Expression of human FOXP3 in gene-edited T cells

Abstract

This invention provides compositions and materials that constitutively or regulatedly express FOXP3 in human primary lymphocytes by targeting FOXP3 cDNA to a FOXP3 locus or a non-FOXP3 locus. [Solution] The composition and materials provide specificity for CRISPR / Cas-mediated regulation of the mouse, non-human primate, or human FOXP3 gene. Using guide RNA sequences, the FOXP3 locus, AAVS1 locus, and other candidate loci are targeted to perform CRISPR / Cas-mediated gene regulation, and a gene delivery cassette for gene modification based on homologous recombination repair is provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 663,561, entitled “EXPRESSION OF MRNA ENCODING HUMAN FOXP3 FROM A NON-FOXP3 OR A FOXP3 GENETIC LOCI IN GENE EDITED T CELLS,” filed on 27 April 2018, and to U.S. Provisional Application No. 62 / 773,414, entitled “EXPRESSION OF HUMAN FOXP3 IN GENE EDITED T CELLS,” filed on 30 November 2018, both of which are incorporated herein by reference in their entirety for any purpose.

[0002] Sequence listing reference This application was filed together with an electronic sequence listing. This sequence listing was provided as a file of approximately 496kb created on April 25, 2019, with the filename SCRI187WOSEQLIST. The information contained herein is incorporated herein by reference in its entirety.

[0003] Aspects of the present invention described herein relate to constitutively or underregulated expression of FOXP3 in gene-edited lymphocytes (such as T cells) by incorporating the FOXP3 coding sequence into the FOXP3 locus or a non-FOXP3 locus of lymphocytes. [Background technology]

[0004] Lentiviral gene transfer of FOXP3 (also known as forkheadbox protein P3, forkheadbox P3, AAID, DIETER, IPEX, JM2, PIDX, XPID, or scurfin) has been previously reported in Chen, C. et al. (2011). Transplant. Proc. 43(5):2031-2048, Passerini, L. et al. (2013). Sci. Transl. Med., 5(215):215ra174, and Passerini, L. et al. (2017). Front. Immunol. 8:1282 (all of these publications are explicitly incorporated herein by citation). Furthermore, Passerini et al. (2017) reported that T lymphocytes obtained from patients with FOXP3 mutations exhibited T reg We have previously reported on the development of methods to restore function. As reported by Passerini et al. (2017), lentiviral gene transfer was used in CD4+ T cells and effector T cells, thereby converting these T cells into regulatory T cells. reg The characteristics of the cells were demonstrated, and potent in vitro and in vivo inhibitory activity was conferred. Furthermore, Passerini et al. (2013) showed that the introduction of the FOXP3 gene via lentivirus conferred CD4+ T cells to T reg It has been demonstrated that it can be converted into cells, and this T reg The cells were shown to be stable in an inflammatory state. Furthermore, Chen et al. (2011) reported adoptive transfer of recombinant T cells infected with a lentiviral vector encoding the FOXP3-IRES-GFP fragment. These cells were shown to prevent GVHD in mouse model recipients. Novel approaches are needed to express and regulate FOXP3 in primary human lymphocytes.

[0005] Because regulatory T cells have the potential to induce antigen-specific immune tolerance, many researchers are interested in treating autoimmune diseases with regulatory T cells. regThere are various forms of "), and according to the current nomenclature, regulatory T cells that arise in the thymus during the development of T cells (thymus-derived regulatory T cells or "tT reg ") and regulatory T cells induced peripherally (peripherally-derived regulatory T cells or "pT reg ") are classified.

[0006] An important aspect of the ecology of regulatory T cells is the expression of the transcription factor FOXP3 (FOXP3 is also known as forkhead box protein P3, forkhead box P3, AAID, DIETER, IPEX, JM2, PIDX, XPID or scurfin). FOXP3 is thought to be necessary for the induction of the differentiation of the regulatory T cell lineage. This concept is based on the observation that severe autoimmune diseases develop in neonates in humans lacking FOXP3. Since the expression of FOXP3 is thought to be subject to epigenetic regulation, using tT reg or pT reg in the treatment of autoimmune diseases may not be the best strategy. In tT reg , the upstream region known as the "thymus-specific demethylated region" of the FOXP3 gene is completely demethylated, which is thought to result in stable expression of FOXP3. Normally, complete demethylation is not observed in pT reg . FOXP3 is thought to be epigenetically silenced in inflammatory pT reg , and probably also in tT reg , and as a result, pT reg may change into pro-inflammatory CD4+ T cells. Since using pT reg that revert to an inflammatory phenotype in intravenous drip may exacerbate the symptoms of autoimmune diseases, the lack of stability of pT reg is a serious problem.

