herein, discontinuous stimulation of regulatory T cells (DSORT™) can achieve rapid Treg proliferation by alternating periods of stimulation and rest. An exemplary DSORT™ process is shown in Figure 8. Despite an expansion rate of over 1000-fold, such methods can create a pure, stable, highly functional Treg product. Advantageously, these methods do not require the use of rapamycin to slow cell proliferation during the first 1-2 weeks of culture.

[0011] Provided herein is a method for expanding a regulatory T cell (Treg) population, the method comprising: (a) a first stimulation step comprising culturing a Treg population in the presence of a first stimulating agent to generate a first stimulated Treg population; and (b) a first resting step comprising continuing to culture the first stimulated Treg population in the absence of the stimulating agent to generate a first resting Treg population. In some embodiments, the method further comprises a second stimulation step comprising (c) culturing the first resting Treg population in the presence of a second stimulating agent to generate a second stimulated Treg population. In some embodiments, the method further comprises a second resting step comprising (d) continuing to culture the second stimulated Treg population in the absence of the stimulating agent to generate a second resting Treg population. In some embodiments, the method further comprises a third stimulation step comprising (e) culturing the second resting Treg population in the presence of a third stimulating agent to generate a third stimulated Treg population. In some embodiments, the method further comprises a third resting step comprising (f) continuing to culture the third stimulated Treg population in the absence of the stimulatory agent to generate a third resting Treg population. In some embodiments, the method further comprises one or more additional stimulation step(s) to generate a further stimulated Treg population, and / or one or more additional resting step(s) to generate a further resting Treg population.

[0012] In some embodiments, the method further comprises genetically engineering the first rested Treg population, the second stimulated Treg population, the second rested Treg population, the third stimulated Treg population, the third rested Treg population, the further stimulated Treg population, or the further rested Treg population.

[0013] In some embodiments, the method further comprises recovering the first rested Treg population, the second stimulated Treg population, the second rested Treg population, the third stimulated Treg population, the third rested Treg population, the further stimulated Treg population, the further rested Treg population, or the genetically engineered Treg population.

[0014] Provided herein is a method for expanding a regulatory T cell (Treg) population, the method comprising: (a) a stimulation step comprising culturing a Treg population in a medium comprising a stimulating agent to generate a first stimulated Treg population; (b) washing the stimulated Treg population to remove the medium comprising the stimulating agent to generate a washed Treg population; and (c) culturing the washed Treg population in a fresh medium to generate a first resting Treg population. In some embodiments, the fresh medium does not comprise any stimulating factor. In some embodiments, the method further comprises genetically manipulating the first resting Treg population. In some embodiments, the method further comprises culturing the first resting Treg population in a medium comprising a stimulating agent to generate a second stimulated Treg population.

[0015] Provided herein is a method for expanding a population of regulatory T cells (Tregs), the method comprising: (a) a resting step comprising culturing a previously stimulated population of Tregs in the absence of a stimulating agent to generate a first population of resting Tregs; and (b) a stimulation step comprising adding a stimulating agent to the first population of resting Tregs to generate a stimulated population of Tregs.

[0016] In some embodiments, the method further comprises genetically engineering the Tregs. In some embodiments, the method further comprises genetically engineering the first population of resting Tregs.

[0017] In some embodiments, the method further comprises culturing the engineered Treg population.

[0018] In some embodiments, culturing the engineered Treg population is performed in the presence of a stimulatory agent. In some embodiments, the method further comprises continuing to culture the engineered Treg population in the absence of the stimulatory agent.

[0019] In some embodiments, culturing the engineered Treg population is performed in the absence of a stimulatory agent. In some embodiments, the method further comprises continuing to culture the engineered Treg population in the presence of a stimulatory agent.

[0020] Provided herein is a method for expanding a regulatory T cell (Treg) population, the method comprising: (a) a stimulation step comprising genetically engineering a Treg population to produce an engineered Treg population; (b) culturing the engineered Treg population in the presence of a stimulant to produce a stimulated engineered Treg population; and (c) a resting step comprising continuing to culture the stimulated engineered Treg population in the absence of the stimulant to produce a resting engineered Treg population.

[0021] Provided herein are methods for expanding a regulatory T cell (Treg) population, the method comprising: (a) genetically engineering a Treg population to produce an engineered Treg population; (b) a resting step comprising culturing the engineered Treg population in the absence of a stimulating agent to produce a resting engineered Treg population; and (c) a stimulation step comprising culturing the resting engineered Treg population in the presence of a stimulating agent to produce a stimulated engineered Treg population.

[0022] In some embodiments, prior to genetic engineering, the Treg population is subjected to a stimulation step comprising culturing the population in the presence of a stimulatory agent.

[0023] In some embodiments, prior to genetic manipulation, the Treg population is subjected to a stimulation step comprising culturing the population in the presence of a stimulating agent, followed by a resting step comprising culturing the population in the absence of the stimulating agent.

[0024] In some embodiments, prior to genetic manipulation, the Treg population is subjected to a stimulation step comprising culturing the population in the presence of a stimulating agent, followed by a resting step comprising culturing the population in the absence of the stimulating agent, followed by another stimulation step comprising culturing the population in the presence of the stimulating agent.

[0025] In some embodiments, the genetic manipulation is performed in the absence of a stimulating agent.

[0026] In some embodiments, the method further comprises harvesting the Treg population.

[0027] In some embodiments, the method further comprises obtaining a Treg population from the subject.

[0028] In some embodiments, the method further comprises obtaining a Treg population from a thymus, peripheral blood, umbilical cord blood, or tissue sample of the subject.

[0029] In some embodiments, the method further comprises obtaining a Treg population from the subject's peripheral blood prior to the culturing in step (a).

[0030] In some embodiments, the Treg population is obtained from the subject's thymus, peripheral blood, umbilical cord blood or tissue sample. In some embodiments, the Treg population is obtained from the subject's peripheral blood.

[0031] In some embodiments, the subject is a human.

[0032] In some embodiments, the tetrameric antibody complex is a stimulatory agent in at least one of the stimulation steps and / or culture of the engineered population. In some embodiments, the tetrameric antibody complex specifically binds to CD3, CD28, CD2, or a combination thereof.

[0033] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof, and / or the anti-CD28 antibody or antigen-binding fragment thereof is a stimulatory agent in at least one of the stimulation steps and / or culture of the engineered population.

[0034] In some embodiments, CD3-bound and / or CD28-bound supermagnetic beads are stimulatory agents in at least one of the stimulation steps and / or cultures of the engineered population.

[0035] In some embodiments, the method does not use supermagnetic beads.

[0036] In some embodiments, the same stimulating agent is used throughout the method. In some embodiments, at least two different stimulating agents are used in the method.

[0037] In some embodiments, the stimulating agent is present at the same concentration throughout all of the stimulating steps of the method. In some embodiments, at least two different concentrations of the stimulating agent are used in the method.

[0038] In some embodiments, the Tregs are cultured in the presence of IL-2. In some embodiments, the IL-2 concentration is decreased during the method. In some embodiments, IL-2 is present at a concentration of about 800 units / mL for about 7 days, and then at about 300 units / mL.

[0039] In some embodiments, the Treg population is cultured in the presence of a stimulating agent for about 1 to about 5 days, then cultured in the absence of the stimulating agent for about 1 to about 5 days, then cultured in the presence of the stimulating agent for about 1 to about 5 days, and then genetically engineered.

[0040] In some embodiments, the Treg population is cultured in the presence of a stimulatory agent for about 3 to about 4 days, then in the absence of the stimulatory agent for about 3 to about 4 days, then in the presence of the stimulatory agent for about 1 to about 4 days, and then genetically engineered. In some embodiments, the Treg population is cultured in the presence and / or absence of a stimulatory agent according to Table A.

[0041] In some embodiments, the Tregs are cultured in the presence of N-acetyl-cysteine. In some embodiments, N-acetyl-L-cysteine ​​is present in the culture at a concentration of about 5 mM.

[0042] In some embodiments, the Treg population is engineered when the Treg population has expanded about 250-fold. In some embodiments, the Treg population is engineered about 6 to about 10 days after the Tregs are obtained from the subject. In some embodiments, the Treg population is engineered about 7 days after the Tregs are obtained from the subject.

[0043] In some embodiments, the genetic engineering comprises introducing a nucleic acid into the Treg population. In some embodiments, the nucleic acid is a viral nucleic acid. In some embodiments, the nucleic acid is not a viral nucleic acid. In some embodiments, the nucleic acid encodes a protein. In some embodiments, the protein is a heterologous protein. In some embodiments, the heterologous protein is a chimeric antigen receptor (CAR).

[0044] In some embodiments, the genetic engineering comprises introducing a gene regulatory system into the Treg population. In some embodiments, the gene regulatory system comprises: (i) a nucleic acid molecule; (ii) an enzyme protein; or (iii) a nucleic acid molecule and an enzyme protein. In some embodiments, the gene regulatory system comprises a nucleic acid molecule selected from siRNA, shRNA, microRNA (miR), antagomir, or antisense RNA. In some embodiments, the gene regulatory system comprises an enzyme protein, and the enzyme protein is engineered to specifically bind to a target sequence of one or more genes in the Treg. In some embodiments, the enzyme protein is a transcription activator-like effector nuclease (TALEN), zinc finger nuclease, or meganuclease. In some embodiments, the gene regulatory system comprises a nucleic acid molecule and an enzyme protein, where the nucleic acid molecule is a guide RNA (gRNA) molecule and the enzyme protein is a Cas protein or a Cas orthologue. In some embodiments, the Cas protein is a Cas9 protein.

[0045] In some embodiments, the introduction uses electroporation or ribonucleoprotein (RNP) mediated methods.

[0046] In some embodiments, the method does not use artificial antigen presenting cells.

[0047] In some embodiments, the methods do not use rapamycin. In some embodiments, the methods include using rapamycin.

[0048] In some embodiments, the method increases the number of Tregs by at least 1000 fold in 11 days.

[0049] In some embodiments, the method results in Tregs with a smaller surface area than Tregs cultured in the presence of a stimulant for 6 days. In some embodiments, the method expands the Helios+FoxP3+ Treg population compared to Tregs cultured in the presence of a stimulant for 6 days. In some embodiments, the method results in Tregs with an increased ability to suppress the proliferation of effector T cells (Teffs) compared to Tregs cultured in the presence of a stimulant for 6 days. In some embodiments, the increased ability to suppress the proliferation of Teffs is at least 8-fold increased ability.

[0050] In some embodiments, the methods prevent overstimulation of Treg populations.

[0051] In some embodiments, the method reduces activation-induced cell death compared to Tregs cultured in the presence of a stimulatory agent for 6 days.

[0052] In some embodiments, at least 75% of Helios expression in Tregs is maintained.

[0053] In some embodiments, the Tregs are Helios+.

[0054] In some embodiments, Tregs have a fully demethylated Treg-specific demethylation region (TSDR).

[0055] In some embodiments, the method further comprises cryopreserving the Treg population.

[0056] Provided herein are Treg populations produced by any of the methods provided herein. In some embodiments, provided herein are cryopreserved populations of Tregs produced by any of the methods provided herein.

[0057] In some embodiments, the method further comprises administering to the subject a population of Tregs.

[0058] Provided herein is a method of treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject an effective amount of a Treg population obtained using any of the methods provided herein or any of the Tregs provided herein.

[0059] Provided herein is a method of treating or preventing graft-versus-host disease (GVHD) in a subject, comprising administering to the subject an effective amount of a Treg population obtained using any of the methods provided herein or any of the Tregs provided herein.

[0060] Provided herein is a method of reducing an immune response in a subject, comprising administering to the subject an effective amount of a Treg population obtained using any of the methods provided herein or any of the Tregs provided herein.

[0061] In some embodiments, the Treg population is allogeneic to the subject. In some embodiments, the Treg population is autologous to the subject. [Brief description of the drawings]

[0062] [Figure 1] Figure 1 shows the proliferation of regulatory T cells (Treg) following continuous or discontinuous stimulation with CD3 / 28 / 2 tetramer monoclonal antibody (mAb) or Dynabeads (see Example 2). [Diagram 2]Relative size is shown as a measure of activation of Tregs following continuous ("Baseline") or discontinuous ("DSORT™") stimulation (see Example 3). [Diagram 3] Figure 1 shows the expansion of Tregs following continuous stimulation or discontinuous stimulation with a long non-stimulation period. Stimulation was performed with either CD3 / 28 / 2 tetramer monoclonal antibody (mAb) or Dynabeads. (See Example 4) [Figure 4] Shown is the proliferation of CRISPR engineered Tregs following continuous ("Cont") or discontinuous (DSORT™) stimulation. Open boxes represent the mean of n=3. (See Example 5) [Diagram 5]Provided are graphs (top) showing Treg cell counts (left y-axis) and fold expansion (right y-axis) obtained using DSORT™ and a table (bottom) showing fold expansion obtained using DSORT™ ("KSQ Tx") in addition to the alternating expansion protocol. "Thiel" study refers to THEIL, A., et al., "Adoptive transfer of allogeneic regulatory T cells into patients with chronic graft-versus-host disease," Cytotherapy, 17(4): P473-486 (April, 2015). "Bluestone" study refers to BLUESTONE, JA, et al., "Type 1 diabetes immunotherapy using polyclonal regulatory T cells," Sci. Transl. med. 7(315): 315ra189 (November, 2015). "Canavan" study refers to CANAVAN,JB,et al.,"Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease,"Gut,65(4):584-594(published online February,2015). "Safina" study refers to SAFINIA,N.,et al.,"Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation,"Oncotarget,7(7):7563-7577(February,2016). "Trzonkowska" study refers to MAREK-TRZONKOWSKA,N.,et al.,"Mild hypothermia provides Treg stability,"Scientific Reports,7:11915(September,2017)."Lombardi" study refers to FRASER,H.,et al.,"A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials,"Molecular Therapy-Methods & Clinical Development,8:198-209(January,2018). "Leventhal" study refers to MATHEW,JM,et al.,"A Phase I Clinical Trial with Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants,"Scientific Reports,8:7428(May,2018). "Thonhoff" study refers to THONHOFF,JR,et al.,"Expanded autologous regulatory T-lymphocyte infusions in ALS,"Neurology Neuroimmunology & Neuroinflammation,5(4):1-7(July,2018). "MacDonald" study refers to MACDONALD, KN, et al., "Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol," Cytotherapy, 21(12):1216-1233 (December, 2019). (See Example 6.) [Figure 6](A) Methylation status of the Treg-specific demethylated region (TSDR) in cells with and without expression of Helios and Foxp3. (B) Ability of cell populations with different percentages of cells expressing Helios to suppress effector T cell proliferation. (C) Expression of Helios in cells expanded using discontinuous ("DSORT™") or continuous ("Baseline") stimulation. (See Example 7) [Figure 7] Figure 1 shows the ability of Tregs stimulated continuously (basal expansion) or discontinuously (DSORT™) to suppress the proliferation of CD4+ effector T cells (see Example 8). [Figure 8] Schematic diagram of an exemplary DSORT™ process in which Tregs are subjected to alternating stimulation and resting cycles and genetically engineered. "Flask" indicates the transfer of Tregs from plate to flask. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] 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 pertains. In the event of a conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated herein by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0064] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0065] In order to further define this disclosure, the following terms and definitions are provided.

[0066] Whenever an embodiment is described herein using the term "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0067] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0068] The use of the term "or" herein does not imply mutual exclusion of alternatives. In this application, the use of "or" means "and / or" unless expressly stated or understood by the skilled artisan. In the context of multiple dependent claims, the use of "or" refers back to more than one preceding independent or dependent claim. The term "and / or" as used herein should be interpreted as a separate disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0069] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. The headings provided herein are not intended to be limiting of the various aspects of the disclosure, but may be had by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0070] As used herein, the term "about" includes ±10% of the stated number. Thus, "about 10" means 9 to 11. As will be understood by one of ordinary skill in the art, reference herein to "about" a value or parameter includes (and describes) specific examples directed to the value or parameter itself. For example, a statement referring to "about X" encompasses the statement of "X."

[0071] The term "regulatory T cells" or "Tregs" as used herein refers to a subpopulation of T cells that constitutively express the forkhead box P3 (Foxp3) transcription factor, which regulates the immune system, maintains tolerance to self-antigens, and suppresses autoimmune and inflammatory diseases. These cells generally suppress or downregulate the induction and proliferation of effector T cells and regulate the function of antigen-presenting cells. Tregs are cells capable of suppressive activity (i.e., inhibition of proliferation of conventional T cells) by cell-to-cell contact or through the release of immunosuppressive cytokines.

[0072] As used herein, the term "population" refers to a population of cells, where a majority of the cells, i.e., at least 50% (optionally at least 60%, at least 70%, or at least about 80%) of the total number of cells, have a particular characteristic of a cell of interest (e.g., a functional characteristic and / or marker of interest). Thus, a "Treg population" refers to a population of cells where the majority of the cells are Tregs, but some cells may not be Tregs (e.g., some cells may be Teffs).

[0073] As used herein, "culturing" refers to growing one or more cells in a test tube under defined or controlled conditions. Examples of culture conditions that can be defined include temperature, gas mixture, time, and media composition.

[0074] As used herein, the terms "culture medium" and "cell culture medium" and "feed medium" and "fermentation medium" refer to a nutrient solution used for the growth and / or maintenance of cells, particularly mammalian cells. Without limitation, these solutions usually provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of carbohydrates such as glucose; (2) all essential amino acids, usually a basis set of 20 amino acids; (3) vitamins and / or other organic compounds required in low concentrations; (4) free fatty acids or free lipids, such as linoleic acid; and (5) trace elements, where a trace element is defined as an inorganic compound or naturally occurring element required in very low concentrations, usually in the micromolar range. Nutrient solutions can be selectively supplemented with one or more components derived from any of the following categories: (1) hormones and other growth factors, e.g., serum, insulin, transferrin, and epidermal growth factor; (2) salts, e.g., magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) peptones or peptone mixtures that can be obtained from protein and tissue hydrolysates, e.g., purified gelatin, plant material, or animal by-products; (6) antibiotics, such as gentamicin; (7) cytoprotectants, e.g., Pluronic® polyols; and (8) galactose. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980) can be used as culture media for the host cells. Any other necessary supplements may also be included at appropriate concentrations.