[0007] On the other hand, many approaches using lentiviral constructs induce random integration into the cell genome, which can disrupt tumor suppressor genes or activate proto-oncogenes. Furthermore, integration sites may be located in genomic regions characterized by low expression, which can result in unstable expression of FOXP3. [Overview of the Initiative] [Means for solving the problem]

[0008] One aspect of the present invention is, Deoxyribonucleic acid (DNA) endonuclease, or nucleic acid encoding said DNA endonuclease; Guide RNA (gRNA) containing a spacer sequence complementary to the sequence in the FOXP3 locus, AAVS1 locus, or TCRa(TRAC) locus of lymphocyte cells (e.g., T cells), or nucleic acid encoding said gRNA; and Donor templates containing nucleic acid sequences encoding the FOXP3 protein or its functional derivatives. It is a system that includes this. In some embodiments, the gRNA is i) A spacer sequence shown in any of sequence numbers 1-7, 15-20, 27-29, 33, and 34, or a variant of the spacer sequence having three or fewer mismatches compared to any of sequence numbers 1-7, 15-20, 27-29, 33, and 34; ii) A spacer sequence shown in any of sequence numbers 1 to 7, or a variant of the spacer sequence having three or fewer mismatches compared to any of sequence numbers 1 to 7; or iii) A spacer sequence shown in any of sequence numbers 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of sequence numbers 2, 3, and 5. Includes. In some embodiments, the FOXP3 or its functional derivative is human wild-type FOXP3. In some embodiments, the DNA endonuclease is a Cas endonuclease. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the DNA endonuclease or the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in liposomes or lipid nanoparticles.

[0009] Furthermore, this specification describes a method for editing the genome of lymphocyte cells, comprising the step of providing the lymphocyte cells with one of the systems described herein. In some embodiments, the lymphocyte cells are not germ cells.

[0010] Furthermore, this disclosure describes recombinant lymphocytes whose genomes have been edited by any one of the methods described herein, and compositions comprising such recombinant lymphocytes.

[0011] Furthermore, this specification describes a method for treating a FOXP3-related disease or FOXP3-related condition in a subject, comprising the step of providing one of the systems described herein to lymphocyte cells in the subject. The disease or condition may be an inflammatory disease or an autoimmune disease, for example, IPEX syndrome or graft-versus-host disease (GVHD). Some embodiments include pharmaceuticals for use in the treatment of a FOXP3-related disease or FOXP3-related condition in a subject. Further embodiments relate to genome-edited recombinant lymphocyte cells by one of the methods described herein for use in the suppression or treatment of a FOXP3-related disease or FOXP3-related condition, such as an inflammatory disease or autoimmune disease, such as IPEX syndrome or graft-versus-host disease (GVHD). Further embodiments relate to the use of genome-edited recombinant lymphocyte cells by one of the methods described herein as pharmaceuticals. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the design of multiple AAV5 donor templates, each containing a GFP coding sequence within a frame and having a different promoter element.

[0013] [Figure 2] This figure shows designs for multiple AAV5 donor templates, each containing LNFGR and P2A code sequences within the frame and having MND, sEF1a, or PGK as the promoter element.

[0014] [Figure 3] This bar graph shows the FOXP3 MFI in each experiment.

[0015] [Figure 4] This is the result of gene editing of non-human primate-derived T cells: rhesus monkey CD4+ electroporation.

[0016] [Figure 5] This is the result of gene editing in non-human primate-derived T cells: Rhesus macaque CD4+ AAV serotyping. Two different guide RNAs and their variants targeting the last exon of the human TRAC gene were designed. The editing (NHEJ and HDR) efficiency was determined when each of these guide RNAs was used alone or in combination with one of the three different gene trap (GT) AAV donor templates shown in Figure 6.

[0017] [Figure 6] This figure shows an exemplary TCRa gene trap construct.

[0018] [Figure 7] This report summarizes the results of intracellular flow cytometry measurements that measured the expression levels of inflammatory cytokines IL-2, IFNγ, and TNFα. p-values ​​were calculated using the unpaired Student's t-test.

[0019] [Figure 8] These are Kaplan-Meier curves showing the survival rate (%) of each group over time (number of days). The number of animals in each group is as indicated in the legend, and the data were obtained from two experiments conducted using healthy T-cell donors (N=2). The p-values ​​for the mock-edited group and the edTreg group are relative to the Teff-only group.

[0020] [Figure 9] This is a schematic diagram of AAV donor mold #1303, FWD 07UCOE, RVS 07UCOE, and control (without 07UCOE).

[0021] [Figure 10] This shows the GVHD scores of mice treated with different edTreg preparations in the in vivo mouse xenoGVHD experiment of Example 19.

[0022] [Figure 11]This shows the results of the immunophenotypic analysis of animals in the mouse xenoGVHD experiment of Example 19, indicating the proportion of each cell type in the LNGFR- cell population and the LNGFR+ cell population.

[0023] [Figure 12] These are the data from the in vivo xenoGVHD experiment in Example 19. The survival rates (%) are shown for mice that received Teff alone, Teff + mock-edited T cells, or Teff + edTreg intraperitoneally (IP) or intravenously (IV).

[0024] [Figure 13] This shows the results of an experiment in which CD4+ T cells derived from IPEX targets were edited using Cas9 / gRNA-T9 (ratio 1:2.5) RNP and AAV donor template #3066, according to Example 20. The bar graph shows HDR efficiency (%) and cytokine profile.

[0025] [Figure 14] This shows the results of an experiment in which CD4+ T cells derived from IPEX targets were edited using Cas9 / gRNA-T9 (ratio 1:2.5) RNP and AAV donor template #3080, according to Example 20. The bar graph shows HDR efficiency (%) and cytokine profile.

[0026] [Figure 15-17]Figure 15 shows the in vitro and in vivo results of edTreg-mediated suppression assays performed using three different batches of edTreg. The results for mock-edited CD4+ cells, CD4+ cells edited with AAV donor template #3066 according to Example 10 ("3066"), or CD4+ cells edited with AAV donor template #3080 according to Example 10 ("3080") in in vitro suppression experiments performed using the assay protocol of Method 1 (left and center graphs). Irradiation and Treg:Teff ratios are as shown on the X-axis. Furthermore, the results of in vivo experiments using the same batch of edTreg in the mouse CATI model described in Example 13 are shown (right graph). Figure 16 shows the results for mock-edited CD4+ cells or batch #2 CD4+ cells edited with AAV donor template #3066 according to Example 10 in in vitro suppression experiments performed using the assay protocol of Method 2 (left and center graphs). The Treg:Teff ratio is as shown on the X-axis. Furthermore, the results of in vivo experiments using batch #2 edTreg in the mouse CATI model described in Example 13 are shown (right graph). Figure 17 shows the results of in vitro suppression experiments performed using the assay protocol of Method 2 for mock-edited CD4+ cells, or batch #3 CD4+ cells edited with AAV donor template #3066 according to Example 10 (left graph). The Treg:Teff ratio is as shown on the X-axis. Furthermore, the results of in vivo experiments using batch #3 edTreg in the mouse CATI model described in Example 13 are shown (right graph). [Modes for carrying out the invention]

[0027] This specification describes the expression of FOXP3 from a DNA sequence (e.g., a codon-optimized DNA sequence, e.g., a codon-optimized DNA sequence for expression in human cells) incorporated into a FOXP3 locus or a non-FOXP3 locus. A guide RNA is used to target the FOXP3 locus or a non-FOXP3 locus (e.g., in mouse, human, or non-human primates) and perform genome editing via CRISPR / Cas. Therefore, aspects of the invention relate to cleaving DNA at a FOXP3 locus or a non-FOXP3 target locus and promoting the incorporation of the FOXP3 coding sequence by using a novel guide RNA in combination with a Cas protein. In some embodiments, this incorporation is performed by non-homologous end joining (NHEJ) or homologous recombination repair (HDR) associated with a donor template containing the FOXP3 coding sequence. The embodiments described herein can be used in combination with a wide range of selection markers such as LNGFR, RQR8, and CISC / DISC / μDISC, and can be multiplexed in combination with editing of another target locus or co-expression of another gene product (such as cytokines).

[0028] As will be explained in more detail later, the applicants have developed a novel AAV donor template containing a gene delivery cassette and, in combination with a Cas protein, to deliver T cells (for example, T reg Cells (referred to as "edT" in this specification) reg cells”, “edT reg " or "edT reg We identified a guide RNA that can frequently induce on-target cleavage in human T cells (which generate genome-edited T cells with the phenotype of edT) and incorporate the gene delivery cassette into the FOXP3 locus. reg Using this cell generation approach, we successfully induced an immunosuppressive phenotype in CD4+ T cells obtained from subjects with IPEX syndrome. Furthermore, we introduced edT cells into NSG recipient mice. regThe cells were able to engraft sustainably, and a high survival rate was obtained in the treated mice. These findings demonstrate that genome editing systems such as the CRISPR / Cas system described herein can perform efficient editing, thereby enabling the expression of human wild-type FOXP3 in human hematopoietic stem cells and sustaining engraftment at a level that is predicted to provide clinical utility for diseases or disorders involving abnormalities in FOXP3 function, for example, after administration as autologous cell adoptive therapy to subjects with IPEX syndrome.

[0029] The use of a CRISPR / Cas system, including gRNA and donor templates configured to insert the FOXP3 coding sequence into an endogenous or non-FOXP3 locus, is promising as a treatment for IPEX syndrome. IPEX syndrome can result from various mutations spread throughout the gene; therefore, insertion of the entire FOXP3 cDNA (e.g., the entire FOXP3 cDNA optimized for human codons) into the start codon may be desirable. By utilizing the endogenous FOXP3 promoter, it is expected that the transcriptional signaling necessary to obtain acceptable FOXP3 expression levels in edited lymphocytes can be provided.