[0075] As used herein, "stimulatory agent" or "stimulatory agent" refers to an agent that activates (directly or indirectly) the T cell receptor and / or costimulatory molecules such as CD28 or GITR on Tregs and / or has mitogenic activity. Stimulatory agents can enhance the proliferation of Tregs. The ability of an agent to act as a stimulatory agent can be demonstrated by its ability to increase phosphorylation of ITAM motifs on the CD3 zeta subunit.

[0076] As used herein, "in the presence of a stimulatory agent" or culture "in the presence of a stimulatory agent" refers to culturing Tregs in the presence of a sufficient amount of a stimulatory agent to activate the T cell receptor and / or costimulatory molecules, such as CD28 or GITR, on Tregs.

[0077] As used herein, "in the absence of a stimulant" or culturing in the "absence of a stimulant" refers to culturing in the absence of a stimulant in an amount sufficient to activate the T cell receptor on Tregs and / or costimulatory molecules such as CD28 and GITR. Culturing in the "absence of a stimulant" includes culturing without the use of any stimulant.

[0078] As used herein, the term "seeding" refers to the addition of cells to culture medium to initiate the culture.

[0079] The term "genetic engineering" as used herein refers to the process of altering (adding, suppressing, or replacing) the genetic makeup of a cell (i.e., of at least one nucleic acid) by directly manipulating the nucleic acid within the cell using biotechnology. Genetic engineering includes, for example, introducing a heterologous nucleic acid into a cell or cell population. The nucleic acid can be introduced into a cell or cell population, for example, by transduction or transfection.

[0080] The term "transduction" or "transducing" refers to the introduction of genetic material by a virus and its expression in a recipient cell.

[0081] The term "transfection" or "transfecting" as used herein refers to the process of introducing DNA (e.g., a formulated DNA expression vector) into a cell, thereby enabling transformation of the cell.

[0082] As used herein, the term "vector" refers to a nucleic acid molecule that allows the insertion of foreign nucleic acid without destroying the ability of the vector to replicate and / or integrate in a host cell.

[0083] The term "recombinant vector" as used herein refers to a polynucleotide molecule capable of transferring or transporting another polynucleotide inserted into the vector. The inserted polynucleotide may be an expression cassette. In some embodiments, the recombinant vector may be a viral vector or a non-viral vector (e.g., a plasmid).

[0084] "Expression cassette" or "expression construct" refers to a DNA polynucleotide sequence operably linked to a promoter. "Operably linked" refers to the juxtaposition of the components so described in a relationship that allows them to function in their intended manner. For example, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0085] As used herein, the terms "expression" and "expressing" are used to refer to transcription and translation that occurs in a cell. The expression level of a product gene in a host cell may be determined based on either the amount of corresponding mRNA present in the cell, or the amount of protein encoded by the product gene that is produced by the cell, or both.

[0086] The term "sample" refers to a biological composition (e.g., a portion of a cell or tissue). In some embodiments, a sample is a "primary sample" in that it is obtained directly from a subject; in some embodiments, a "sample" is the result of processing of the primary sample, e.g., to remove particular components and / or to isolate or purify a particular component of interest. In some embodiments, the sample is a blood sample.

[0087] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this disclosure, "polypeptide" refers to proteins with modifications (generally conservative in nature) such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or errors due to PCR amplification.

[0088] The term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target, e.g., a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule so long as the antibody exhibits the desired biological activity. Antibodies can be of any of the immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional structures. Antibodies can be naked or can be conjugated to other molecules, such as toxins or radioisotopes.

[0089] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment", "antigen-binding domain" or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigen-determining variable regions (e.g., complementarity-determining regions (CDRs)) of an intact antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially generated.

[0090] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a portion of an antibody, generally a portion of a light or heavy chain, generally the amino-terminal 110-120 or 110-125 amino acids of the mature heavy chain and approximately 90-115 amino acids of the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to a particular antigen. The variability in sequence is concentrated in regions called complementarity determining regions (CDRs), while the more conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

[0091] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0092] In some embodiments, the antigen-binding fragment of an antibody is an scFv. As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. The heavy (VH) and light (VL) chains are directly linked or linked by a peptide-encoding linker (e.g., 10, 15, 20, or 25 amino acids) that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. The linker can be rich in glycine for flexibility, as well as rich in serine or threonine for solubility. Despite the removal of the constant regions and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from a nucleic acid that includes a VH-encoding sequence and a VL-encoding sequence.

[0093] The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded polynucleotides (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiment.

[0094] The term "engineered antigen receptor" refers to a non-naturally occurring antigen-specific receptor, such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). The term "chimeric antigen receptor" or "CAR" has its general meaning in the art and refers to an artificially constructed hybrid polypeptide that contains an antigen binding domain, for example, of an antibody (e.g., scFv), linked to a T cell signaling domain. The antigen binding domain and the T cell signaling domain can be linked via a hinge. "TCR" has its general meaning in the art and refers to a protein complex that recognizes a specific target and includes a TCR alpha chain and / or a TCR beta chain.

[0095] The term "hinge" or "hinge region" refers to a flexible connector region, e.g., in natural or synthetic polypeptides, that confers structural flexibility and spacing to adjacent polypeptide regions.

[0096] The term "modified" refers to a substance or compound (e.g., a cell, a polynucleotide sequence, and / or a polypeptide sequence) that has been altered or changed compared to the corresponding unmodified substance or compound.

[0097] As used herein, the term "naturally occurring" when applied to a nucleic acid, polypeptide, cell, or organism refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0098] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants). Thus, "substantially pure" also includes a material that is at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0099] The term "in vivo" refers to events that take place within a mammalian subject, and not to events that take place in cell culture (including mammalian cell culture).

[0100] The term "in vitro" refers to events that take place outside a mammalian subject's body, in an artificial environment.

[0101] The term "in vitro" refers to events that take place within a test system. In vitro assays include cell-based assays, in which live or dead cells may be employed, and may also include cell-free assays, in which intact cells are not employed.

[0102] As used herein, "treatment" is an approach to obtain beneficial or desired clinical outcomes. As used herein, "treatment" refers to the administration or application of a therapeutic agent to a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of the spread of the disease (e.g., metastasis), prevention or delay of recurrence of the disease, delay or slowing of disease progression, amelioration of the disease state, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, prevention of its onset, and remission (whether partial or total). Reduction of the pathological consequences of a proliferative disease is also encompassed by "treatment". The methods described herein contemplate any one or more of these aspects of treatment. In line with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0103] As used herein, "delaying" means to postpone, hinder, slow, retard, stabilize, inhibit, and / or postpone the onset or progression of a disease. This delay can be of varying duration depending on the history of the disease and / or the individual being treated.

[0104] A "therapeutically effective amount" of a substance may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic effect.

[0105] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic outcome (eg, prevention of infection or onset of disease).

[0106] "Decrease" or "reduce" refers to a decrease or decrease in a particular value by at least 5%, e.g., a 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% decrease compared to a baseline value.

[0107] By "increase" it is meant an increase in a particular value of at least 5%, e.g., a 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, 500% or more increase in the particular value as compared to a baseline value.

[0108] The terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of a therapeutic agent to a desired site of biological action. Administration techniques that can be employed with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. Administration of two or more therapeutic agents includes simultaneous (concurrent) administration, and sequential administration in any order.

[0109] The terms "individual" or "subject" are used interchangeably herein to refer to animals, e.g., mammals such as humans. In some cases, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock animals, mammalian sport animals, and mammalian pets. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some cases, the subject receiving treatment can be a patient, which indicates the fact that the subject has been identified as having or at particular risk of suffering from a disorder relevant to the treatment.

[0110] For the purposes of this disclosure, the regulatory T cells used in therapy can be isolated from the subject ("autologous cells") or from another individual ("allogeneic cells") to which they are subsequently administered. As used herein, "allogeneic cells" refers to cells that are isolated from one subject (donor) and infused into another subject (recipient or host). As used herein, "autologous cells" refers to cells that are isolated and infused back into the same subject (recipient or host).

[0111] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in which the biological activity of the active ingredient(s) is effective and which does not contain any additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations may be sterile.

[0112] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions and / or formulations that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of safe medical judgment and commensurate with a reasonable benefit / risk ratio.

[0113] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a patient. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. Pharmaceutically acceptable carriers are appropriate for the formulation in which they are used.

[0114] A "sterile" preparation is aseptic or substantially free of living microorganisms and their spores.

[0115] General methods of molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press, 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons, 1999); Protein Methods (Bollag et al., John Wiley & Sons, 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press, 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press, 1995); Immunology Methods Manual, (I. Lefkovits ed., Academic Press, 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons, 1998).

[0116] Tregs and Treg populations Tregs that express the transcription factor forkhead box P3 (FoxP3) are naturally present in the immune system. Thus, in some embodiments provided herein, Tregs express FoxP3 + CD4 + T cells. The transcription factor Helios is also expressed in many Tregs that naturally occur in the immune system. Thus, in some embodiments provided herein, Tregs are FoxP3 + Helios + or FoxP3 + CD4 + Helios + T cells.

[0117] In some embodiments, Tregs have a fully demethylated Treg-specific demethylation region (TSDR).

[0118] Demethylation of FoxP3, Helios, and TSDR are intracellular Treg markers. However, CD25 hi (polymer density) and CD127 lo Cell surface markers including (low molecular density) also serve as markers for Tregs and can be used to identify Tregs, for example, using flow cytometry. Thus, in some embodiments, Tregs are CD25 + In some cases, Tregs are CD25 + CD4 + In some embodiments, Tregs are CD25 hi CD4 + In some embodiments, Tregs are CD127 lo In some embodiments, Tregs are CD127 lo CD4 + In some embodiments, Tregs are CD25 hi CD127 lo CD4 + T cells.

[0119] In some embodiments, Tregs are CD25 + FoxP3 +In some embodiments, Tregs are CD25 + CD4 + FoxP3 + In some embodiments, Tregs are CD25 hi CD4 + FoxP3 + In some embodiments, Tregs are CD127 lo FoxP3 + In some embodiments, Tregs are CD127 lo CD4 + FoxP3 + In some embodiments, Tregs are CD25 hi CD127 lo CD4 + FoxP3 + T cells.

[0120] In some embodiments, Tregs are CD45RA hi CD25 hi CD127 lo CD4 + In some embodiments, Tregs are CD45RA hi CD25 hi CD127 lo CD4 + FoxP3 + T cells.

[0121] In some embodiments, Tregs are CD25 + FoxP3 + Helios + In some embodiments, Tregs are CD25 + CD4 + FoxP3 + Helios + In some embodiments, Tregs are CD25 hi CD4 + FoxP3 + Helios + In some embodiments, Tregs are CD127 lo FoxP3 + Helios +In some embodiments, Tregs are CD127 lo CD4 + FoxP3 + Helios + In some embodiments, Tregs are CD25 hi CD127 lo CD4 + FoxP3 + Helios + T cells.

[0122] In some embodiments, Tregs are CD45RA hi CD25 hi CD127 lo CD4 + Helios + In some embodiments, Tregs are CD45RA hi CD25 hi CD127 lo CD4 + FoxP3 + Helios + T cells.

[0123] In some aspects, Tregs express SOCS, PD-1, CLTA4, neuropilin, TRAIL, and / or GITR.

[0124] In some embodiments, Tregs express IL-10 and / or TGFβ.

[0125] In some embodiments, the Tregs are human Tregs.

[0126] Activated cells can be characterized based on forward scatter (FSC) profile. Higher FSC value, which is proportional to the relative size of the cells, indicates greater activation. The cell size (FSC-A) of Tregs generated according to the methods provided herein may be smaller than Tregs generated according to conventional methods, such as Tregs stimulated continuously for 6 days (about 144 hours). (See, e.g., Example 3). In some embodiments, Tregs generated according to the methods provided herein have a relative cell size (FSC-A) that is less than 0.75 times that of Tregs stimulated continuously for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a relative cell size (FSC-A) that is less than 0.8 times that of Tregs stimulated continuously for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a relative cell size (FSC-A) that is less than 0.75 times that of Tregs stimulated continuously for 6 days (about 144 hours).

[0127] Also provided herein are populations of Tregs. In some embodiments, the population comprises at least 1×10 3 cells, at least 1 x 10 4 cells, at least 1 x 10 5 cells, at least 1 x 10 6 cells, at least 1 x 10 7 cells, at least 1 x 10 8 cells, at least 1 x 10 9 cells, or at least 1 x 10 10 In some embodiments, the population comprises at least 1 x 10 cells. 3 Tregs, at least 1 x 10 4 Tregs, at least 1 × 10 5 Tregs, at least 1 x 10 6 Tregs, at least 1 x 10 7 Tregs, at least 1 x 10 8 Tregs, at least 1 x 10 9 Tregs, or at least 1 × 1010 Contains Tregs.

[0128] In some embodiments, at least 60% of the cells in the Treg population are Treg. In some embodiments, at least 70% of the cells in the Treg population are Treg. In some embodiments, at least 75% of the cells in the Treg population are Treg. In some embodiments, at least 80% of the cells in the Treg population are Treg. In some embodiments, at least 85% of the cells in the Treg population are Treg. In some embodiments, at least 90% of the cells in the Treg population are Treg. In some embodiments, at least 95% of the cells in the Treg population are Treg. In some embodiments, at least 96% of the cells in the Treg population are Treg. In some embodiments, at least 97% of the cells in the Treg population are Treg. In some embodiments, at least 98% of the cells in the Treg population are Treg. In some embodiments, at least 99% of the cells in the Treg population are Treg.

[0129] In some embodiments, less than 1% of the cells in the Treg population are effector T cells. In some embodiments, less than 2% of the cells in the Treg population are effector T cells. In some embodiments, less than 3% of the cells in the Treg population are effector T cells. In some embodiments, less than 4% of the cells in the Treg population are effector T cells. In some embodiments, less than 5% of the cells in the Treg population are effector T cells. In some embodiments, less than 10% of the cells in the Treg population are effector T cells.

[0130] In some embodiments, less than 1% of the cells in the Treg population are CD25 - CD4 + or CD8 + In some embodiments, less than 2% of the cells in the Treg population are CD25 - CD4 + or CD8 +In some embodiments, less than 3% of the cells in the Treg population are CD25 - CD4 + or CD8 + In some embodiments, less than 4% of the cells in the Treg population are CD25 - CD4 + or CD8 + In some embodiments, less than 5% of the cells in the Treg population are CD25 - CD4 + or CD8 + In some embodiments, less than 10% of the cells in the Treg population are CD25 - CD4 + or CD8 + T cells. CD25 in the population - CD4 + or CD8 + The percentage of cells that are T cells can be determined using flow cytometry.

[0131] In some embodiments, Treg populations generated according to the methods provided have an average relative cell size (FSC-A) that is less than 0.75 times that of a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein have an average relative cell size (FSC-A) that is less than 0.8 times that of a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein have a relative cell size (FSC-A) that is less than 0.75 times that of a Treg population stimulated continuously for 6 days (about 144 hours).

[0132] Helios in Treg populations generated according to the methods provided herein + FoxP3 +The proportion of Tregs may be less than a Treg population generated according to conventional methods, e.g., a Treg population stimulated continuously for 6 days (about 144 hours). (See, e.g., Example 7.) In some embodiments, a Treg population generated according to the methods provided herein has at least 1.5 times the proportion of Helios Tregs as a Treg population stimulated continuously for 6 days (about 144 hours). + FoxP3 + CD4 + In some embodiments, the Treg population generated according to the methods provided herein can have at least twice the proportion of Helios T cells as the Treg population stimulated continuously for 6 days (about 144 hours). + FoxP3 + CD4 + In some embodiments, the Treg populations generated according to the methods provided herein have 1.5-3 times the proportion of Helios T cells compared to the Treg populations stimulated continuously for 6 days (about 144 hours). + FoxP3 + CD4 + In some embodiments, the Treg populations generated according to the methods provided herein have 1.5-2.5 times the proportion of Helios T cells compared to the Treg populations stimulated continuously for 6 days (about 144 hours). + FoxP3 + CD4 + Helios in the Treg population. + FoxP3 + The percentage of Tregs can be determined using flow cytometry.

[0133] In some embodiments, at least 50% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 60% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 70% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 75% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 80% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 85% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 90% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 95% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 96% of the cells in the Treg population generated by the methods described herein express Helios. In some embodiments, at least 97% of cells in a Treg population generated by the methods described herein express Helios. In some embodiments, at least 98% of cells in a Treg population generated by the methods described herein express Helios. The percentage of cells in a Treg population that express Helios can be determined using flow cytometry.

[0134] In some embodiments, the percentage of Tregs expressing Helios is not reduced by more than 50% when Tregs are expanded (and optionally genetically engineered) according to the methods provided herein. In some embodiments, the percentage of Tregs expressing Helios is not reduced by more than 40% when Tregs are expanded (and optionally genetically engineered) according to the methods provided herein. In some embodiments, the percentage of Tregs expressing Helios is not reduced by more than 30% when Tregs are expanded (and optionally genetically engineered) according to the methods provided herein. In some embodiments, the percentage of Tregs expressing Helios is not reduced by more than 25% when Tregs are expanded (and optionally genetically engineered) according to the methods provided herein.

[0135] In some embodiments, at least 60% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 70% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 75% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 80% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 85% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 90% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 95% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 96% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 97% of the cells in the Treg population generated by the methods described herein express FoxP3. In some embodiments, at least 98% of the cells in a Treg population generated by the methods described herein express FoxP3. The percentage of cells in a Treg population that express FOXP3 can be determined using flow cytometry.

[0136] In some embodiments, at least 50% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 60% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 70% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 75% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 80% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 85% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 90% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 95% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 96% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 97% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. In some embodiments, at least 98% of the cells in a Treg population generated by the methods described herein express FoxP3 and Helios. The percentage of cells in a Treg population expressing FOXP3 and Helios can be determined using flow cytometry.

[0137] As demonstrated herein, Treg populations generated according to the methods provided herein can have an increased ability to suppress effector T cell (Teff) proliferation compared to Treg populations generated according to conventional methods, e.g., Treg populations stimulated continuously for 6 days (about 144 hours) (see, e.g., Example 8).

[0138] The ability of a Treg population to suppress Teff proliferation can be determined, for example, using an in vitro suppression assay such as that provided herein in Example 1. The ability of a Treg population to suppress Teff proliferation can be detected in vitro.