[0030] Previous techniques for expressing FOXP3 have relied on expression via the endogenous FOXP3 gene or introduction of the FOXP3 gene by lentiviral. More specifically, FOXP3 expression has been achieved by delivery of lentiviral vectors or expression from gene-edited endogenous FOXP3 loci. Existing lentiviral delivery methods for FOXP3 expression have problems, such as the ability to control expression levels because expression depends on random viral integration, and the problem of expression disappearing due to viral silencing. As disclosed in some embodiments described herein, it has been possible to induce DNA cleavage at the endogenous FOXP3 locus of lymphocytes by utilizing site-specific gene editing techniques (e.g., using TALEN or CRISPR / Cas systems). Therefore, the gene editing method provided in the embodiments described herein allows for site-specific targeting and integration of the FOXP3 coding sequence, and this method is considered to be a safer and more controlled approach.

[0031] In systems using ribonucleoprotein (RNP) complexes containing Cas polypeptides associated with guide RNA (gRNA), targeted integration can be performed with higher efficiency than TALEN or Cas mRNA-based approaches because the RNPs can immediately exert their function upon delivery to cells. In some embodiments described herein, components of the CRISPR / Cas system are delivered to cells in the form of RNPs and used to target the human and / or non-human primate FOXP3 locus, or other loci such as AAVS1 (adeno-associated virus integration site 1) or TCR (TRAC).

[0032] The embodiments described herein may be used to express the full length of functional foxp3 in human T cells to obtain a regulatory or repressive phenotype. Such cell therapies may be useful in treating a wide range of conditions, including but not limited to IPEX syndrome, autoimmune diseases, graft-versus-host diseases, and solid organ transplantation. Other anticipated applications include, for example, disruption and / or site-specific integration of the foxp3 gene within the foxp3 locus or non-foxp3 locus in mouse, human, or non-human primate; constitutive or regulatory expression of the target gene by integration into one or both alleles at the AAVS1 site or another locus; use of any of the above approaches in the treatment of patients with IPEX syndrome; and T cells for the treatment or palliative care of autoimmune diseases using any of the above approaches. reg One example is its preparation from the CD34 cell population.

[0033] Furthermore, by generating FOXP3-expressing human T cells using the embodiments described herein, the phenotype of T cells can be modified, for example, by conferring a regulatory or repressive phenotype to the T cells. One advantage of this approach is that FOXP3 can be associated with the expression of endogenous genes. Another advantage is that, in vitro or in vivo, by using CISC / DISC, FOXP3 expression can be associated with the co-expression of gene products that enable the enrichment of gene-edited cells or mediate their expansion and proliferation. Moreover, the changes obtained by gene editing both alleles can be used to enrich or enhance the function of cell therapies.

[0034] This specification also describes the transcription of FOXP3 mRNA from DNA sequences integrated into the FOXP locus or non-FOXP3 locus and optimized for human codons. Using guide RNA sequences, FOXP3 in the mouse, human, and non-human primate FOXP3 genes is targeted for CRISPR / Cas-mediated gene regulation. Thus, aspects of the present invention relate to inducing DNA breaks at the human and non-human primate FOXP3 locus and the human AAVS1 locus by utilizing novel guide RNA sequences in combination with Cas proteins, thereby promoting gene disruption via non-homologous end joining (NHEJ) in the absence of a repair donor template, or gene integration via homology-dependent repair (HDR) in the presence of a repair donor template. Some of the embodiments described herein can be used in combination with a wide range of selection markers such as LNGFR, RQR8, and CISC / DISC / μDISC, and can be multiplexed in combination with editing of other target loci or co-expression of other gene products (such as cytokines).

[0035] As will be described in more detail later, the aforementioned reagents can be delivered using ribonucleoprotein (RNP) to target human and / or non-human primate FOXP3. In some embodiments, the reagents include a unique guide RNA sequence, which, when used in combination with a novel gene delivery cassette containing FOXP3 cDNA and / or other cis-configured gene products, as well as a Cas protein, can induce on-target cleavage at a high frequency.

[0036] Genetic transfer of FOXP3 using lentiviruses has been reported in the past. Lentiviral constructs are randomly integrated into the genome and can disrupt tumor suppressor genes or activate proto-oncogenes. Furthermore, the integration site may be silenced, preventing stable expression of FOXP3. In contrast, gene editing allows for site-specific targeting and integration. Therefore, gene editing is considered a safer and better controlled approach. RNPs are more efficient than TALENs and Cas mRNA because they exert their function immediately upon delivery to cells.