[0139] In one embodiment provided herein, the ability of a Treg population to suppress Teff proliferation is determined using the following assay: (i) stimulating overnight rested Tregs in Treg medium containing 10 μl / mL ImmunoCult CD3 / 28 / 2 tetramers (StemCell Technologies, Cat#10970)) and IL-2 (300 units / mL); (ii) washing the Tregs to remove the tetramers and IL-2; (iii) resuspending the Tregs in Treg medium; (iv) mixing the Tregs with cell tracing violet (CTV) labeled PBMCs (e.g., at a ratio of 1:1 to 1:16); (v) adding 0.1 mL of ImmunoCult CD3 / 28 / 2 tetramers (3 μl / mL) for 4 days (about 96 hours); and (vi) staining the cells for CD3, CD4, and CD8. Lower CTV signals from CD4+ or CD8+ effector T cells in the presence of Tregs compared with their absence indicate that Tregs suppress Teff proliferation.

[0140] In some embodiments, the Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least twice as much as a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, the Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least three times as much as a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, the Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least four times as much as a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, the Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least five times as much as a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, the Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least six times as much as a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least 7-fold more than Treg populations stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation at least 8-fold more than Treg populations stimulated continuously for 6 days (about 144 hours).

[0141] In some embodiments, Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation about 2- to about 10-fold more than a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation about 4- to about 10-fold more than a Treg population stimulated continuously for 6 days (about 144 hours). In some embodiments, Treg populations generated according to the methods provided herein can have the ability to suppress Teff proliferation about 6- to about 10-fold more than a Treg population stimulated continuously for 6 days (about 144 hours).

[0142] In some embodiments, the steps of the methods described herein (e.g., all stimulating steps, all resting steps, and all editing steps, if present) are completed in less than 30 days, or less than 29 days, or less than 28 days, or less than 27 days, or less than 26 days, or less than 25 days, or less than 24 days, or less than 23 days, or less than 22 days, or less than 21 days, or less than 20 days, or less than 19 days, or less than 18 days, or less than 17 days, or less than 16 days, or less than 14 days, or less than 14 days, or between 14 and 30 days, or between 14 and 25 days, or between 15 and 28 days, or between 15 and 25 days.

[0143] Stimulation of Tregs As provided herein, the method of expanding a Treg population can include a stimulation step in which the Treg population is cultured in the presence of a stimulatory agent, which can generate a stimulated Treg population.

[0144] In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 40% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 50% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 60% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 70% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 80% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 90% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 95% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulating agent activates at least 96% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulatory agent activates at least 97% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population.In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulatory agent activates at least 98% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population. In some embodiments of the methods provided herein, culturing the Treg population in the presence of a stimulatory agent activates at least 99% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population.

[0145] Throughout each stimulation step, Tregs are continuously cultured in the presence of the stimulating agent. In some embodiments, each stimulation step involves a single addition of the stimulating agent to the Treg culture (at the beginning of the stimulation step), so that the concentration of the stimulating agent can vary (e.g., decrease) throughout the stimulation step. In some embodiments, each stimulation step involves repeated addition of the stimulating agent to the Treg culture to maintain a desired concentration of the stimulating agent.

[0146] Some methods provided herein begin with a stimulating step. The stimulating step can also occur after the resting step and / or before the resting step. In some embodiments, the stimulating step occurs between two resting steps.

[0147] The stimulatory agent can be an antigen-nonspecific stimulator (e.g., an anti-CD3 antibody) or an antigen-specific stimulator, e.g., an MHC peptide multimer. In some embodiments, the stimulatory agent activates the T cell receptor on Tregs, e.g., by binding to the TCR. In some embodiments, the stimulatory agent activates CD28 on Tregs, e.g., by binding to CD28. In some embodiments, the stimulatory agent activates the T cell receptor and CD28 on Tregs, e.g., by binding to the TCR and CD28. In some embodiments, the stimulatory agent is a mitogen such as PHA or ConA.

[0148] In some embodiments, the stimulatory agent is an antibody or antigen-binding fragment thereof. In some embodiments, the stimulatory agent is an antibody complex. In some embodiments, the stimulatory agent is a tetrameric antibody complex. In some embodiments, the tetrameric antibody complex specifically binds to CD3, CD28, and / or CD2. In some embodiments, the tetrameric antibody complex specifically binds to CD3, CD28, and CD2. An exemplary tetrameric antibody complex that specifically binds to CD3, CD28, and CD2 is available as ImmunoCult CD3 / 28 / 2 tetramer from StemCell Technologies, catalog number 10970.

[0149] In some embodiments, the tetrameric antibody complex is present at a concentration of at least 0.5 ng / ml.

[0150] In some embodiments, the stimulatory agent is an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is OKT3. Such stimulatory agents may be present at a concentration of at least 0.5 ng / ml.

[0151] The term "anti-CD3 antibody" refers to an antibody, e.g., a monoclonal antibody, including a human antibody, a humanized antibody, a chimeric antibody, and a murine antibody, directed against the CD3 receptor in the T cell antigen receptor of a mature T cell. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelizumab, teplizumab, and visilizumab.

[0152] The "OKT-3" antibody (also referred to herein as "OKT3") is commercially available, for example, as OKT-3 30 ng / mL, MACS GMP CD3 pure from Miltenyi Biotech, Inc., San Diego, Calif., USA. The amino acid sequences of the heavy and light chains of muromonab are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTT LTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 2)

[0153] In some embodiments, the stimulatory agent is an anti-CD28 antibody or antigen-binding fragment thereof. Such a stimulatory agent may be present at a concentration of at least 0.5 ng / ml.

[0154] The stimulating agent can be present on a substrate such as a cell or a bead. The beads can be plastic, glass, or other suitable material and are typically in the range of 1 to 20 microns. In some embodiments, the beads are paramagnetic beads. In some embodiments, the beads are supermagnetic beads. Thus, for example, the stimulating agent can be anti-CD3 coated supermagnetic beads and / or anti-CD28 coated supermagnetic beads. Such stimulating agents can be present, for example, at a concentration of about 5 μg / ml or about 10 μg / ml. Such stimulating agents can be present, for example, at a concentration of about 2.5 μg / ml to about 15 μg / ml.

[0155] Cells suitable for use as substrates include antigen presenting cells (APCs) and artificial antigen presenting cells (aAPCs).

[0156] In some embodiments, the stimulatory agent is an antigen presenting cell (APC) or an artificial antigen presenting cell (aAPC). The aAPC can be an irradiated aAPC.

[0157] In some embodiments, the stimulant is not on the bead.

[0158] In some embodiments, the stimulatory agent is a tetrameric antibody complex that specifically binds to CD3, CD28, and / or CD2, an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, and / or an antigen-presenting cell or an artificial antigen-presenting cell.

[0159] In some embodiments, the stimulatory agent is present at a concentration sufficient to increase Treg proliferation, e.g., at least 1.5-fold within 72 hours. In some embodiments, the stimulatory agent is present at a concentration sufficient to increase Treg proliferation, e.g., at least 2-fold within 72 hours. In some embodiments, the stimulatory agent is present at a concentration sufficient to increase Treg proliferation, e.g., at least 3-fold within 72 hours. Treg proliferation can be measured using a luminescent cell viability assay such as Cell Titer Glo®, which determines the number of metabolically active viable cells in culture based on quantification of ATP.

[0160] In some embodiments, the stimulatory agent is present at a concentration sufficient to increase the forward scatter (FSC) profile of cells, a commonly used indicator of the activation state of T cells cultured in vitro.

[0161] As provided herein, the method of expanding a Treg population can include multiple stimulation steps. In such embodiments, the stimulation steps can use the same stimulating agent, or the stimulation steps can use different stimulating agents. When multiple stimulation steps use the same stimulating agent, the stimulating agents can use the same concentration of the stimulating agent, or they can use different concentrations of the stimulating agent. Stimulating agents can be used in combination, such that a single stimulation step can include the use of two stimulating agents (simultaneously and / or sequentially). Thus, in some embodiments, the stimulating agent is a superparamagnetic bead coated with CD3 and CD28.

[0162] In some embodiments, the methods provided herein include two stimulation steps (separated by a rest step). The methods provided herein include at least two stimulation steps (each separated by a rest step). The methods provided herein include at least three stimulation steps (separated by rest steps).

[0163] In some embodiments, the stimulating step is at least 1 day (about 24 hours). In some embodiments, the stimulating step is at least 2 days (about 48 hours). In some embodiments, the stimulating step is at least 3 days (about 72 hours). In some embodiments, the stimulating step is at least 4 days (about 96 hours).

[0164] In some embodiments, the stimulation step is about 1 day (about 24 hours). In some embodiments, the stimulation step is about 2 days (about 48 hours). In some embodiments, the stimulation step is about 3 days (about 72 hours). In some embodiments, the stimulation step is about 4 days (about 96 hours).

[0165] In some embodiments, the stimulation step is about 1 day (about 24 hours) to about 5 days (about 120 hours). In some embodiments, the stimulation step is about 1 day (about 24 hours) to about 4 days (about 96 hours). In some embodiments, the stimulation step is about 1 day (about 24 hours) to about 3 days (about 72 hours). In some embodiments, the stimulation step is about 2 days (about 48 hours) to about 5 days (about 120 hours). In some embodiments, the stimulation step is about 2 days (about 48 hours) to about 4 days (about 96 hours). In some embodiments, the stimulation step is about 3 days (about 72 hours) to about 4 days (about 96 hours).

[0166] Depending on the day, the stimulation step will not last longer than 6 days (about 144 hours), or will not last longer than 5 days (about 120 hours).

[0167] In some embodiments, the methods provided herein include at least two (e.g., two) stimulation steps (each separated by a rest step), where the first stimulation step is longer than the second stimulation step. In some embodiments, the first stimulation step is at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours longer than the second stimulation step. The first stimulation step can be, for example, about 1 day (about 24 hours) to about 4 days (about 96 hours), and the second stimulation step can be, for example, about 1 day (about 24 hours) to about 3 days (about 72 hours). In some embodiments, the first stimulation step is about 3 days (about 72 hours), and the second stimulation step is about 2 days (about 48 hours).

[0168] In some embodiments, the methods provided herein include at least one stimulation step prior to genetically engineering the Tregs. In some embodiments, the methods provided herein include at least two stimulation steps (each separated by a resting step) prior to genetically engineering the Tregs.

[0169] In some embodiments, the methods provided herein include at least one stimulation step after genetically engineering the Tregs.

[0170] Quiescing Tregs As provided herein, the method of expanding a Treg population can include a resting step in which the Treg population is cultured in the absence of a stimulatory agent. The resting step can generate a resting Treg population. As used herein, resting refers to the absence of a stimulatory agent (and does not refer to the absence of, e.g., cell growth, division, proliferation, or other activity of Tregs).

[0171] In some embodiments of the methods provided herein, a significant portion of Tregs in a Treg population cultured in the absence of a stimulating agent do not contain active T cell receptors and / or costimulatory molecules such as CD28 or GITR. In some embodiments of the methods provided herein, culturing a Treg population in the absence of a stimulating agent activates no more than 20% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on Tregs in the population. In some embodiments of the methods provided herein, culturing a Treg population in the absence of a stimulating agent activates no more than 15% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on Tregs in the population. In some embodiments of the methods provided herein, culturing a Treg population in the absence of a stimulating agent activates no more than 10% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on Tregs in the population. In some embodiments of the methods provided herein, culturing a Treg population in the absence of a stimulatory agent activates no more than 5% of the T cell receptors and / or costimulatory molecules (e.g., CD28 or GITR) on the Tregs in the population.

[0172] The skilled artisan will know methods to determine whether a Treg population is cultured in the absence of a stimulating agent. For example, removal / absence of a stimulating agent may be quantified using methods known to the skilled artisan, for example, by determining changes in cell size and / or granularity, by determining downregulation of Treg activation markers, and / or by downregulation of immunosuppressive cytokine production.

[0173] In some embodiments, the absence of a stimulating agent is determined by determining changes in cell size and / or granularity. In some embodiments, changes in cell size and / or granularity are measured using a flow cytometer (i.e., FSC / SSC measurement). As mentioned herein, activated cells can be characterized based on their forward scatter (FSC) profile. In some embodiments, the cell size (FSC-A) of the Treg population in the absence of a stimulating agent is less than about 40%, or less than about 50%, or less than about 60%, or less than about 70%, or less than about 75%, or less than about 80%, or less than about 90%, or less, compared to the cell size (FSC-A) of the Treg population before the removal of the stimulating agent. In some embodiments, the cell size (FSC-A) of the Treg population in the absence of a stimulating agent is determined about 12 hours, or about 18 hours, or about 24 hours, or about 36 hours, or about 48 hours, or about 72 hours after the removal of the stimulating agent. In some embodiments, the cell size (FSC-A) of the Treg population prior to removal of the stimulating agent is determined at about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or less prior to removal of the stimulating agent.

[0174] Other non-limiting examples for determining the absence of a stimulating agent include determining downregulation of Treg activation markers (e.g., FoxP3, Helios, CTLA4, CD25, CD69, HLA-DR) and / or determining downregulation of immunosuppressive cytokine production (e.g., IL-10, TGFb). In some embodiments, in the absence of a stimulating agent, expression of Treg activation markers and / or production of immunosuppressive cytokines is less than about 40%, or less than about 50%, or less than about 60%, or less than about 70%, or less than about 75%, or less than about 80%, or less than about 90%, or less than or equal to, compared to the expression of Treg activation markers and / or production of immunosuppressive cytokines of the Treg population before the stimulating agent is removed. In some embodiments, the expression of Treg activation markers and / or production of immunosuppressive cytokines of the Treg population in the absence of the stimulating agent is determined about 12 hours, or about 18 hours, or about 24 hours, or about 36 hours, or about 48 hours, or about 72 hours after removal of the stimulating agent. In some embodiments, the expression of Treg activation markers and / or production of immunosuppressive cytokines of the Treg population prior to removal of the stimulating agent is determined about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or less prior to removal of the stimulating agent.

[0175] The resting step can be initiated by removing stimulating agents from the Treg culture. The stimulating agents can be removed, for example, by washing and / or centrifugation steps. As demonstrated herein, removal of the stimuli for the resting step after, for example, 2 days (about 48 hours), or 3 days (about 72 hours), or 4 days (about 96 hours), or more of stimulation, can result in improved expansion of Tregs.

[0176] In some embodiments, the concentration of the stimulant is reduced by at least 25% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 50% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 60% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 70% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 80% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 90% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 91% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulant is reduced by at least 92% in the resting step compared to the concentration of the stimulant present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 93% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 94% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 95% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 96% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 97% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 98% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, the concentration of the stimulating agent is reduced by at least 99% in the resting step compared to the concentration of the stimulating agent present in the stimulating step. In some embodiments, no stimulating agent is present in the resting step, i.e., Tregs are cultured without any stimulating agent. Some methods provided herein begin with a resting step. A resting step can occur after the stimulating step and / or before the stimulating step, hi some embodiments, a resting step occurs between two stimulating steps.

[0177] In some embodiments, the methods provided herein include at least one resting step. In some embodiments, the methods provided herein include at least two resting steps.

[0178] In some embodiments, the resting step is at least one day (about 24 hours).

[0179] In some embodiments, the resting step is at least 2 days (about 48 hours).

[0180] In some embodiments, the resting step is about 3 days (about 72 hours).

[0181] In some embodiments, the resting step is about 4 days (about 96 hours).

[0182] In some embodiments, the resting step is about 5 days (about 120 hours).

[0183] In some embodiments, the resting step is about 1 day (about 24 hours) to about 5 days (about 120 hours). In some embodiments, the resting step is about 1 day (about 24 hours) to about 4 days (about 96 hours). In some embodiments, the resting step is about 1 day (about 24 hours) to about 3 days (about 72 hours). In some embodiments, the resting step is about 2 days (about 48 hours) to about 4 days (about 96 hours). In some embodiments, the resting step is about 2 days (about 48 hours) to about 5 days (about 120 hours). In some embodiments, the resting step is about 3 days (about 72 hours) to about 4 days (about 96 hours).

[0184] In some embodiments, the methods provided herein comprise at least one resting step before genetically engineering Treg.As used herein, a resting step "before" genetic engineering does not require that the resting step is performed immediately before genetic engineering.Thus, a method that comprises a stimulation step, then a resting step, then a stimulation step, then genetic engineering is a method that comprises a resting step before genetic engineering.

[0185] In some embodiments, the methods provided herein include at least one resting step after genetically engineering the Tregs.

[0186] In some embodiments, the methods provided herein include a resting step after genetically engineering Tregs, hi some embodiments, Tregs are genetically engineered and then rested without a stimulation step between the genetic engineering and resting steps.

[0187] In some aspects, the methods provided herein include (i) at least one resting step before the Tregs are genetically engineered, and (ii) at least one resting step after the Tregs are genetically engineered, e.g., before the Tregs are restimulated.

[0188] Treg expansion As provided herein, the methods of expanding Tregs can include alternating periods of stimulating Tregs and resting Tregs, or "discontinuous stimulation." Thus, in some embodiments, the methods provided herein include at least one stimulation step and at least one resting step. In some embodiments, the methods provided herein include at least two stimulation steps separated by a resting step. In some embodiments, the methods provided herein include at least three stimulation steps, each separated by a resting step. In some embodiments, Tregs can be genetically engineered (as discussed elsewhere herein), for example, during the resting steps (i.e., Tregs can be genetically engineered in the absence of a stimulating agent). In some embodiments, the methods provided herein do not include genetic engineering.

[0189] In some embodiments, the methods provided herein include a first stimulation step, followed by a first resting step, followed by a second stimulation step, followed by a second resting step. In some embodiments, Tregs are harvested after the second resting step.

[0190] In some embodiments, the methods provided herein include a first stimulation step, followed by a first resting step, followed by a second stimulation step, followed by a second resting step, followed by a third stimulation step, followed by a third resting step. In some embodiments, Tregs are harvested after the third resting step.

[0191] In some embodiments, the methods provided herein include a first stimulation step, followed by a first resting step, followed by a second stimulation step, followed by a second resting step during which Tregs are genetically engineered, followed by a third stimulation step, followed by a third resting step. In some embodiments, Tregs are harvested after the third resting step.

[0192] Non-limiting examples of the duration of the stimulation step and resting step are described herein. In a non-limiting embodiment, the first stimulation step can be about 2 days (about 48 hours) to about 5 days (about 120 hours) (e.g., about 3 days (about 72 hours)); the first resting step can be about 2 days (about 48 hours) to about 5 days (about 120 hours) (e.g., about 3 days (about 72 hours)); the second stimulation step can be about 2 days (about 48 hours) to about 5 days (about 120 hours) (e.g., about 2 days (about 48 hours)). the second resting step can be from about 2 days (about 48 hours) to about 5 days (about 120 hours) (e.g., about 3 days (about 72 hours)); the third stimulation step can be from about 2 days (about 48 hours) to about 5 days (about 72 hours) (e.g., about 2 days (about 48 hours)); and the third resting step can be from about 2 days (about 48 hours) to about 5 days (about 120 hours) (e.g., about 2 days (about 48 hours)). Non-limiting examples of resting step and stimulation step times are provided in Table A. [Table 1]

[0193] In some embodiments, the Tregs are cryopreserved.

[0194] As provided herein, Tregs can be cultured in a medium suitable for Tregs. Exemplary T cell media can include, for example, (i) X-VIVO15 T cell growth medium (Lonza, Cat#04-418Q) supplemented with 10% human inactivated serum, (ii) RPMI1640 medium supplemented with 5 mM HEPES, 2 mM L-glucamine, 50 mg / ml penicillin, 50 mg / ml streptomycin, 5 mM non-essential amino acids, 5 mM sodium pyruvate, and 10% FBS, or (iii) DMEM base containing 10% heat-inactivated fetal bovine serum (Biosource International), non-essential amino acids, 0.5 mM sodium pyruvate, 5 mM Hepes, 1 mM glutaMaxI, and 55 μM β-mercaptoethanol.

[0195] Treg culture medium can further include cytokines, such as interleukins, for example interleukin-2 (IL-2), interleukin-15 (IL-15), and / or interleukin-7 (IL-7).

[0196] The term "IL-2" (or "IL2") refers to the cytokine and T-cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, forms with conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the entire disclosures of which are incorporated herein by reference. The term IL-2 encompasses human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available from multiple suppliers at 22 million IU per single-use vial), as well as recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b), and other commercially available equivalents from other suppliers. Aldesleukin (desalanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses PEGylated forms of IL-2, including the PEGylated IL-2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference in their entirety. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference in their entirety. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosures of which are incorporated herein by reference in their entirety.The human IL2 gene is identified by NCBI Gene ID 3558. An exemplary nucleotide sequence of the human IL2 gene is NCBI Reference Sequence: NG_016779.1.

[0197] The amino acid sequence of a recombinant human IL-2 suitable for use herein is as follows: MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 3).

[0198] Aldesleukin (desalanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of an aldesleukin suitable for use in the methods provided herein is as follows: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4).

[0199] Interleukin 2 (IL-2) is a type of cytokine signaling molecule in the immune system, and is reported to be a 15.5-16 kDa protein that regulates the activity of immune-responsible white blood cells (leukocytes, often lymphocytes). IL-2 is part of the body's natural response to microbial infections and is understood to mediate its effects by binding to the IL-2 receptor expressed by lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.

[0200] IL-2 is believed to play an essential role in the immune system, tolerance, and key functions of immunity, primarily through its direct effects on T cells. In the thymus, where T cells mature, IL-2 prevents autoimmune diseases by promoting the differentiation of certain immature T cells into regulatory T cells that suppress other T cells that are ready to attack normal healthy cells in the body. IL-2 enhances activation-induced cell death (AICD). IL-2 also helps the body fight off infections, as it promotes the differentiation of T cells into effector and memory T cells when the first T cells are stimulated by antigen. IL-2, together with other polarizing cytokines, stimulates the differentiation of naive CD4+ T cells into Th1 and Th2 lymphocytes, while preventing the differentiation into Th17 and follicular Th lymphocytes. Its expression and secretion are tightly regulated and function as part of both temporal positive and negative feedback loops in enhancing and suppressing immune responses. Through its role in the development of T cell immunological memory, which depends on the expansion in number and function of antigen-selected T cell clones, IL-2 plays a role in the persistence of cell-mediated immunity.

[0201] In some embodiments, the Tregs are cultured in the presence of an interleukin (e.g., IL-2). The interleukin (e.g., IL-2) can be a recombinant interleukin (e.g., IL-2). In some embodiments, the concentration of the interleukin (e.g., IL-2) is at least 400 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is at least 500 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is at least 550 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is at least 600 units / mL.

[0202] In some embodiments, the concentration of the interleukin (e.g., IL-2) is 1,000 units / mL or less. In some embodiments, the concentration of the interleukin (e.g., IL-2) is 900 units / mL or less. In some embodiments, the concentration of the interleukin (e.g., IL-2) is 800 units / mL or less.

[0203] In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 200 units / mL to about 2,500 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 500 units / mL to about 1,000 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 500 units / mL to about 900 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 500 units / mL to about 800 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 550 units / mL to about 1,000 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 550 units / mL to about 900 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 550 units / mL to about 800 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 600 units / mL to about 1,000 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 600 units / mL to about 900 units / mL. In some embodiments, the concentration of the interleukin (e.g., IL-2) is about 600 units / mL to about 800 units / mL.

[0204] The concentration of interleukins (such as IL-2) can be altered during the methods provided herein. For example, the concentration of interleukins (e.g., IL-2) can be reduced. In some embodiments, the interleukin (e.g., IL-2) is present at a concentration of about 800 units / mL and then reduced to a concentration of about 300 units / mL. In some embodiments, the interleukin (e.g., IL-2) is present at a concentration of about 800 units / mL for about 7 days (about 168 hours) and then at about 300 units / mL.

[0205] The term "IL-7" (also referred to herein as "IL7") is a cytokine secreted by stromal cells of the bone marrow and thymus. It is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but not by conventional lymphocytes. IL-7 stimulates the differentiation of multipotent (multiple lineage) hematopoietic stem cells into lymphoid progenitor cells (as opposed to bone marrow progenitor cells, whose differentiation is stimulated by IL-3). IL-7 has been reported to stimulate the proliferation of all cells of the lymphoid lineage (B cells, T cells, NK cells). It is believed to be important for B cell maturation, T cell and NK cell survival, development, and proliferation at certain stages of homeostasis. An example of the nucleotide sequence of the human IL7 gene is NCBI Reference Sequence: AH006906.2. The amino acid sequence of recombinant human IL-7 suitable for use in the methods provided herein is as follows: MDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO:5).

[0206] When utilized in the methods provided herein, the concentration of IL-7 can be from about 10 U / ml to about 7,000 U / ml, hi some embodiments, the concentration of IL-7 can be from about 5 ng / ml to about 3,500 ng / ml.

[0207] The term "IL-15" (also referred to as "IL15") refers to the cytokine and T cell growth factor known as interleukin-15, and as used herein includes all forms of IL-15, including human and other mammalian forms, forms with conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Steel JC, Waldmann TA, Morris JC (January 2012), "Interleukin-15 biology and its therapeutic implications in cancer," Trends in Pharmacological Sciences, 33(1):35-41, and Waldmann TA, Tagaya Y (1999), "The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens," Annual Review of Immunology, 17:19-49, the disclosures of which are incorporated herein by reference in their entireties. The term IL-15 also encompasses recombinant forms of IL-15. As used herein, the term IL-15 also encompasses pegylated forms of IL-15. The human IL15 gene is identified by NCBI gene ID 3600. An example of the nucleotide sequence of the human IL15 gene is NCBI reference sequence: NG_029605.2. The amino acid sequence of recombinant human IL-15 suitable for use in the methods provided herein is as follows: MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6)

[0208] IL-15 may be utilized in the methods provided herein at concentrations greater than 0.5 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 1 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 2 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 10 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 50 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 75 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 100 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 150 ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 200 ng / ml. In some embodiments, the concentration of IL-15 utilized is less than 10,000ng / ml, optionally less than 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, or 1000ng / ml. In some embodiments, the concentration of IL-15 utilized is about 300ng / ml. In some embodiments, the concentration of IL-15 utilized is about 1000ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 1000ng / ml. In some embodiments, the concentration of IL-15 utilized is greater than 100ng / ml. In some embodiments, the concentration of IL-15 is about 100ng / ml to about 1000ng / ml. In some embodiments, the concentration of IL-15 is about 300ng / ml.

[0209] IL-15 may be utilized in the methods provided herein at a concentration of greater than 1 U / ml. In some embodiments, the concentration of IL-15 utilized is greater than 2 U / ml. In some embodiments, the concentration of IL-15 utilized is greater than 4 U / ml. In some embodiments, the concentration of IL-15 utilized is greater than 20 U / ml. In some embodiments, the concentration of IL-15 utilized is greater than 200 U / ml. In some embodiments, the concentration of IL-15 utilized is less than 20,000 U / ml, optionally less than 18,000, 16,000, 14,000, 12,000, 10,000, 8000, 6000, 4000, or 2000 ng / ml. In some embodiments, the concentration of IL-15 utilized is about 600 U / ml. In some embodiments, the concentration of IL-15 utilized is about 2000 U / ml. In some embodiments, the concentration of IL-15 utilized is greater than 2000 U / ml. In some embodiments, the concentration of IL-15 is greater than 200 U / ml. In some embodiments, the concentration of IL-15 is about 200 U / ml to about 2000 U / ml. In some embodiments, the concentration of IL-15 is about 600 U / ml.

[0210] In some embodiments, Tregs are cultured in the presence of N-acetyl-L-cysteine ​​(NAC). In some embodiments, NAC is present in the culture at a concentration of about 5 mM. In some embodiments, NAC is present in the culture at a concentration of about 8.2 mg / ml.

[0211] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml.

[0212] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 5,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 5,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 5,000,000 cells per ml.

[0213] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 3,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 3,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 3,000,000 cells per ml.

[0214] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 2,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 2,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 2,500,000 cells per ml.

[0215] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 2,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 2,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 2,000,000 cells per ml.

[0216] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 1,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 1,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 1,500,000 cells per ml.

[0217] In some embodiments, Tregs are cultured at a concentration of about 125,000 cells per ml to about 1,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 250,000 cells per ml to about 1,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration of about 500,000 cells per ml to about 1,000,000 cells per ml.

[0218] In some embodiments, Tregs are cultured at a concentration not exceeding 5,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration not exceeding 3,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration not exceeding 2,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration not exceeding 2,000,000 cells per ml. In some embodiments, Tregs are cultured at a concentration not exceeding 1,500,000 cells per ml. In some embodiments, Tregs are cultured at a concentration not exceeding 1,000,000 cells per ml.

[0219] In some embodiments, Tregs are cultured at a temperature suitable for T cell proliferation, for example at least 25 degrees Celsius, at least 30 degrees Celsius, or about 37 degrees Celsius.

[0220] In some embodiments, the method includes the use of rapamycin, which can limit the proliferation of effector T cells (Teff). Rapamycin can be used, for example, in the first week of culture. In some embodiments, rapamycin can be used in the first two weeks of culture. In some embodiments, the method does not include the use of rapamycin. In some embodiments, the method does not include the use of rapamycin prior to genetic manipulation. In some embodiments, the method does not include the use of rapamycin in the first two weeks of culture. In some embodiments, the method does not include the use of rapamycin in the first week of culture.

[0221] In some embodiments, the methods provided herein include the use of supermagnetic beads. In some embodiments, the methods do not include the use of supermagnetic beads. In some embodiments, the methods do not include the use of beads.

[0222] In some aspects, the methods provided herein include the use of artificial antigen presenting cells. In some aspects, the methods provided herein do not include the use of artificial antigen presenting cells.

[0223] In some aspects, the methods of expanding Tregs provided herein include: (a) culturing Tregs for about 3 days (about 72 hours) (e.g., days 0 to 3) in the presence of a stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2, optionally at a concentration of 10 μl / ml); (b) optionally removing the stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2) by washing; optionally reseeding the cells at 500,000 cells per ml; and culturing the cells for about 3 days (about 72 hours) (e.g., days 3 to 6) in the absence of the stimulatory agent, wherein the methods result in at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, or about 80-fold expansion of Tregs.

[0224] In some aspects, the methods of expanding Tregs provided herein include: (a) culturing the Tregs in the presence of a stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2, optionally at a concentration of 10 μl / ml) for about 3 days (about 72 hours) (e.g., days 0 to 3); (b) optionally removing the stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2) by washing; optionally reseeding the cells at 500,000 cells per ml; culturing the cells in the absence of the stimulatory agent for about 3 days (about 72 hours) (e.g., days 3 to 6); (c) culturing the Tregs for about 2 days (about 48 hours) (e.g., days 6 to 8) in the presence of a stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2, optionally at a concentration of 10 μl / ml); adjusting to a concentration of 500,000 cells per ml; (d) removing the stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2), optionally by washing; genetically engineering the Tregs; culturing the cells in the absence of the stimulatory agent for about 3 days (about 72 hours) (e.g., days 8 to 11); (e) culturing the Tregs in the presence of a stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2, optionally at a concentration of 10 μl / ml) for about 2 days (about 48 hours) (e.g., days 11 to 13); (f) optionally removing the stimulatory agent (e.g., a tetrameric antibody complex that specifically binds CD3, CD28, and CD2) by washing; optionally reseeding the cells at 500,000 cells per ml; culturing the cells in the absence of the stimulatory agent for about 2 days (about 48 hours) (e.g., days 13 to 15); (g) harvesting the cells.

[0225] Such methods comprising steps (a)-(g) can include culturing Tregs in the presence of IL-2 (optionally at a concentration of 400 IU / ml) and / or NAC (optionally at a concentration of 8.2 mg / ml). In some embodiments, the concentration of IL-2 is adjusted to 400 IU / ml on days 2-9, 11, 13, and 14.

[0226] Such methods comprising steps (a)-(g) may include splitting the cells multiple times, for example on days 4, 5, 7, 11, 13, and 14.

[0227] In some embodiments, the Tregs have expanded at least 60-fold, or at least 70-fold, by the end of step (b), e.g., by day 6, or 80-fold, by the end of step (b), e.g., by day 6.

[0228] In some embodiments, Tregs have expanded about 80 fold by the end of step (b), for example by day 6.

[0229] In some embodiments, step (c) further comprises transferring the Tregs into a T25 flask.

[0230] Using the expansion methods provided herein, the number of Tregs in a Treg population can be expanded by at least 500-fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 2500-fold, at least 3000-fold, at least 3500-fold, or at least 4000-fold.

[0231] In some embodiments, the number of Tregs is increased by at least 500-fold within 10 days.

[0232] In some embodiments, the number of Tregs is increased at least 1000-fold within 20 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 19 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 18 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 17 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 16 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 15 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 14 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 13 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 12 days. In some embodiments, the number of Tregs is increased at least 1000-fold within 11 days.

[0233] In some embodiments, the number of Tregs is increased by at least 2500 fold within 20 days. In some embodiments, the number of Tregs is increased by at least 3000 fold within 20 days. In some embodiments, the number of Tregs is increased by at least 3500 fold within 20 days. In some embodiments, the number of Tregs is increased by at least 4000 fold within 20 days.

[0234] Tregs generated according to the methods provided herein have improved properties, for example, because the methods provided herein prevent overstimulation of Treg populations and / or prevent activation-induced cell death. For example, in some embodiments, Tregs generated according to the methods provided herein have a smaller surface area, for example, compared to Tregs cultured in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a surface area that is less than 0.9 times, less than 0.85 times, less than 0.8 times, or less than 0.75 times the surface area of ​​Tregs cultured in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a higher ratio of Helios+FoxP3+Tregs, for example, compared to Tregs cultured in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a higher fold expansion, for example, compared to Tregs cultured in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have an expansion fold that is at least 1.5-fold or at least 2-fold greater than that of Tregs cultured, for example, in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a higher ability to suppress the proliferation of effector T cells (Teffs), for example, compared to Tregs cultured, for example, in the presence of a stimulant for 6 days (about 144 hours). In some embodiments, Tregs generated according to the methods provided herein have a higher ability to suppress the proliferation of Teffs, for example, compared to Tregs cultured, for example, in the presence of a stimulant for 6 days (about 144 hours).

[0235] In some embodiments, Tregs expanded according to the methods provided herein maintain at least 60%, at least 70%, at least 80%, or at least 90% of Helios expression compared to the Treg population prior to expansion.

[0236] In some embodiments, Tregs generated according to the methods provided herein continue to expand after genetic manipulation. In some embodiments, Tregs generated according to the methods provided herein expand at least 1.5-fold after genetic manipulation (e.g., within 3 days (about 72 hours) or within 4 days (about 96 hours) after genetic manipulation). In some embodiments, Tregs generated according to the methods provided herein expand at least 2-fold after genetic manipulation (e.g., within 3 days (about 72 hours) or within 4 days (about 96 hours) after genetic manipulation). In some embodiments, Tregs generated according to the methods provided herein expand at least 3-fold after genetic manipulation (e.g., within 3 days (about 72 hours) or within 4 days (about 96 hours) after genetic manipulation).

[0237] In some embodiments, the methods provided herein include harvesting the expanded (and optionally genetically engineered) Treg population. In some embodiments, harvesting occurs immediately after a stimulation step. In some embodiments, harvesting occurs immediately after a resting step.

[0238] In some embodiments, the stimulating step occurs after the genetic manipulation and before the harvesting. In some embodiments, the resting step occurs after the genetic manipulation and before the harvesting. In some embodiments, both the stimulating step and the resting step occur after the genetic manipulation and before the harvesting.

[0239] In some embodiments, the methods provided herein include cryopreserving the expanded (and optionally genetically engineered) Treg population. In some embodiments, cryopreservation occurs immediately after the stimulation step. In some embodiments, cryopreservation occurs immediately after the resting step.

[0240] Genetic manipulation of Tregs In some embodiments of the methods provided herein, Tregs are genetically engineered. Genetic engineering can include, for example, introducing a nucleic acid or a gene regulatory system into Tregs or a Treg population. The nucleic acid or gene regulatory system can be introduced using methods known in the art, including, for example, by electroporation and / or ribonucleoprotein (RNP)-mediated methods.

[0241] The nucleic acid that can be introduced into Tregs via genetic engineering can be a viral or non-viral nucleic acid.