[0037] Furthermore, this specification also envisions a method for designing AAV constructs in which homologous arms are shortened to enable efficient packaging within the AAV. While this may result in slightly lower editing efficiency, the edited cells can be enriched with a selection marker such as LNGFR, or other approaches can be used to overcome the reduced editing efficiency.

[0038] The cells produced by this invention are recombinant regulatory T cells, produced using a CRISPR system combined with a repair donor DNA template, for adoptive immunotherapy for a wide range of clinical conditions such as cancer, autoimmune diseases, and organ transplantation, or for the treatment of IPEX syndrome, a hereditary immune disorder. Furthermore, this specification also describes a method for disrupting the expression of the endogenous FOXP3 gene using a CRISPR system.

[0039] This specification presents evidence that an engineering approach to stabilize FOXP3 expression in T cells can generate a proliferative population of suppressive T cells that are no longer sensitive to epigenetic modifications involved in suppressive function. These cells may thus possess improved therapeutic properties.

[0040] In the embodiments described herein, FOXP3 is stably expressed by modifying the regulatory elements of the FOXP3 gene locus using a gene editing nuclease, thereby creating therapeutic cells that stably express FOXP3. In the representative data provided herein, FOXP3 expression was induced by placing a promoter (examples of constitutive promoters include the EF1α promoter, PGK promoter, and / or MND promoter) upstream of the coding exon of FOXP3. However, it is assumed that various approaches can be used to modify the regulatory elements in order to stably express FOXP3. By modifying the endogenous regulatory elements using several approaches, the endogenous FOXP3 gene can be constitutively expressed in the therapeutic cells of the present invention, resulting in a loss of sensitivity to regulation that causes silencing of the FOXP3 gene or conversion to a non-repressive cell phenotype. Therefore, the methods described herein solve the problem of deletion of FOXP3 expression due to epigenetic effects on endogenous regulatory sequences and endogenous promoters.

[0041] In some embodiments, methods for enhancing FOXP3 expression in a bulk population of CD34+ cells are also envisioned. The endogenous TCR repertoire of inflammatory T cell populations in patients with autoimmune diseases or those who have experienced organ graft rejection contains TCRs with specificity to recognize and precisely bind to inflamed or foreign tissues in organs. Such T cells are thought to mediate autoinflammatory responses or organ rejection. By converting a portion of the bulk T cell population to a regulatory phenotype, the TCR specificity of the pro-inflammatory population can be utilized in a therapeutically effective cell population. In this respect, the method of the present invention is superior to other therapies using thymic-derived regulatory T cells (which are thought to have a different TCR repertoire that does not overlap with inflammatory T cells). Furthermore, in patients with autoimmune diseases or those who have experienced organ rejection, it is likely that the in vivo tT regThe population is thought to be failing to induce the immune tolerance necessary to avoid inflammation. The methods described herein can be used for the treatment of autoimmune diseases and for inducing immune tolerance to transplanted organs.

[0042] A significant drawback is the need to use a gene editing tool capable of efficiently performing recombination at the FOXP3 locus. While the method provided herein demonstrates that this reaction can be efficiently carried out using TALEN or CAS / CRISPR nucleases, it is generally believed that recombination can be performed similarly and successfully with any nuclease platform.

[0043] Regulatory T-cell therapy can be used to induce immune tolerance in transplantation and autoimmune diseases. Currently, T reg The infusion is cultured ex vivo. In the Phase 1 trial, only limited efficacy was observed for type 1 diabetes (T1D), but there were some cases where a benefit against GVHD was observed after transplantation. In some embodiments, the next generation of recombinant regulatory T cells are endogenous T cells that have been given targeting by a chimeric antigen receptor (CAR). reg It may also be possible to express FOXP3, which can be used to transform effector T cells into T cells. reg It can also be converted to [this format].

[0044] However, endogenous T used in treatment methods reg and genetically modified T reg Therefore, it is thought that there is some kind of difference. Endogenous T reg The therapy is considered safe, but endogenous T reg The number is too small, which triggers an autoimmune response. reg Human T1 is thought to play an extremely important role in various autoimmune diseases such as IPEX syndrome, type 1 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. regVarious approaches to enhance the number or function of cells are currently in clinical trials, such as low-dose IL-2 and expanded culture of autologous T cells. reg A treatment combining adoption and T1 is currently in clinical trials. IL-2 therapy has limited effectiveness due to its multifaceted activity and potential "off-target" effects that can enhance inflammation. reg Therapy also involves large-scale culture of T reg Due to problems with in vivo stability and viability, and a lack of therapeutically useful antigen specificity, its use will likely be limited.