[0242] The nucleic acid introduced into the Treg or Treg population can be a nucleic acid encoding a protein. The protein can be a protein that is heterologous to the Treg(s). For example, the protein can be an engineered antigen receptor, such as a chimeric antigen receptor (CAR) or an engineered TCR.

[0243] In some embodiments, the engineered antigen receptor is a CAR that comprises an extracellular antigen-binding domain fused via a hinge and transmembrane domain to a cytoplasmic domain that comprises a signaling domain. In some embodiments, the extracellular domain of the CAR comprises an antigen-binding fragment derived from an antibody. Antigen-binding domains useful in the present disclosure include, for example, scFvs. In some embodiments, the intracellular signaling domain of the CAR can be derived from the TCR complex zeta chain (e.g., CD3ξ signaling domain), FcγRIII, FcεRI, or a T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of the CAR further comprises a costimulatory domain, e.g., a 4-1BB, CD28, CD40, MyD88, or CD70 domain. In some embodiments, the intracellular signaling domain of the CAR further comprises two costimulatory domains, e.g., any two of the 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structures and intracellular signaling domains are known in the art (see, e.g., WO2009 / 091826, US20130287748, WO2015 / 142675, WO2014 / 055657, and WO2015 / 090229, which are incorporated by reference herein).

[0244] CARs specific for antigens associated with autoimmune diseases (e.g., GVHD, colitis, and multiple sclerosis) are discussed, for example, in Zhang et al., Frontiers in Immunology, 9:1-8(2018); Int'l Publ. No. WO2017218850A1; and MacDonald et al., JCI, 2016; 126(4):1413-1424, each of which is incorporated by reference herein in its entirety.

[0245] In some embodiments, the engineered antigen receptor is an engineered TCR. An engineered TCR comprises a TCR alpha chain and / or a TCR beta chain that have been isolated and cloned from a T cell population that recognizes a specific target antigen. The engineered TCR recognizes the antigen through the same mechanism as its endogenous counterpart (e.g., by recognition of cognate antigen presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of the target cell). Binding of this antigen stimulates endogenous signaling pathways, resulting in activation and proliferation of the TCR engineered cells.

[0246] As used herein, the term "gene regulatory system" refers to a protein, nucleic acid, or combination thereof that can modify an endogenous target DNA sequence when introduced into a cell, thereby regulating the expression or function of the encoded gene product. Numerous gene editing systems suitable for use in the methods of the present disclosure are known in the art, including, but not limited to, shRNA, siRNA, zinc finger nuclease systems, TALEN systems, and CRISPR / Cas systems. Gene regulatory systems useful in the methods of the present disclosure are provided, for example, in International Publication No. WO 2020 / 160489, the entire contents of which are incorporated herein by reference.

[0247] As used herein, the term "modulate," when used in reference to the effect of a gene regulatory system on an endogenous target gene, encompasses any change in the sequence of the endogenous target gene, any change in the epigenetic state of the endogenous target gene, and / or any change in the expression or function of a protein encoded by the endogenous target gene.

[0248] In some embodiments, the gene regulation system can mediate the sequence change of endogenous target gene, for example, by introducing one or more mutations into endogenous target sequence, for example, by inserting or deleting one or more nucleic acids into endogenous target sequence.Exemplary mechanisms that can mediate the change of endogenous target sequence include, but are not limited to, non-homologous end joining (NHEJ) (for example, classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (for example, via endogenous donor template), SDSA (synthesis-dependent strand annealing), single-strand annealing or single-strand invasion.

[0249] In some embodiments, the gene regulatory system can mediate changes in the epigenetic state of endogenous target sequences. For example, in some embodiments, the gene regulatory system can mediate covalent modifications of endogenous target gene DNA (e.g., cytosine methylation and hydroxymethylation) or associated histone proteins (e.g., lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitination and sumoylation).

[0250] In some embodiments, the gene regulatory system can mediate changes in the expression of a protein encoded by an endogenous target gene. In such embodiments, the gene regulatory system can regulate the expression of the encoded protein by modifying the endogenous target DNA sequence or by acting on the mRNA product encoded by the DNA sequence. In some embodiments, the gene regulatory system can result in the expression of a modified endogenous protein. In such embodiments, the modification to the endogenous DNA sequence mediated by the gene regulatory system results in the expression of an endogenous protein that exhibits reduced function compared to the corresponding endogenous protein in non-engineered Tregs. In such embodiments, the expression level of the modified endogenous protein can be increased or decreased, and can be the same or substantially similar to the expression level of the corresponding endogenous protein in non-engineered Tregs.

[0251] A gene regulatory system that can be introduced into Tregs via genetic engineering can include (i) a nucleic acid molecule; (ii) an enzyme protein; or (iii) a nucleic acid molecule and an enzyme protein. Such a gene regulatory system can include a nucleic acid molecule selected from siRNA, shRNA, microRNA (miR), antagomir, or antisense RNA. Such a gene regulatory system can include an enzyme protein that is engineered to specifically bind to a target sequence of one or more genes in Tregs. The enzyme protein can be, for example, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or a meganuclease. Such a gene regulatory system can include a nucleic acid molecule and an enzyme protein, where the nucleic acid molecule is a guide RNA (gRNA) molecule and the enzyme protein is a Cas protein or a Cas orthologue. The Cas protein can be a Cas9 protein.

[0252] Nucleic acid-based gene regulation systems As used herein, nucleic acid-based gene regulation system is a system that comprises one or more nucleic acid molecules that can regulate the expression of endogenous target genes without the need for exogenous protein.In some embodiments, nucleic acid-based gene regulation system comprises RNA interference molecule or antisense RNA molecule that is complementary to target nucleic acid sequence.

[0253] "Antisense RNA molecule" refers to an RNA molecule, regardless of length, that is complementary to an mRNA transcript.Antisense RNA molecule refers to a single-stranded RNA molecule that can be introduced into a cell, tissue, or subject, and that results in reduced expression of an endogenous target gene product through a mechanism that does not rely on endogenous gene silencing pathways, but rather on RNase H-mediated degradation of the target mRNA transcript.In some embodiments, the antisense nucleic acid may contain modified backbones, such as phosphorothioates, phosphorodithioates, or others known in the art, or may contain non-natural internucleoside linkages.In some embodiments, the antisense nucleic acid may contain locked nucleic acids (LNAs).

[0254] As used herein, "RNA interference molecule" refers to an RNA polynucleotide that mediates the decreased expression of an endogenous target gene product by degradation of the target mRNA via an endogenous gene silencing pathway (e.g., Dicer and RNA-induced silencing complex (RISC)). Exemplary RNA interference agents include microRNAs (also referred to herein as "miRNAs"), short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), RNA aptamers, and morpholinos.

[0255] In some embodiments, the nucleic acid-based gene regulation system comprises one or more miRNAs. miRNA refers to a naturally occurring small non-coding RNA molecule of about 21-25 nucleotides in length. miRNAs are at least partially complementary to one or more target mRNA molecules. miRNAs can downregulate (e.g., decrease) expression of endogenous target gene products through translational repression, mRNA cleavage, and / or deadenylation.

[0256] In some embodiments, the nucleic acid-based gene regulation system comprises one or more shRNAs. shRNAs are single-stranded RNA molecules, approximately 50-70 nucleotides in length, that form a stem-loop structure and trigger degradation of complementary mRNA sequences. shRNAs can be cloned into plasmids or non-replicating recombinant viral vectors and introduced into cells, resulting in integration of the shRNA-encoded sequence into the genome. Thus, shRNAs can stably and consistently suppress translation and expression of endogenous target genes.

[0257] In some embodiments, the nucleic acid-based gene regulation system comprises one or more siRNAs. siRNA generally refers to a double-stranded RNA molecule about 21-23 nucleotides in length. siRNA associates with a multiprotein complex called the RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cleaved. The antisense "guide" strand contained in the activated RISC guides the RISC to the corresponding mRNA due to sequence homology, and the same nuclease cleaves the target mRNA, resulting in specific gene silencing. Optimally, the siRNA is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and has a 2-base overhang at its 3' end. siRNA can be introduced into individual cells and / or culture systems, resulting in degradation of the target mRNA sequence. siRNAs and shRNAs are further described in Fire et al., Nature, 391:19, 1998 and U.S. Patent Nos. 7,732,417; 8,202,846; and 8,383,599.

[0258] In some embodiments, the nucleic acid-based gene regulation system comprises one or more morpholinos. As used herein, "morpholino" refers to a modified nucleic acid oligomer in which a standard nucleobase is bound to a morpholine ring and linked via a phosphorodiamidate bond. Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences. However, rather than targeting and degrading complementary mRNA sequences, morpholinos function through steric inhibition of mRNA translation or alteration of mRNA splicing.

[0259] In some embodiments, the nucleic acid-based gene regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90% identical to the RNA encoded by the DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the nucleic acid-based gene regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by the DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the nucleic acid-based gene regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is 100% identical to the RNA encoded by the DNA sequence of a target gene selected from those listed in Table 1. [Table 2]

[0260] In some embodiments, the nucleic acid-based gene regulation system comprises an siRNA or shRNA molecule selected from those known in the art, such as siRNA and shRNA constructs available from suppliers such as Sigma Aldrich, Dharmacon, ThermoFisher, etc.

[0261] In some embodiments, the gene regulation system comprises two or more nucleic acid molecules (e.g., two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or two or more morpholinos), where at least one of the nucleic acid molecules binds to a target RNA sequence that is at least 90% identical to an RNA sequence encoded by a DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more nucleic acid molecules (e.g., two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or two or more morpholinos), where at least one of the nucleic acid molecules binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more nucleic acid molecules (e.g., two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or more morpholinos), where at least one of the nucleic acid molecules binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by the DNA sequence of a target gene selected from Table 1.

[0262] Protein-based gene regulation systems In some embodiments, a protein-based gene regulation system is a system comprising one or more proteins that can regulate the expression of an endogenous target gene in a sequence-specific manner without the need for a nucleic acid guide molecule. In some embodiments, a protein-based gene regulation system comprises a protein comprising one or more zinc finger binding domains and an enzymatic domain. In some embodiments, a protein-based gene regulation system comprises a protein comprising a transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain. Such embodiments are referred to herein as "TALENs."

[0263] 1. Zinc Finger System Zinc finger-based systems include fusion proteins that contain two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A "zinc finger DNA binding domain", "zinc finger protein", or "ZFP" is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner via one or more zinc fingers, which is a region of amino acid sequence within the binding domain whose structure is stabilized via the coordination of a zinc ion. The zinc finger domain binds to a target DNA sequence, thereby directing the activity of the enzymatic domain to the vicinity of that sequence, thereby inducing modification of an endogenous target gene in the vicinity of the target sequence. Zinc finger domains can be engineered to bind to virtually any desired sequence. Thus, after identifying a target locus (e.g., a target locus in a target gene referenced in Table 1) that contains a target DNA sequence for which cleavage or recombination is desired, one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences within the target locus. Expression of a fusion protein containing a zinc finger binding domain and an enzymatic domain in a cell affects modification of the target locus.

[0264] In some embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985), EMBO J. 4:1609-1614; Rhodes (1993), Scientific American February:56-65; U.S. Patent No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. Each zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence that may overlap the four-nucleotide binding site of an adjacent zinc finger by one nucleotide). Thus, the length of the sequence (e.g., target sequence) that the zinc finger binding domain is engineered to bind will determine the number of zinc fingers in the engineered zinc finger binding domain. For example, for ZFPs whose finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence will be bound by two finger binding domains; a nine-nucleotide target sequence will be bound by three finger binding domains, and so on. The binding sites (i.e., subsites) of individual zinc fingers within a target site need not be contiguous, but can be separated by one or several nucleotides depending on the length and nature of the amino acid sequences between the zinc fingers (i.e., inter-finger linkers) in a multiple finger binding domain. In some embodiments, the DNA binding domain of an individual ZFN contains between 3-6 individual zinc finger repeats, each capable of recognizing between 9-18 base pairs.

[0265] Zinc finger binding domains can be engineered to bind to a selected sequence. See, e.g., Beerli et al. (2002), Nature Biotechnol. 20:135-141; Pabo et al. (2001), Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001), Nature Biotechnol. 19:656-660; Segal et al. (2001), Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000), Curr. Opin. Struct. Biol. 10:411-416. Engineered zinc finger binding domains can have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection.

[0266] Selection of target DNA sequences for binding by zinc finger domains can be achieved, for example, according to the methods disclosed in U.S. Patent No. 6,453,242. It will be apparent to those skilled in the art that simple visual inspection of nucleotide sequences can also be used to select target DNA sequences. Thus, any means for target DNA sequence selection can be used in the methods described herein. Target sites generally have a length of at least 9 nucleotides, and are therefore bound by zinc finger binding domains that contain at least three zinc fingers. However, for example, binding of a 4-finger binding domain to a 12-nucleotide target site, binding of a 5-finger binding domain to a 15-nucleotide target site, or binding of a 6-finger binding domain to a 18-nucleotide target site is also possible. Obviously, binding of even larger binding domains (e.g., 7-finger, 8-finger, 9-finger, etc.) to even longer target sites is also possible.

[0267] In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the zinc finger system is selected from those known in the art, such as those available from suppliers such as Sigma Aldrich.

[0268] In some embodiments, the gene regulation system comprises two or more ZFP fusion proteins each comprising a zinc finger binding domain, where at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more ZFP fusion proteins each comprising a zinc finger binding domain, where at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more ZFP fusion proteins each comprising a zinc finger binding domain, where at least one of the zinc finger binding domains binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from Table 1.

[0269] The enzyme domain portion of the zinc finger fusion protein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the enzyme domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997), Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., 51 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.), Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as the source of the cleavage domain.

[0270] Exemplary restriction endonucleases (restriction enzymes) suitable for use as the enzymatic domain of the ZFPs described herein exist in many species and can bind to DNA in a sequence-specific manner (at a recognition site) and cleave the DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes a double-stranded cleavage of DNA 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994, as well as Li et al. (1992), Proc. Natl. Acad. Sci. USA, 89:4275-4279; Li et al. (1993), Proc. Natl. Acad. Sci. USA, 90:2764-2768; Kim et al. (1994a), Proc. Natl. Acad. Sci. USA, 91:883-887; Kim et al. (1994b), J. Biol. Chem. 269:31,978-31,982. Thus, in some embodiments, the fusion protein comprises at least one enzyme domain derived from a type IIS restriction enzyme and one or more zinc finger binding domains.

[0271] An exemplary type IIS restriction enzyme whose cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998), Proc. Natl. Acad. Sci. USA, 95:10,570-10,575. Thus, for targeted cleavage of double-stranded DNA using zinc finger-FokI fusions, two fusion proteins, each containing a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used. Exemplary ZFPs containing a FokI enzymatic domain are described in U.S. Pat. No. 9,782,437.

[0272] 2.TALEN System TALEN-based systems include proteins that contain TAL effector DNA binding domains and enzyme domains. They are created by fusing the TAL effector DNA binding domains to DNA cleavage domains (nucleases that cleave DNA strands). The FokI restriction enzyme mentioned above is an exemplary enzyme domain suitable for use in TALEN-based gene regulation systems.

[0273] TAL effectors are proteins secreted by bacteria via a type III secretion system when Xanthomonas bacteria infect plants. The DNA-binding domain contains a highly conserved repeated 33-34 amino acid sequence that differs at the 12th and 13th amino acids. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly correlate with specific nucleotide recognition. Thus, TAL effector domains can be engineered to bind specific target DNA sequences by selecting combinations of repeat segments containing appropriate RVDs. Nucleic acid specificities for RVD combinations are as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (although in some embodiments, NN can also bind adenine with even lower specificity).

[0274] In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the TAL effector domain binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from those listed in Table 1.

[0275] In some embodiments, the gene regulation system comprises two or more TAL effector fusion proteins each comprising a TAL effector domain, where at least one of the TAL effector domains binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more TAL effector fusion proteins each comprising a TAL effector domain, where at least one of the TAL effector domains binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more TAL effector fusion proteins each comprising a TAL effector domain, where at least one of the TAL effector domains binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from Table 1.

[0276] Methods and compositions for assembling TAL effector repeats are known in the art. See, for example, Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for the construction of TAL effector repeats are commercially available from Addgene.

[0277] Combined nucleic acid / protein-based gene regulation systems The combined gene regulation system comprises a site-specific modifying polypeptide and a nucleic acid guide molecule. As used herein, a "site-specific modifying polypeptide" refers to a polypeptide that binds to a nucleic acid guide molecule, is directed to a target nucleic acid sequence (e.g., an endogenous target DNA or RNA sequence) by the nucleic acid guide molecule to which it is bound, and modifies the target nucleic acid sequence (e.g., truncates, mutates, or methylates the target nucleic acid sequence).

[0278] The site-specific modifying polypeptide comprises two parts: a part that binds to the nucleic acid guide and an active part. In some embodiments, the site-specific modifying polypeptide comprises an active part that exhibits a site-specific enzymatic activity (e.g., DNA methylation, DNA or RNA cleavage, histone acetylation, histone methylation, etc.), where the site of the enzymatic activity is determined by the guide nucleic acid. In some cases, the site-specific modifying polypeptide comprises an active part that has an enzymatic activity that modifies an endogenous target nucleic acid sequence (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity). In other cases, the site-directed modifying polypeptide comprises an active portion having an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity) that modifies a polypeptide (e.g., histone) associated with an endogenous target nucleic acid sequence. In some embodiments, the site-directed modifying polypeptide comprises an active portion that modulates transcription (e.g., to increase or decrease transcription) of a target DNA sequence. In some embodiments, the site-directed modifying polypeptide comprises an active portion that modulates expression or translation (e.g., to increase or decrease transcription) of a target RNA sequence.

[0279] A nucleic acid guide comprises two portions: a first portion (herein referred to as the "nucleic acid binding segment") that is complementary to and capable of binding to an endogenous target nucleic acid sequence, and a second portion (herein referred to as the "protein binding segment") that can interact with a site-directed modifying polypeptide. In some embodiments, the nucleic acid binding segment and the protein binding segment of the nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the nucleic acid binding segment and the protein binding segment of the nucleic acid guide are each comprised within a separate polynucleotide molecule, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form a functional guide.