[0045] endogenous T reg (nT reg There are also potential drawbacks to the use of ). For example, patients with autoimmune diseases may have T reg They have a genetic predisposition to instability. For example, CAR expression nT reg Conversion from CAR-expressing effector T cells can occur. Furthermore, nT reg Cells may be subject to epigenetic regulation of FOXP3, which can lead to downregulation of FOXP3 induction, and this is related to nT reg This means that the function of a group cannot be fully predicted. Furthermore, endogenous T reg It may not have precise specificity for TCR (T cell receptor). reg Its function is also related to selection markers, and it is associated with the expanded and proliferating endogenous T reg It is thought that inflammatory cells are always present in cell populations. Therefore, since the method provided herein uses recombinant cells, it is possible to induce the function of regulatory T cells by CAR expression, and the engrafted CAR T cells reg This avoids the possibility of them being converted into pro-inflammatory CAR T cells, and in this respect, natural T reg This method is an improved version compared to the transplantation method.

[0046] Regulatory T cells (tT) originating from the thymus reg or nT reg) is T reg This method stably expresses FOXP3, which plays a crucial role in the repressive function of tT. In representative studies described herein, stably expressing FOXP3 by knocking in a constitutive promoter upstream of the FOXP3 gene was achieved. reg CD4+T has an inhibitory function similar to that of conv It has been shown that cells can be obtained. This is also described in PCT / US2016 / 059729 (this document is incorporated herein by reference in its entirety).

[0047] Approaches that induce endogenous FOXP3 expression may not be suitable for donors with FOXP3 mutations because editing of the FOXP3 locus is limited (see, for example, Example 1). To further broaden the application of this technique, FOXP3 mRNA was expressed by introducing promoter and codon-optimized FOXP3 cDNA sequences into either a FOXP3 or non-FOXP3 locus. Cell therapies can then be enriched using selection markers such as LNGFR or DISC / μDISC.

[0048] Definition of Terms In this specification, “nucleic acid” or “nucleic acid molecule” includes, for example, polynucleotides or oligonucleotides, and includes, but is not limited to, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease activity, exonuclease activity, and synthesis. Nucleic acid molecules may consist of monomers made from natural nucleotide monomers (DNA, RNA, etc.) or analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically similar or electronically similar, such as aza sugars and carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. "Nucleic acid molecules" also include so-called "peptide nucleic acids," which contain native or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded.

[0049] The "coding strand" includes, but is not limited to, a DNA strand having the same base sequence as the RNA transcript being produced (where thymine is replaced by uracil). The coding strand contains codons, and the non-coding strand contains anticodons.

[0050] "Regulatory elements" include, for example, segments of nucleic acid molecules capable of increasing or decreasing the expression of specific genes in an organism, and are not limited to, segments of nucleic acid molecules capable of influencing the transcription and / or translation of operably linked transcribed DNA molecules. Regulatory elements such as promoters (e.g., MND promoters), leaders, introns, and transcription termination regions are DNA molecules that possess gene regulatory activity and play an essential role in the overall expression of genes in living cells. Therefore, isolated regulatory elements (such as promoters) that function in plants are useful for modifying the phenotype of plants by genetic engineering methods. The regulation of gene expression is a fundamental function of all organisms and viruses. Regulatory elements include, but are not limited to, CAAT boxes, CCAAT boxes, Pribno boxes, TATA boxes, SECIS elements, mRNA polyadenylation signals, hormone response elements such as A boxes, Z boxes, C boxes, E boxes, G boxes, and insulin gene regulatory sequences, DNA binding regions, activation regions, and / or enhancer regions.

[0051] In some embodiments, the guide RNA includes a further segment at either the 5' or 3' end that provides any of the aforementioned features. For example, suitable third segments include a 5' end cap (e.g., a 7-methylguanylate cap (m7G)); a 3' end polyadenylated tail (e.g., a 3' end poly(A) tail); a riboswitch sequence (e.g., one that stabilizes under control and / or allows access by a protein or protein complex under control); a stability control sequence; a sequence that forms a dsRNA double strand (e.g., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a traceability modification or sequence (e.g., direct binding to a fluorescent molecule, binding to a region that facilitates fluorescence detection, a sequence that enables fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a DNA-acting protein such as a transcription activator, transcription repressor, DNA methyltransferase, DNA methyl-degrading enzyme, histone acetyltransferase, histone deacetylase, etc.); and combinations thereof.

[0052] Guide RNA and Cas proteins may form a ribonucleoprotein complex (for example, by binding via non-covalent interactions). The guide RNA provides target specificity to the ribonucleoprotein complex by containing a nucleotide sequence complementary to the target DNA sequence. Site-directed modifying enzymes of the ribonucleoprotein complex provide endonuclease activity. In other words, site-directed modifying enzymes, upon association with the protein-binding segment of the guide RNA, are led to the target DNA sequence (e.g., target sequence in chromosomal nucleic acids; target sequence in extrachromosomal nucleic acids (e.g., episomal nucleic acids, minicircles, etc.); target sequence in mitochondrial nucleic acids; target sequence in chloroplast nucleic acids; target sequence in plasmids, etc.).

[0053] In this specification, "FOXP3" includes, but is not limited to, proteins involved in immune system responses. The FOXP3 gene contains 11 coding exons. Foxp3 is involved in endogenous regulatory T cells (nT). reg (T cell line)) and adoptively transferred / induced regulatory T cells (a / iT reg It is a specific marker for ). Animal studies have shown that inducing or administering FOXP3-positive T cells significantly reduces the severity of disease (autoimmune disease) in diabetes models, multiple sclerosis models, asthma models, inflammatory bowel disease models, thyroiditis models, or kidney disease models. However, T cells have been reported to exhibit plasticity. Therefore, the therapeutic use of regulatory T cells is complicated because regulatory T cells transferred to a subject may change into pro-inflammatory helper T17 (Th17) cells instead of regulatory cells. With this in mind, this specification provides a method to avoid complications resulting from the change from regulatory cells to pro-inflammatory cells. For example, iT reg FOXP3, expressed from [source], is used as a master regulator of the immune system, as well as in immune tolerance and immunosuppression. reg Human T1 is thought to play an extremely important role in various autoimmune diseases such as IPEX syndrome, type 1 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. reg Various approaches to enhance the number or function of cells are currently in clinical trials, such as low-dose IL-2 and expanded culture of autologous T cells. reg A treatment combining adoption and T1 is currently in clinical trials. IL-2 therapy has limited effectiveness due to its multifaceted activity and potential "off-target" effects that can enhance inflammation. reg Therapy also involves large-scale culture of T reg Due to problems with in vivo stability and viability, and a lack of therapeutically useful antigen specificity, its use will likely be limited.

[0054] A “nuclease” includes, but is not limited to, proteins or enzymes capable of cleaving phosphate diester bonds between nucleotide subunits of nucleic acids. The nucleases described herein are used in “gene editing.” Gene editing is a type of genetic engineering technique that uses one or more types of nucleases to insert, delete, or replace DNA in the genome of an organism. Examples of nucleases include, but are not limited to, nucleases used in the CRISPR / CAS system, zinc finger nucleases, and TALEN nucleases. Nucleases can be used to target gene loci or specific nucleic acid sequences.

[0055] A "coding exon" includes, but is not limited to, a portion of a gene that codes for a part of the final mature RNA produced by a gene after introns have been removed by RNA splicing. An "exon" refers to both the DNA sequence within a gene and its corresponding RNA transcript sequence. After introns are removed by RNA splicing, the remaining exons are covalently linked to each other, forming part of the mature messenger RNA.

[0056] In this specification, “Cas endonuclease” or “Cas nuclease” includes, for example, RNA-induced DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. In this specification, “Cas endonuclease” refers to both natural Cas endonucleases and recombinant Cas endonucleases. “Cas9” includes, for example, RNA-induced DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system.

[0057] In this specification, "zinc finger nuclease" includes, but is not limited to, artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA-degrading domain. The zinc finger domain can be modified to target a specific DNA sequence of interest, thereby enabling the zinc finger nuclease to target unique sequences within a complex genome.

[0058] In this specification, "TALEN," or "transcription activator-like effector nuclease," includes, but is not limited to, restriction enzymes that can be modified to cleave specific DNA sequences. TALENs are created by fusing a TAL effector DNA-binding domain to a DNA-degrading domain (a nuclease that cleaves DNA strands). Since transcription activator-like effectors (TALEs) can be modified to bind to virtually any desired DNA sequence, they can be combined with nucleases to cleave DNA at specific locations. Restriction enzymes can be introduced into cells for the purpose of in-situ gene editing or genome editing, a technique known as genome editing using recombinant nucleases. TALENs, along with zinc finger nucleases and CRISPR / Cas9, are among the leading tools in the field of genome editing.

[0059] "Knock-in" includes, but is not limited to, genetic engineering methods for replacing DNA sequence information with different copies within a gene locus on a one-to-one basis, or for inserting sequence information that is not present within a gene locus.