[0280] The nucleic acid guide mediates the target specificity of the combined protein / nucleic acid gene regulatory system by specifically hybridizing to the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is an RNA sequence, such as an RNA sequence contained within the mRNA transcript of the target gene. In some embodiments, the target nucleic acid sequence is a DNA sequence contained within the DNA sequence of the target gene. Reference to a target gene herein encompasses the full-length DNA sequence of that particular gene, including multiple target loci (i.e., portions (e.g., exons or introns) of the particular target gene sequence). Within each target locus is an even shorter stretch of DNA sequence, referred to herein as a "target DNA sequence," that may be modified by the gene regulatory system described herein. Additionally, each target locus includes a "target modification site," which refers to the exact location of the modification induced by the gene regulatory system (e.g., the location of an insertion, deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification).

[0281] The gene regulatory systems described herein may include a single nucleic acid guide or may include multiple nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).

[0282] In some embodiments, the combination protein / nucleic acid gene regulatory system comprises a site-directed modifying polypeptide from an Argonaute (Ago) protein (e.g., Ago or TtAgo of T. thermophiles). In some embodiments, the site-directed modifying polypeptide is Ago DNA endonuclease of T. thermophiles and the nucleic acid guide is guide DNA (gDNA) (see Swarts et al., Nature, 507 (2014), 258-261). In some embodiments, the disclosure provides a polynucleotide encoding a gDNA. In some embodiments, the nucleic acid encoding the gDNA is included in an expression vector, e.g., a recombinant expression vector. In some embodiments, the disclosure provides a polynucleotide encoding a TtAgo site-directed modifying polypeptide or a variant thereof. In some embodiments, the polynucleotide encoding a TtAgo site-directed modifying polypeptide is included in an expression vector, e.g., a recombinant expression vector.

[0283] In some embodiments, the gene editing system described herein is a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) nuclease system. In some embodiments, the CRISPR / Cas system is a class 2 system. Class 2 CRISPR / Cas systems are classified into three types: type II, type V, and type VI systems. In some embodiments, the CRISPR / Cas system is a class 2 type II system that utilizes a Cas9 protein. In some embodiments, the site-directed modifying polypeptide is a Cas9 DNA endonuclease (or a variant thereof), and the nucleic acid guide molecule is a guide RNA (gRNA). In some embodiments, the CRISPR / Cas system is a class 2 type V system that utilizes a Cas12 protein (e.g., Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (also known as CasY), and Cas12e (also known as CasX)). In some embodiments, the site-directed modifying polypeptide is a Cas12 DNA endonuclease (or a variant thereof) and the nucleic acid guide molecule is a gRNA. In some embodiments, the CRISPR / Cas system is a class 2 and type VI system that utilizes Cas13 proteins (e.g., Cas13a (also known as C2c2), Cas13b, and Cas13c) (see Pyzocha et al., ACS Chemical Biology, 13(2), 347-356). In some embodiments, the site-directed modifying polypeptide is a Cas13 RNA endonuclease and the nucleic acid guide molecule is a gRNA.

[0284] A Cas polypeptide refers to a polypeptide that can interact with and cooperate with a gRNA molecule to home to or localize to a target DNA or RNA sequence. Cas polypeptides include naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ from naturally occurring Cas sequences by one or more amino acid residues.

[0285] A guide RNA (gRNA) comprises two segments: a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of the gRNA is contained in one RNA molecule and the DNA-binding segment is contained in another separate RNA molecule. Such embodiments are referred to herein as "dual-molecule gRNA" or "bi-molecule gRNA" or "dual gRNA". In some embodiments, the gRNA is a single RNA molecule, referred to herein as "single guide RNA" or "sgRNA". The terms "guide RNA" or "gRNA" are inclusive and refer to both bi-molecule guide RNAs and sgRNAs.

[0286] The protein-binding segment of the gRNA is composed, in part, of two complementary stretches of nucleotides that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex) and facilitate binding to a Cas protein. The nucleic acid-binding segment of the gRNA (or "nucleic acid-binding sequence") comprises a nucleotide sequence that is complementary to and capable of binding to a specific target nucleic acid sequence. The protein-binding segment of the gRNA interacts with a Cas polypeptide, and the interaction of the gRNA molecule with the site-directed modifying polypeptide allows Cas to bind to an endogenous nucleic acid sequence and create one or more modifications in or around the target nucleic acid sequence. The exact location of the target modification site is determined by both (i) the base-pairing complementarity between the gRNA and the target nucleic acid sequence; and (ii) the location of a short motif in the target DNA sequence called the protospacer adjacent motif (PAM) (in the target RNA sequence, called the protospacer flanking sequence (PFS)). The PAM / PFS sequence is required for Cas to bind to the target nucleic acid sequence. A variety of PAM / PFS sequences are known in the art and are suitable for use with certain Cas endonucleases (e.g., Cas9 endonuclease) (see, e.g., Nat Methods. 2013, Nov;10(11):1116-1121 and Sci Rep. 2014;4:5405). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site in the target DNA sequence. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site in the target DNA sequence. The DNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence that mediates sequence-specific gRNA-mediated Cas binding. In some embodiments, the PFS sequence is located at the 3' end of the target RNA sequence. In some embodiments, the target modification site is located at the 5' end of the target locus. In some embodiments, the target modification site is located at the 3' end of the target locus. In some embodiments, the target modification site is located within an intron or exon of the target locus.

[0287] In some aspects, the disclosure provides a polynucleotide encoding a gRNA. In some aspects, the nucleic acid encoding the gRNA is included in an expression vector, e.g., a recombinant expression vector. In some aspects, the disclosure provides a polynucleotide encoding a site-directed modifying polypeptide. In some aspects, the polynucleotide encoding a site-directed modifying polypeptide is included in an expression vector, e.g., a recombinant expression vector.

[0288] 1. Cas proteins In some embodiments, the site-directed modifying polypeptide is a Cas protein. Any Cas protein can be used, including those provided herein. S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterospoxus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputomm, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii、Nitrosomonas sp.、Parvibaculum lavamentivorans、Pasteurella multocida、Phascolarctobacterium succinatutens、Ralstonia syzygii、Rhodopseudomonas palustris、Rhodovulum sp.、Simonsiella muelleri、Sphingomonas sp.Cas molecules from various species can be used in the methods and compositions described herein, including Cas molecules from Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

[0289] In some embodiments, the Cas protein is a naturally occurring Cas protein. In some embodiments, the Cas endonuclease is selected from the group consisting of C2C1, C2C3, Cpf1 (also known as Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csx12, Csx13 ... syl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.

[0290] In some embodiments, the Cas protein is an endoribonuclease, such as a Cas13 protein. In some embodiments, the Cas13 protein is a Cas13a (Abudayyeh et al., Nature, 550 (2017), 280-284), Cas13b (Cox et al., Science, (2017) 358: 6336, 1019-1027), Cas13c (Cox et al., Science, (2017) 358: 6336, 1019-1027), or Cas13d (Zhang et al., Cell, 175 (2018), 212-223) protein.

[0291] In some embodiments, the Cas9 protein is any Cas9 protein, including any of the Cas9 proteins specifically provided herein. In some embodiments, the Cas protein is a wild-type or naturally occurring Cas protein or Cas9 orthologue. Wild-type Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC-like domain to cleave the non-target strand. WT Cas9 binds to DNA based on gRNA specificity, resulting in double-stranded DNA breaks that can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology, 201310:5,727-737, and additional Cas9 orthologues are described in International PCT Publication No. WO2015 / 071474. Such Cas9 molecules include cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, cluster 7 bacterial family, cluster 8 bacterial family, cluster 9 bacterial family, cluster 10 bacterial family, cluster 11 bacterial family, cluster 12 bacterial family, cluster 13 bacterial family, cluster 14 bacterial family, cluster 15 bacterial family, cluster 16 bacterial family, cluster 17 bacterial family, cluster 18 bacterial family, cluster 19 bacterial family, cluster 20 bacterial family, cluster 21 bacterial family, cluster 22 bacterial family, cluster 23 bacterial family, cluster 24 bacterial family, cluster 25 bacterial family, cluster 26 bacterial family, cluster 27 bacterial family, cluster 28 bacterial family, cluster 29 bacterial family, cluster 30 bacterial family, cluster 31 bacterial family, cluster 32 bacterial family, cluster 33 bacterial family, cluster 34 bacterial family, cluster 35 bacterial family, cluster 36 bacterial family, cluster 37 bacterial family, cluster 38 bacterial family, cluster 39 bacterial family,The Cas9 molecule may be from a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.

[0292] In some embodiments, the naturally occurring Cas9 polypeptide is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the Cas9 protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the Cas9 amino acid sequence set forth in Chylinski et al., RNA Biology, 2013, 10:5, 727-737; Hou et al., PNAS Early Edition, 2013, 1-6).

[0293] In some embodiments, the Cas polypeptide has the following activity: a. Nickase activity, i.e., the ability to cleave a single strand of a nucleic acid molecule, e.g., a non-complementary or complementary strand; b. double-stranded nuclease activity, i.e., in one embodiment, the presence of two nickase activities, the ability to cleave both strands of a double-stranded nucleic acid and create a double-stranded break; c. endonuclease activity; d. exonuclease activity; and / or e. Helicase activity, i.e., the ability to unwind the helical structure of double-stranded nucleic acid.

[0294] In some embodiments, the Cas polypeptide is fused to a heterologous protein that recruits a DNA damage signaling protein, an exonuclease, or a phosphatase to further increase the likelihood or rate of repair of the target sequence by some repair mechanism. In some embodiments, the WT Cas polypeptide is co-expressed with a nucleic acid repair template to promote the integration of exogenous nucleic acid sequences by homology directed repair.

[0295] In some embodiments, different Cas proteins (i.e., Cas9 proteins from different species) may be advantageous for use in the various methods provided to take advantage of different enzymatic properties of the different Cas proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for increased or decreased levels of cytotoxicity; for altering the balance between NHEJ, homology directed repair, single-strand breaks, and double-strand breaks).

[0296] In some embodiments, the Cas protein is the Cas9 protein from S. pyogenes, which recognizes the PAM sequence motifs NGG, NAG, NGA (Mali et al, Science, 2013; 339(6121): 823-826). In some embodiments, the Cas protein is the Cas9 protein from S. thermophiles, which recognizes the PAM sequence motifs NGGNG and / or NNAGAAW (W = A or T) (see, e.g., Horvath et al, Science, 2010; 327(5962): 167-170, and Deveau et al, J. Bacteriol. 2008; 190(4): 1390-1400). In some embodiments, the Cas protein is a Cas9 protein from S. mutans and recognizes the PAM sequence motifs NGG and / or NAAR (R=A or G) (see, e.g., Deveau et al, J. BACTERIOL. 2008;190(4):1390-1400). In some embodiments, the Cas protein is a Cas9 protein from S. aureus and recognizes the PAM sequence motif NNGRR (R=A or G). In some embodiments, the Cas protein is a Cas9 protein from S. aureus and recognizes the PAM sequence motif NGRRT (R=A or G). In some embodiments, the Cas protein is a Cas9 protein from S. aureus and recognizes the PAM sequence motif NGRRV (R=A or G). In some embodiments, the Cas protein is a Cas9 protein from N. meningitidis, which recognizes the PAM sequence motifs N GATT or N GCTT (R=A or G, V=A, G or C) (see, e.g., Hou et al., PNAS, 2013, 1-6). In the foregoing embodiments, N can be any nucleotide residue, e.g., A, G, C or T. In some embodiments, the Cas protein is a Cas13a protein from Leptotrichia shahii, which recognizes a single 3' A, U or C PFS sequence motif.

[0297] In some embodiments, polynucleotides are provided that encode Cas proteins. In some embodiments, the polynucleotides encode Cas proteins that are at least 90% identical to the Cas proteins described in International PCT Publication No. WO2015 / 071474 or Chylinski et al., RNA Biology, 2013, 10:5, 727-737. In some embodiments, the polynucleotides encode Cas proteins that are at least 95%, 96%, 97%, 98% or 99% identical to the Cas proteins described in International PCT Publication No. WO2015 / 071474 or Chylinski et al., RNA Biology, 2013, 10:5, 727-737. In some embodiments, the polynucleotide encodes a Cas protein that is 100% identical to a Cas protein described in International PCT Publication No. WO2015 / 071474 or Chylinski et al., RNA Biology, 2013, 10:5, 727-737.

[0298] 2. Cas mutant type In some embodiments, the Cas polypeptide is engineered to alter one or more properties of the Cas polypeptide. For example, in some embodiments, the Cas polypeptide comprises an altered enzymatic property, such as an altered nuclease activity (relative to a naturally occurring or other reference Cas molecule) or an altered helicase activity. In some embodiments, the engineered Cas polypeptide can have a modification that alters its size, such as a deletion of an amino acid sequence that reduces its size without significantly affecting another property of the Cas polypeptide. In some embodiments, the engineered Cas polypeptide comprises a change that affects PAM recognition. For example, the engineered Cas polypeptide can be altered to recognize a PAM sequence other than the PAM sequence recognized by the corresponding wild-type Cas protein.

[0299] Cas polypeptides with desired properties can be generated in a number of ways, including modification of a naturally occurring or parent Cas polypeptide to provide a mutant or modified Cas polypeptide with desired properties. For example, one or more mutations can be introduced into the sequence of a parent Cas polypeptide (e.g., a naturally occurring or engineered Cas polypeptide). Such mutations and differences can include substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In some embodiments, a mutant Cas polypeptide contains one or more mutations (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations) compared to the parent Cas polypeptide.

[0300] In some embodiments, the mutant Cas polypeptide comprises a cleavage characteristic different from a naturally occurring Cas polypeptide. In some embodiments, the Cas is an inactivated Cas (dCas) mutant. In some embodiments, the Cas polypeptide does not comprise any inherent enzymatic activity and cannot mediate cleavage of a target nucleic acid. In some embodiments, the dCas may be fused to a heterologous protein capable of modifying a target nucleic acid in a non-cleavage-based manner. For example, in some embodiments, the dCas protein is fused to a transcription activator domain or a transcription repressor domain (e.g., Krüppel-associated box (KRAB or SKD); Mad mSIN3-interacting domain (SID or SID4X); ERF repressor domain (ERD)), MAX interacting protein 1 (MXI1), methyl-CpG binding protein 2 (MECP2), etc.). In such cases, the dCas fusion protein is directed by the gRNA to a specific location (i.e., sequence) within the target nucleic acid and exerts locus-specific regulation, such as, for example, blocking RNA polymerase binding to the promoter (selectively inhibiting the function of a transcriptional activator) and / or altering the local chromatin state (e.g., when a fusion sequence is used that modifies the target DNA or modifies a polypeptide associated with the target DNA). In some cases, the change is transient (e.g., repression or activation of transcription). In some cases, the change is heritable (e.g., when epigenetic modifications are made to the target DNA or to proteins associated with the target DNA, such as nucleosomal histones).

[0301] In some embodiments, the dCas is a dCas13 mutant (Konermann et al., Cell, 173 (2018), 665-676). These dCas13 mutants can then be fused to enzymes that modify RNA, including adenosine deaminases (e.g., ADAR1 and ADAR2). Adenosine deaminases convert adenine to inosine, which the translation machinery treats like guanine, thereby creating a functional A→G change in the RNA sequence. In some embodiments, the dCas is a dCas9 mutant.

[0302] In some embodiments, the mutant Cas9 is a Cas9 nickase mutant. The Cas9 nickase mutant contains only one catalytically active domain (either the HNH domain or the RuvC domain). The Cas9 nickase mutant retains DNA binding based on the specificity of the gRNA, but can only cleave one strand of DNA, resulting in a single-stranded break (e.g., a "nick"). In some embodiments, two complementary Cas9 nickase mutants (e.g., one Cas9 nickase mutant with an inactivated RuvC domain and one Cas9 nickase mutant with an inactivated HNH domain) are expressed in the same cell with two gRNAs corresponding to two respective target sequences; one target sequence is on the sense DNA strand and the other is on the antisense DNA strand. This dual nickase system can result in staggered double-stranded breaks, increasing target specificity, since it is unlikely that two off-target nicks will be generated close enough to generate a double-stranded break. In some embodiments, the Cas9 nickase mutant is co-expressed with a nucleic acid repair template to promote integration of the exogenous nucleic acid sequence by homology directed repair.

[0303] In some embodiments, the Cas polypeptides described herein can be engineered to alter the PAM / PFS specificity of the Cas polypeptide. In some embodiments, a mutant Cas polypeptide has a PAM / PFS specificity that differs from that of the parent Cas polypeptide. For example, a naturally occurring Cas protein can be modified to change the PAM / PFS sequence recognized by the mutant Cas polypeptide to reduce off-target sites, improve specificity, or eliminate the PAM / PFS recognition requirement. In some embodiments, the Cas protein can be modified to increase the length of the PAM / PFS recognition sequence. In some embodiments, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids in length. Cas polypeptides that recognize different PAM / PFS sequences and / or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas polypeptides are described, for example, in Esvelt et al. Nature, 2011, 472(7344):499-503.

[0304] Exemplary Cas mutants are described in International PCT Publication No. WO2015 / 161276 and Konermann et al., Cell, 173 (2018), 665-676, which are incorporated by reference in their entireties.

[0305] 3.gRNA The present disclosure provides a guide RNA (gRNA) that guides a site-specific modifying polypeptide to a specific target nucleic acid sequence. The gRNA comprises a nucleic acid-targeting segment and a protein-binding segment. The nucleic acid-targeting segment of the gRNA comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid sequence. Thus, the nucleic acid-targeting segment of the gRNA interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing), and the nucleotide sequence of the nucleic acid-targeting segment determines the location within the target nucleic acid to which the gRNA will bind. The nucleic acid-targeting segment of the gRNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence within the target nucleic acid sequence.

[0306] The protein-binding segment of the guide RNA interacts with a site-specific modifying polypeptide (e.g., Cas protein) to form a complex. The guide RNA directs the bound polypeptide to a specific nucleotide sequence in the target nucleic acid via the nucleic acid-targeting segment described above. The protein-binding segment of the gRNA is complementary to each other and includes two stretches of nucleotides that form a double-stranded RNA duplex.

[0307] In some embodiments, the gRNA comprises two separate RNA molecules. In some embodiments, each of the two RNA molecules comprises a stretch of nucleotides that are complementary to each other, such that the complementary nucleotides of the two RNA molecules hybridize to form a double-stranded RNA duplex of the protein-binding segment. In some embodiments, the gRNA comprises a single RNA molecule (sgRNA).

[0308] The specificity of the gRNA for the target locus is mediated by the sequence of the nucleic acid binding segment comprising about 20 nucleotides that are complementary to the target nucleic acid sequence in the target locus. In some embodiments, the corresponding target nucleic acid sequence is about 20 nucleotides in length. In some embodiments, the nucleic acid binding segment of the gRNA sequence of the present disclosure is at least 90% complementary to the target nucleic acid sequence in the target locus. In some embodiments, the nucleic acid binding segment of the gRNA sequence of the present disclosure is at least 95%, 96%, 97%, 98% or 99% complementary to the target nucleic acid sequence in the target locus. In some embodiments, the nucleic acid binding segment of the gRNA sequence of the present disclosure is 100% complementary to the target nucleic acid sequence in the target locus.

[0309] In some embodiments, the target nucleic acid sequence is an RNA target sequence. In some embodiments, the target nucleic acid sequence is a DNA target sequence. In some embodiments, the nucleic acid binding segment of the gRNA sequence binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the nucleic acid binding segment of the gRNA sequence binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from those listed in Table 1. In some embodiments, the nucleic acid binding segment of the gRNA sequence binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from those listed in Table 1.

[0310] In some embodiments, the gene regulation system comprises two or more gRNA molecules each comprising a DNA binding segment, where at least one of the nucleic acid binding segments binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more gRNA molecules each comprising a nucleic acid binding segment, where at least one of the nucleic acid binding segments binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from Table 1. In some embodiments, the gene regulation system comprises two or more gRNA molecules each comprising a nucleic acid binding segment, where at least one of the nucleic acid binding segments binds to a target DNA sequence that is 100% identical to a target DNA sequence of a target gene selected from Table 1.

[0311] In some embodiments, the nucleic acid binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas-OFF finder) to identify target sequences unique to a particular target locus or target gene.

[0312] In some embodiments, the gRNAs described herein can include one or more modified nucleosides or nucleotides that introduce stability against nucleases. In some embodiments, these modified gRNAs may induce a reduced innate immune response compared to unmodified gRNAs. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single-stranded nucleic acids, generally of viral or bacterial origin, involving the expression and release of cytokines, particularly interferon expression and release, and the induction of cell death.

[0313] In some embodiments, gRNAs described herein are modified at or near their 5' ends (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5' ends). In some embodiments, the 5' ends of gRNAs are modified by inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., G(5')ppp(5')G cap analog, m7G(5')ppp(5')G cap analog, or 3'-0-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA)). In some embodiments, in vitro transcribed gRNAs are modified by treatment with a phosphatase (e.g., calf intestinal alkaline phosphatase) to remove the 5' triphosphate group. In some embodiments, the gRNA includes a modification at or near its 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3' end). For example, in some embodiments, the 3' end of the gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues.

[0314] In some embodiments, modified nucleosides and nucleotides can be present in gRNA, but may also be present in other gene regulation systems, such as mRNA, RNAi, or siRNA based systems. In some embodiments, modified nucleosides and nucleotides are: a. an alteration, e.g., substitution, of one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens in the phosphodiester backbone linkages; b. A modification, e.g., substitution, of a component of the ribose sugar, e.g., the 2' hydroxyl on the ribose sugar; c. Large-scale replacement of the phosphate moiety with a "dephospho" linker; d. Modifications or substitutions of naturally occurring nucleobases; e. ribose-phosphate backbone substitutions or modifications; f. modification of the 3' or 5' end of an oligonucleotide, such as removal, modification or replacement of a terminal phosphate group, or attachment of a moiety; and g. Sugar modifications may be included.

[0315] In some embodiments, the modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, in some embodiments, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In some embodiments, every base of the gRNA is modified. In some embodiments, each of the phosphate groups of the gRNA molecule is replaced with a phosphorothioate group.

[0316] In some embodiments, the software tool can be used to optimize the selection of gRNAs within a user's target sequence, e.g., to minimize overall off-target activity across the genome. Off-target activity can be other than cleavage. For example, for each possible gRNA selection using Cas9 in S.pyogenes, the software tool can identify all potential off-target sequences (preceding NAG or NGGPAM) across the genome that contain up to a certain number of mismatched base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally derived weighting scheme. Each possible gRNA can then be ranked according to the total predicted off-target cleavage; the top ranked gRNAs represent those most likely to have the greatest on-target cleavage and the least off-target cleavage. Other features can also be included in the tool, e.g., automated reagent design for gRNA vector construction, primer design for precise Surveyor assays, and primer design for high-throughput detection and quantification of off-target cleavage via next-generation sequencing.

[0317] Some methods provided herein begin with genetic engineering. Such genetic engineering can be followed, for example, by one or more resting steps and / or one or more stimulating steps. For example, Tregs can be genetically engineered, rested, and then stimulated. Alternatively, Tregs can be genetically engineered, stimulated, and then rested.

[0318] In other embodiments of the methods provided herein, Tregs can be genetically engineered after one or more resting steps and / or one or more stimulation steps, for example, Tregs can be genetically engineered after a first stimulation step, a resting step, and a second stimulation step.

[0319] In some embodiments, Tregs are cultured in the presence of stimulatory factors immediately prior to genetic engineering. In some embodiments, Tregs are genetically engineered in the absence of stimulatory agents. In some embodiments, Tregs are cultured in the absence of stimulatory agents (resting) immediately after genetic engineering. In some embodiments, Tregs are genetically engineered during the resting step (e.g., in the absence of stimulatory agents).

[0320] In some embodiments, Tregs are cultured in the absence of stimulatory agents immediately prior to genetic manipulation.

[0321] In some embodiments, Tregs are genetically engineered after about 6 to about 12 days in culture. In some embodiments, Tregs are genetically engineered after about 6 to about 11 days in culture. In some embodiments, Tregs are genetically engineered after about 6 to about 10 days in culture. In some embodiments, Tregs are genetically engineered after about 6 to about 9 days in culture. In some embodiments, Tregs are genetically engineered after about 6 to about 8 days in culture. In some embodiments, Tregs are genetically engineered after about 7 to about 12 days in culture. In some embodiments, Tregs are genetically engineered after about 7 to about 11 days in culture. In some embodiments, Tregs are genetically engineered after about 7 to about 10 days in culture. In some embodiments, Tregs are genetically engineered after about 7 to about 9 days in culture. In some embodiments, Tregs are genetically engineered after about 7 to about 8 days in culture.

[0322] In some embodiments, Tregs are genetically engineered after at least 6 days in culture. In some embodiments, Tregs are genetically engineered after at least 7 days in culture. In some embodiments, Tregs are genetically engineered after at least 8 days in culture.

[0323] In some embodiments, Tregs are genetically engineered after about 6 days in culture. In some embodiments, Tregs are genetically engineered after about 7 days in culture. In some embodiments, Tregs are genetically engineered after about 8 days in culture. In some embodiments, Tregs are genetically engineered after about 9 days in culture. In some embodiments, Tregs are genetically engineered after about 10 days in culture.

[0324] In some embodiments, the Tregs are engineered after not more than 10 days of exposure to the stimulant, where the 10 days do not include more than 5 consecutive days. In some embodiments, the Tregs are engineered after not more than 9 days of exposure to the stimulant, where the 9 days do not include more than 5 consecutive days. In some embodiments, the Tregs are engineered after not more than 8 days of exposure to the stimulant, where the 8 days do not include more than 5 consecutive days. In some embodiments, the Tregs are engineered after not more than 7 days of exposure to the stimulant, where the 7 days do not include more than 5 consecutive days. In some embodiments, the Tregs are engineered after not more than 6 days of exposure to the stimulant, where the 6 days do not include more than 5 consecutive days.

[0325] In some embodiments, the Tregs are engineered after at least 4 days total of exposure to a stimulant, where the 4 days total does not include more than 3 consecutive days. In some embodiments, the Tregs are engineered after at least 5 days total of exposure to a stimulant, where the 5 days total does not include more than 3 consecutive days. In some embodiments, the Tregs are engineered after at least 6 days total of exposure to a stimulant, where the 4 days total does not include more than 6 consecutive days. In some embodiments, the Tregs are engineered after at least 7 days total of exposure to a stimulant, where the 7 days total does not include more than 5 consecutive days.

[0326] The days of culture prior to genetic manipulation can include, for example, a first stimulation step, a resting step, and a second stimulation step.

[0327] In some embodiments, Tregs are genetically engineered at a time when the Treg population has expanded by about 100-fold to about 500-fold. In some embodiments, Tregs are genetically engineered at a time when the Treg population has expanded by about 250-fold.

[0328] Isolation of Tregs Before expansion, Treg can be isolated, for example, from a subject or from a sample obtained from a subject.In some embodiments, Treg is obtained from peripheral blood, thymus, lymph node, spleen, bone marrow, umbilical cord blood, or tissue sample of a subject.In some embodiments, Treg is obtained from a sample obtained from peripheral blood, thymus, lymph node, spleen, bone marrow, umbilical cord blood, or tissue sample of a subject.The subject can be, for example, a mammalian subject, such as a human subject.

[0329] In some embodiments, the Tregs are obtained from peripheral blood. In some embodiments, the Tregs are obtained from human peripheral blood.

[0330] In some embodiments, Tregs are obtained from human peripheral blood-derived PBMCs, where CD4 + T cells were isolated by negative immunomagnetic selection (e.g., EasySep human CD4 + T cell isolation kit) and CD4 + T cells were labeled with antibodies against CD25, CD4, CD127, and hCD45RA, and CD4 + CD25 hi CD127 lo CD45RA + Tregs are selected.

[0331] PBMCs can be isolated from blood by density gradient sedimentation and are CD4 + T cells can be enriched by positive selection from PBMCs by magnetic cell sorting. +T cells are then stained with fluorescently labeled antibodies specific for regulatory T cell markers such as CD4, CD25, and CD127, and then separated by fluorescence activated cell sorting (FACS) to identify CD4 + CD25 + CD127 lo Enrich Tregs, e.g., CD4 + CD25 - CD127 + can be isolated from conventional T cells.

[0332] Once a sample containing Tregs has been separated, Tregs can be enriched. Tregs can be enriched, for example, by targeting a selection of cell surface markers specific for immunosuppressive Tregs and isolating using automated cell sorting, such as FACS, solid-phase magnetic beads, and the like. Methods for enriching Tregs are provided, for example, in U.S. Pat. No. 7,722,862, which is incorporated herein by reference in its entirety. Enrichment can include positive and / or negative selection. For example, negative selection can be used to remove cells bearing surface markers specific for non-Treg cell types, such as, for example, CD8, CD11b, CD16, CD19, CD36, and / or CD56.

[0333] In some embodiments, e.g., using FACS of CD4+ T cells, CD45RA hi CD25 hi CD127 lo CD4 + Tregs are isolated using cell sorting for cells. In some embodiments, total CD45RA hi CD25 hi CD127 lo CD4 + Cell sorting for cells is used to isolate Tregs.

[0334] In some embodiments, Tregs are isolated by enriching for CD25+ T cells using magnetic beads. In some embodiments, Tregs are isolated by enriching for CD25+ T cells using fluorescence activated cell sorting (FACS).

[0335] In some embodiments, Tregs are isolated based on the presence of CD4 and CD25, and the absence of the alpha chain of IL-7R (CD127).

[0336] In some embodiments, Tregs are isolated without the use of magnetic beads. In some embodiments, Tregs are isolated without the use of artificial antigen presenting cells. In some embodiments, Tregs are isolated without the use of magnetic beads or artificial antigen presenting cells.

[0337] In some embodiments, Tregs are obtained by isolating peripheral blood mononuclear cells (PBMCs) from a subject using lymphocyte density gradient centrifugation, depleting CD8+ cells using magnetic beads, and then enriching for CD25+ T cells.

[0338] In some embodiments, Tregs are isolated (eg, from a subject) by bead separation.

[0339] Uses of expanded Tregs Also provided herein are methods of using the Tregs expanded according to the methods provided herein. For example, the expanded Tregs can be administered to a subject. The administration can be, for example, to treat an autoimmune or inflammatory disease in a subject, to treat or prevent graft-versus-host disease (GVHD) in a subject, and / or to reduce an immune response in a subject. The subject can be a mammalian subject, for example, a human subject.

[0340] In some embodiments, the autoimmune or inflammatory disease or disorder is selected from the group consisting of psoriasis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ulcerative colitis, Crohn's disease, HCV-associated vasculitis, alopecia areata, ankylosing spondylitis, Sjogren's syndrome, autoimmune hepatitis, inflammatory bowel disease (IBD), colitis, vasculitis, temporal arteritis, lupus, celiac disease, polymyalgia rheumatica, and arthritis.

[0341] In some embodiments, Tregs are obtained from a subject, expanded according to any of the methods provided herein, and then administered to the same subject. Thus, the use of expanded Tregs provided herein can be autologous.

[0342] In some embodiments, Tregs are obtained from a subject, expanded according to any of the methods provided herein, and then administered to a different subject. Thus, the use of the expanded Tregs provided herein can be allogeneic.

[0343] The amount of Treg that will be effective in treating or preventing symptoms will depend on the nature of the disease. The exact dose to be adopted will also depend on the route of administration and the severity of the condition. Usually, the administration of T cell therapy is defined by the number of cells per kilogram of body weight. However, T cells will replicate and expand after administration. Cells can be administered by injection techniques known in the art. EXAMPLES

[0344] The experiments described herein use a cyclic in vitro approach to maximize the expansion of human regulatory T cells (Tregs) for clinical use as an immunotherapy for the treatment of autoimmune disease.

[0345] Example 1: Materials and Methods material gRNA: Where indicated, single molecule gRNA (sgRNA) was used in all experiments. Duplexed gRNA molecules were formed by duplexing 200 μM tracrRNA (IDT Cat. No. 1072534) with 200 μM target-specific crRNA (IDT Cat. No. 11-01-03-01) in nuclease-free duplex buffer at 95°C for 5 min to form a 100 μM tracrRNA:crRNA duplex, where tracrRNA and crRNA are present in a 1:1 ratio.

[0346] Cas9: Cas9 was expressed in target cells by introduction of the Cas9 protein from S. pyogenes (IDT Cat#1074182).

[0347] RNP: gRNA-Cas9 ribonucleoprotein (RNP) was formed by mixing 1.2 μL of 100 μM tracrRNA:crRNA duplex with 1 μL of 20 μM Cas9 protein and 0.8 μL of PBS. The mixture was incubated at room temperature for 20 min to form the RNP complex.

[0348] method Isolation of human Treg cells: Peripheral blood Treg cells were isolated stepwise from fresh leukocyte packs or whole blood from healthy volunteer blood donors. First, peripheral blood mononuclear cells (PBMCs) were isolated using the EasySep Direct Human PBMC Isolation Kit (StemCell Technologies, Cat. No. 19654). Next, EasySep Human CD4 + CD4 T cells were isolated via negative immunomagnetic selection using a T cell isolation kit (StemCell Technologies, catalog no. 17952). + T cells were isolated. + T cells were labeled with antibodies against hCD25 (PE, BioLegend), hCD4 (APC, BD Pharmingen), hCD127 (BV421, BD Pharmingen) and hCD45RA (APC-Cy7, BD Pharmingen) and then analyzed for the following parameters: CD4 + CD25 hi CD127 lo CD45RA + were selected based on.

[0349] Ex vivo expansion of human Treg cells: Isolated Tregs were cultured at 1.25 × 10 cells / mL in X-VIVO 15 T cell expansion medium (Lonza, Cat. No. 04-418Q) supplemented with 10% human heat-inactivated serum, human IL-2 (800 units / mL), N-acetyl-L-cysteine ​​(5 mM), and 10 μl / mL ImmunoCult CD3 / 28 / 2 tetramer (StemCell Technologies, Cat. No. 10970) (hereafter referred to as Treg medium). 5 Cells were seeded at 1.25 × 10 cells / mL (0.2 mL per well) into 96-well U-bottom plates (Falcon, BD Pharmingen). On day 3 of culture, cells were washed and reseeded at the original concentration in Treg medium containing IL-2 and allowed to expand for 3 days. During this expansion phase, cells were split daily to 1.25 × 10 5 On day 6 of culture, cells were restimulated with CD3 / 28 / 2 tetramer (10 μl / mL) at a concentration of 5 × 10 5 The cell density was adjusted to 100 cells / mL (this cell density was maintained for the remainder of the culture period). The concentration of IL-2 was maintained at 800 units / mL for the first 7 days of culture and then reduced to 300 units / mL. Tetramer was added to the cultures only on the first day of each stimulation cycle and was not replenished daily. Cycles of stimulation and expansion were repeated every 3-4 days throughout the remainder of the culture period. The purity of the cultured Treg cells was checked regularly by flow cytometry as outlined below.

[0350] RNP transfection of Treg cells: Where indicated, Cas9-RNP was used to edit Tregs as follows: On day 8 of culture, Treg cells were washed and transfected with 5 × 10 7The cells were resuspended in PBS at 1000 cells / mL. Single guide RNA (sgRNA) targeting OR1A1 (SEQ ID NO: 7: GCTGACCAGTAACTCCCAGG), a gene unrelated to Treg cells, was complexed with transactivating CRISPR RNA (tracrRNA) in a test tube at 95°C for 5 minutes. The newly generated double-stranded molecule was mixed with Cas9 protein as described in Materials (infra) and incubated at room temperature for 20 minutes to form ribonucleoproteins (RNPs). Immediately prior to electroporation, the cells were inoculated into nucleofection buffer (18% supplement 1, 82% P3 buffer from Amaxa P3 Primary Cell 4D-NuclefectorX Kit S (Cat. No. V4XP-3032)) at 8 × 10 7 The cells were resuspended at a concentration of 10 ... 6 Cells were pooled in culture flasks at a density of 100 cells / mL.

[0351] Evaluation of editing efficiency In this method, genomic DNA (gDNA) was isolated and quantified from edited T cells using the Qiagen Blood and Cell Culture DNA Mini Kit (Cat. No.: 13323) following the manufacturer's recommended protocol. Following isolation of gDNA, PCR was performed to amplify the region of edited genomic DNA using locus-specific PCR primers containing the overhangs required for addition of Illumina Next Generation sequencing adapters. The resulting PCR products were run on a 1% agarose gel to ensure specific and proper amplification of the genomic locus, after which PCR cleanup was performed using the Monarch PCR & DNA Cleanup Kit (Cat. No.: T1030S) following the manufacturer's recommended protocol. The purified PCR products were then quantified and a second PCR was performed to anneal the sample-specific index sequences required for multiplexing with the Illumina sequencing adapters. After this, the PCR products were run on a 1% agarose gel to assess size before purification using AMPure XP beads (manufactured in-house). The purified PCR products were then quantified by qPCR using the Kapa Illumina Library Quantification Kit (Cat. No. KK4923) and Kapa Illumina Library Quantification DNA Standards (Cat. No. KK4903). The quantified products were then loaded onto an Illumina NextSeq 500 system using an Illumina NextSeq 500 / 550 Medium Output Reagent Cartridge v2 (Cat. No. FC-404-2003). Analysis of the generated sequencing data was performed to assess insertions and deletions (indels) at the predicted break sites in the DNA of the edited T cell pools.

[0352] Immunophenotyping and TSDR analysis of OR1A1-edited Treg cells: Purity of cultured Treg cells was checked periodically during expansion by surface staining for CD4 (clone SK3, 10 min at room temperature), followed by viability staining (LIVE / DEAD FixableNear-IR stain, ThermoFisher Scientific, Cat. No. L34975, 5 min at room temperature), fixation / permeabilization (eBioscience FoxP3 / Transcription Factor Staining Buffer Set, Cat. No: 00-5523-00, 45 min at room temperature), and finally intracellular staining for Helios (clone 22F6) and FoxP3 (clone 259D / C7) for 30 min at room temperature. Data collection was performed using an LSRFortessa (BD Biosciences) and analysis was performed using FlowJo software (TreeStar). For TSDR analysis, 12 days after editing Treg cells for OR1A1, cells were stained with anti-hCD4 and viability dye as described above, followed by fixation with 0.5% paraformaldehyde (BioLegend, Cat. No. 420801) for 10 min at room temperature. Fixed cells were washed twice with PBS containing 1% BSA, incubated on ice for 30 min in ice-cold methanol (100%) to permeabilize the cell membrane, and then stained intracellularly for Helios and FoxP3 as described above. After staining, cells were washed, resuspended in PBS with 1% BSA, and stained intracellularly for FoxP3 on a BD FACSAria II cell sorter. - , FoxP3 + Helios - , FoxP3 + Helios lo , and FoxP3 + Helios hi (1×10 6Genomic DNA (gDNA) was isolated from each of the sorted populations as described above using the Qiagen Blood and Cell Culture DNA Mini Kit (Cat. No.: 13323) following the manufacturer's recommended protocol. Bisulfite conversion and pyrosequencing of genomic DNA was performed by EpigenDx (Assay ID ADS783-FS2) to quantify the methylation status of the FoxP3 gene region.

[0353] In vitro suppression of allogeneic or autologous T effector cells by Tregs: The suppressive function of fresh or frozen Tregs was determined using a modified version of the FastImmune method developed by Canavan et al. ("A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy" Blood. 2012,Feb,23;119(8)) as well as a conventional in vitro suppression assay developed in-house. For frozen Tregs, cells were thawed 4 days before the start of the assay and rested overnight in Treg medium containing IL-2 (300 units / mL). Overnight rested Tregs were restimulated with Treg medium containing 10 μl / mL ImmunoCult CD3 / 28 / 2 tetramer (StemCell Technologies, Cat. No. 10970) and IL-2 (300 units / mL). One day before the start of the assay, frozen allogeneic or autologous PBMCs were thawed and labeled with cell tracking violet (CTV) as follows: after washing the PBMCs twice with PBS containing 0.1% BSA, the cells were diluted with 2.5 × 10 7 The cells were resuspended in the same buffer at 1 × 10 cells / mL. An equal volume of CTV (12.5 μM) was slowly added to the cell suspension, followed by incubation for 8 min at room temperature in the dark. The labeled cells were washed three times with 10 volumes of Treg medium and resuspended at 1 × 10 cells / mL in the same medium containing IL-2 (100 units / mL). 6 The next day, the labeled PBMCs were washed once to remove residual IL-2 and resuspended in Treg medium at 5 × 10 5The cells were then resuspended at 5 × 10 cells / mL. 50 microliters of this cell suspension was then added per well to two 96-well U-bottom plates (one for each assay). Pre-activated Treg cells were washed twice to remove IL-2 and tetramers and resuspended at 5 × 10 cells / mL in Treg medium. 5 Treg cells were resuspended at 1000 cells / mL. Five two-fold serial dilutions of Treg cells were prepared in duplicate on separate plates, from which 50 μl was transferred to each of two assay plates, resulting in Treg:PBMC ratios ranging from 1 / 1 to 1 / 16, with the last row containing PBMCs only. Dynabeads Human Treg Expander (Thermo Fisher Scientific Cat. No. 11129D, 7'500 beads / well) and APC-conjugated anti-human CD40L mAb (clone 24-31, BioLegend Cat. No. 310805, diluted 1:50) were added to each well of the FastImmune Assay in a final volume of 0.2 mL per well. 0.1 mL of ImmunoCult CD3 / 28 / 2 tetramer (3 μl / mL) was added per well to the in vitro suppression assay plate. After 7–12 h of incubation at 37°C, the FastImmune assay was stopped and cells were stained with mAbs against CD3 (clone HIT3a), CD4 (clone SK3), CD8 (clone SK1) and CD69 (clone FN50) (all mAbs were purchased from BioLegend). The level of Treg suppression was determined by flow cytometry comparing the expression (mean fluorescence intensity) of CD40L and CD69 on T effector cells cultured in the absence and presence of Tregs. Conventional suppression assays were terminated after 4 days of incubation and cells were stained for CD3, CD4 and CD8 (see above for clones and vendors). CTV dilutions for CD4 and CD8 T effector cells were captured by flow cytometry and the Division Index in FlowJo (TreeStar Inc) was used to calculate the average number of cell divisions that cells in the original population had undergone.

[0354] Example 2: Effects of continuous and discontinuous stimulation on Treg proliferation Assays were performed to determine whether Treg proliferation could be accelerated by implementing a discontinuous stimulation regimen in which a period of stimulation (i.e., in the presence of stimulation) is followed by a period of non-stimulatory conditions (i.e., in the absence of stimulation). Human Tregs were derived from CD4+ T cells isolated from peripheral blood by expression of CD45RA + CD25 hi CD127 lo The cells were FACS-sorted as Tregs and then stimulated with CD3 / 28 / 2 tetramer antibody (open symbols) or CD3 / 28-coated magnetic Dynabeads in the presence of IL-2 and NAC. After 3 days of culture, the cells were washed, counted, and then split into two separate wells containing medium and IL-2 with or without tetramer antibody (Ab) / Dynabeads. Cells were counted again on day 6 of culture, at which point discontinuously stimulated Tregs had expanded approximately 40-fold, whereas continuously stimulated Tregs had expanded only approximately 20-fold (see Figure 1). The difference in proliferation between the two conditions was observed regardless of whether the cells were stimulated with tetramer Ab or Dynabeads (see ibid.). Example 3: Effects of continuous and discontinuous stimulation on Treg activation

[0355] An assay was performed to assess how 3 days of non-stimulated conditions affect the activation state of Tregs. Relative cell size, proportional to the cellular forward scatter (FSC) profile, is commonly used as an indicator of the activation state of T cells cultured in vitro: naïve cells have a smaller surface area and therefore a lower FSC value compared to activated cells that have undergone T cell receptor (TCR) ligation. The results of this experiment show that Tregs stimulated for 3 days followed by 3 days of non-stimulated conditions have a significantly smaller surface area than Tregs stimulated for 6 consecutive days. (See Figure 2).

[0356] Example 4: Effects of long-term non-stimulation on Treg proliferation An assay was performed to demonstrate how prolonged non-stimulatory conditions affect Treg proliferation. As shown in Figure 1, stimulation followed by 3 days of non-stimulatory conditions accelerates Treg proliferation. When the period of non-stimulatory conditions is extended to 7 days, proliferation, determined as fold expansion between days 0 and 11, is reduced compared to baseline conditions (continuous stimulation), even when Tregs are restimulated on day 10. (See Figure 3).

[0357] Example 5: Effects of continuous and discontinuous stimulation on the proliferation of engineered Tregs An assay was performed to compare cell recovery after CRISPR manipulation of Tregs subjected to discontinuous (DSORT™) or continuous (baseline) stimulation. Manipulation was performed when cells expanded approximately 250-fold. DSORT™ Tregs expanded approximately 250-fold on day 8; baseline Tregs expanded approximately 250-fold on day 11. Manipulation was performed using a single guide RNA (sgRNA) against the non-Treg associated gene Or1a1 that was transfected (electroporated) into the cells along with Cas9 protein.

[0358] The number of surviving Tregs was measured on the day of transfection, 1 day after transfection, and then every 1-2 days for the remainder of the study. DSORT™ Tregs (open squares) continued to expand an additional 3-fold after genetic manipulation, while baseline Tregs (filled circles) failed to recover to pre-genetic manipulation levels after transfection. (See Figure 4)

[0359] Example 6: Comparison of DSORT™ Growth Protocol with Other Growth Protocols The expansion rate of DSORT™ Tregs was compared to that of Tregs generated using publicly available protocols for in vitro expansion. The results are shown in Figure 5. The top graph shows cell number (left y-axis) and fold expansion (right y-axis) over 11 days of in vitro DSORT™ expansion of Tregs isolated from three different donors and engineered for a control gene (Or1a1) on day 8 of expansion. The bottom table shows the corresponding fold expansion of Tregs derived from various studies published between 2015 and 2019. Tregs in these published studies were cultured for more than 11 days and were not genetically engineered. The average fold expansion of DSORT™ Tregs from the top graph is included in the table (labeled "KSQ Tx 2020") for reference. The results in this table show that the fold expansion of engineered DSORT™ Tregs was nearly twice as high as even the highest fold expansion observed for unengineered Tregs following other expansion protocols.

[0360] Example 7: Effect of continuous versus discontinuous stimulation on Treg stability Assays were performed to address how Tregs maintain lineage stability in vitro upon discontinuous stimulation.

[0361] Helios is a transcription factor that enhances the expression of FoxP3 in Tregs. Tregs that maintain stability from one cell generation to the next have a fully demethylated Treg-specific demethylated region (TSDR, a locus within the FoxP3 gene), whereas the TSDR of Tregs that have been converted from effector T cells in vitro (so-called inducible Tregs) or that are prone to destabilization under inflammatory conditions is partially methylated. Tregs in this experiment were sorted into four subsets based on their expression of FoxP3 and Helios. The sorted cells were then analyzed using a DNA methylation assay (pyrosequencing) to determine the methylation level of the TSDR locus. The results shown in Figure 6A show that only Helios+ Tregs had a fully demethylated TSDR.

[0362] Tregs with various percentages of Helios+ cells were subjected to a conventional in vitro suppression assay to measure the ability of Tregs to suppress the proliferation of effector T cells. In Figure 6B, the level of suppression (x-axis, shown as fold change, FC) is plotted against the percentage of Helios+ Tregs (y-axis). Tregs with a high percentage of Helios+ cells were better at suppressing the proliferation of effector T cells than Tregs with a low percentage of Helios+ cells. This data supports the importance of Helios as a marker of Treg function.

[0363] The percentage of Helios+ Tregs generated using the DSORT™ protocol was compared to the percentage of Helios+ Tregs generated using baseline (uninterruptedly stimulated) Tregs on culture day 12. The results shown in Figure 6C indicate that the majority of Tregs generated using the DSORT™ protocol were Helios+.

[0364] Example 8: Effects of continuous and discontinuous stimulation on the suppressive activity of Tregs An assay was performed to compare the suppressive function of DSORT™ (filled squares) and reference (uninterruptedly stimulated) Tregs (open squares) (Figure 7). The graph presented in Figure 7 shows the proliferation of CD4+ effector T cells at different ratios of Tregs to PBMCs (x-axis) (shown as % of maximum proliferation on the y-axis). The horizontal dashed line in the graph indicates the IC50 (ratio of Tregs:PBMCs at 50% suppression). The table presented in Figure 7 shows the IC50 values ​​of DSORT™ and reference Tregs from multiple donors. Based on these data, DSORT™ Tregs are at least 8-fold more suppressive than reference Tregs. * * *

[0365] All patents and publications cited herein are hereby fully incorporated by reference in their entirety.

Claims

1. 1. A method for expanding a regulatory T cell (Treg) population, the method comprising: (a) a first stimulation step comprising culturing a Treg population in the presence of a first stimulatory agent to generate a first stimulated Treg population; (b) a first resting step comprising continuing to culture the first stimulated Treg population in the absence of a stimulatory agent to generate a first resting Treg population.

2. Furthermore, (c) a second stimulation step comprising culturing the first resting Treg population in the presence of a second stimulatory agent to generate a second stimulated Treg population; (d) a second resting step comprising continuing to culture the second stimulated Treg population in the absence of a stimulatory agent to generate a second resting Treg population; Optionally, the method expands the number of Tregs by at least 1000 fold in 11 days. The method of claim 1.

3. moreover, (e) a third stimulation step comprising culturing the second resting Treg population in the presence of a third stimulatory agent to generate a third stimulated Treg population; 3. The method of claim 2, comprising a third resting step comprising (f) continuing to culture the third stimulated Treg population in the absence of a stimulatory agent to generate a third resting Treg population.

4. The method of any one of claims 1 to 3, wherein the method of claim 1 further comprises genetically engineering the first rested Treg population, the method of claim 2 further comprises genetically engineering the second stimulated Treg population; or the method of claim 3 further comprises genetically engineering the second rested Treg population, the third stimulated Treg population, or the third rested Treg population.

5. 1. A method for expanding a regulatory T cell (Treg) population, the method comprising: (a) a resting step comprising culturing a previously stimulated Treg population in the absence of a stimulatory agent to generate a first resting Treg population; (b) a stimulation step comprising adding a stimulatory agent to the first resting Treg population to generate a stimulated Treg population; The method further comprises genetically engineering the Tregs, and optionally genetically engineering the first resting Treg population; The method further comprises: (i) culturing the engineered Treg population in the presence of a stimulatory agent, and continuing to culture the engineered Treg population in the absence of the stimulatory agent; or (ii) culturing the engineered Treg population in the absence of the stimulatory agent, and continuing to culture the engineered Treg population in the presence of a stimulatory agent. The method.

6. 1. A method for expanding a regulatory T cell (Treg) population, the method comprising: (a) genetically engineering a Treg population to generate an engineered Treg population; (b) (i) a stimulating step comprising culturing said engineered Treg population in the presence of a stimulating agent to produce a stimulated engineered Treg population; and a resting step comprising continuing to culture said stimulated engineered Treg population in the absence of a stimulating agent to produce a resting engineered Treg population; or (ii) a resting step comprising culturing said engineered Treg population in the absence of a stimulating agent to produce a resting engineered Treg population; and a stimulating step comprising culturing said resting engineered Treg population in the presence of a stimulating agent to produce a stimulated engineered Treg population, Prior to genetic manipulation, the Treg population is subjected to a stimulation step comprising culturing the population in the presence of a stimulating agent, followed by a resting step comprising culturing the population in the absence of a stimulating agent, followed by another stimulation step comprising culturing the population in the presence of a stimulating agent; Optionally, the genetic manipulation is performed in the absence of a stimulant. The method.

7. The method of claim 1, wherein: (a) a tetrameric antibody complex is the stimulating agent in at least one of the stimulation steps and / or the culture of the engineered population; optionally, the tetrameric antibody complex specifically binds to CD3, CD28, CD2, or a combination thereof; (b) an anti-CD3 antibody or antigen-binding fragment thereof, and / or an anti-CD28 antibody or antigen-binding fragment thereof is the stimulating agent in at least one of the stimulation steps and / or the culture of the engineered population; or (c) CD3-bound and / or CD28-bound supermagnetic beads are the stimulating agent in at least one of the stimulation steps and / or the culture of the engineered population.

8. The method of any one of claims 1 to 7, wherein the method comprises (a) not using supermagnetic beads, (b) not using artificial antigen presenting cells, (c) not using rapamycin, or (d) using rapamycin.

9. The method of any one of claims 1 to 8, wherein: (a) the Tregs are cultured in the presence of IL-2, optionally reducing the IL-2 concentration during the method, or optionally IL-2 is present at a concentration of about 800 units / mL for about 7 days and then at about 300 units / mL; and / or (b) the Tregs are cultured in the presence of N-acetyl-L-cysteine, optionally wherein the N-acetyl-L-cysteine ​​is present at a concentration of about 5 mM in culture.

10. 10. The method of any one of claims 1 to 9, wherein the Treg population is cultured in the presence of a stimulating agent for about 1 to about 5 days, then in the absence of a stimulating agent for about 1 to about 5 days, then in the presence of a stimulating agent for about 1 to about 5 days, and then genetically engineered; and / or the Treg population is cultured in the presence of a stimulating agent for about 3 to about 4 days, then in the absence of a stimulating agent for about 3 to about 4 days, then in the presence of a stimulating agent for about 1 to about 4 days, and then genetically engineered.

11. The Treg population The method according to any one of claims 1 to 10, wherein the culture is carried out in the presence and / or absence of a stimulant according to the table below.

12. The method of any one of claims 1 to 11, wherein the Treg population is genetically engineered when the Treg population has expanded by about 250 fold.

13. 13. The method of any one of claims 1 to 12, wherein the method results in Tregs with a smaller surface area than Tregs cultured for 6 days in the presence of a stimulant; and / or the method results in an increased proportion of Helios+Foxp3+ Tregs compared to Tregs cultured for 6 days in the presence of a stimulant; and / or the method results in Tregs with a greater ability to suppress effector T cell (Teff) proliferation compared to Tregs cultured for 6 days in the presence of a stimulant; and / or the method prevents over-stimulation of the Treg population; and / or the method reduces activation-induced cell death compared to Tregs cultured for 6 days in the presence of a stimulant.

14. The method of claim 13, wherein the increased ability to inhibit Teff proliferation is at least 8-fold increased ability.

15. The method of any one of claims 1 to 14, wherein (a) at least 75% of Helios expression in the Tregs is maintained, (b) the Tregs are Helios+, and / or (c) the Tregs have a fully demethylated Treg-specific demethylation region (TSDR).

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