[0060] A "promoter" includes, but is not limited to, a nucleotide sequence that induces the transcription of a structural gene. In some embodiments, the promoter is located in the non-coding region at the 5' end of the gene, near the transcription start site of the structural gene. The elements of the promoter sequence that initiate transcription are often characterized by a consensus nucleotide sequence. A promoter is a DNA region that initiates the transcription of a particular gene. The promoter is located near the gene transcription start site upstream (towards the 5' region of the sense strand) within the same DNA strand. The length of the promoter may be 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, 700 base pairs, 800 base pairs, or 1000 base pairs, or approximately 100 base pairs, approximately 200 base pairs, approximately 300 base pairs, approximately 400 base pairs, approximately 500 base pairs, approximately 600 base pairs, approximately 700 base pairs, approximately 800 base pairs, or approximately 1000 base pairs, or within the range defined by any two of these lengths. In this specification, the promoter may be a constitutively active promoter, a repressive promoter, or an inductive promoter. When the promoter is an inductive promoter, the transcription rate increases in response to an inducer. In contrast, when the promoter is a constitutive promoter, the transcription rate is not controlled by an inducer. Repressive promoters are also known. Examples of promoters include, but are not limited to, constitutive promoters, weak heterologous promoters (e.g., endogenous promoters and / or promoters that result in lower expression than constitutive promoters), and inducible promoters. Further examples of promoters include the EF1α promoter, PGK promoter, MND promoter, KI promoter, Ki-67 gene promoter, and / or promoters that can be inducible by drugs such as tamoxifen and / or its metabolites. Commonly used constitutive promoters include, but are not limited to, SV40, CMV, UBC, EF1A, PGK, and / or CAGG, which are used in mammalian systems.

[0061] When the same coding sequence is expressed by a weak promoter and a strong promoter, the weak promoter results in lower mRNA expression than the strong promoter. This can be compared, for example, by analyzing it on an agarose gel. An example of a promoter regulated by adjacent chromatin is the short EF1α promoter, which is highly active at some loci but nearly inactive at others (Eyquem, J. et al. (2013). Biotechnol. Bioeng., 110(8):2225-2235).

[0062] A "transcriptional enhancer domain" is a short DNA region (50–1500 bp) to which a protein (activator) can bind, and which contains, but is not limited to, a DNA region to which the binding of the activator to the transcriptional enhancer domain can increase, promote, or enhance the transcription of a particular gene, or increase its transcription level. Such activator proteins are usually called transcription factors. Enhancers are typically cis-acting, located up to 1 Mbp (1,000,000 bp) away from the target gene, and are located upstream or downstream of the transcription start site, and are forward or reverse. Enhancers may be located upstream or downstream of the gene being regulated. In some embodiments, multiple enhancer domains may be used to increase the transcription level; for example, a multimerized activation-binding domain may be used to further enhance or increase the transcription level. Furthermore, since some researchers have found that enhancers are located upstream or downstream, hundreds of thousands of base pairs away from the transcription start site, it is not necessary for enhancers to be located near the transcription start site to influence transcription. Enhancers do not act on the promoter region itself, but bind to activator proteins. Activator proteins interact with the mediator complex, recruiting polymerase II and basal transcription factors to initiate gene transcription. Enhancers are also located within introns. The direction of the enhancer may be reversed, and this does not affect its function. Furthermore, enhancers may be cleaved or inserted at any location on the chromosome, and such treatments can still affect gene transcription. In some embodiments, enhancers are used to silence repressive mechanisms that inhibit the transcription of the FOXP3 gene. An example of an enhancer-binding domain is the TCRα enhancer. In some embodiments, the enhancer domain in the embodiments described herein is the TCRα enhancer. In some embodiments, the enhancer-binding domain is placed upstream of the promoter, thereby activating the promoter and increasing protein transcription.In some embodiments, the enhancer-binding domain is positioned upstream of the promoter, thereby activating the promoter and increasing the transcription of the FOXP3 gene.

[0063] The “transcriptional activation domain” includes, but is not limited to, a specific DNA sequence to which a transcription factor can bind, and which, by binding to the transcriptional activation domain, can control the rate of transcription of genetic information from DNA to messenger RNA. Specific examples of transcription factors include, but are not limited to, SP1, AP1, C / EBP, heat shock factors, ATF / CREB, c-Myc, Oct-1, and / or NF-1. In some embodiments, the activator domain is used to silence repressive mechanisms that block the transcription of the FOXP3 gene.

[0064] A “Ubiquitous chromatin opening element (UCOE)” includes, but is not limited to, an element featuring a dual promoter located on a non-methylated CpG island derived from a housekeeping gene and transcribed bidirectionally. UCOEs are a promising tool for preventing the silencing of introduced foreign genes and maintaining their expression in various cell models, such as cell lines, pluripotent hematopoietic stem cells, PSCs, and progeny cells differentiated therefrom. “Operatively linked” includes, but is not limited to, a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, thereby enabling the expression of the heterologous nucleic acid sequence. In some embodiments, a first molecule is linked to a second molecule, and these molecules are positioned such that the first molecule influences the function of the second molecule. These two molecules may be part of a contiguous single molecule or may be adjacent to each other. For example, if a promoter regulates the transcription of a transcribable DNA molecule of interest within a cell, the promoter is operationally linked to this transcribable DNA molecule.

[0065] In descriptions of molecules such as peptide fragments, the term "concentration" refers to the amount of molecules present in a given volume of solution, for example, the number of moles of molecules.

[0066] The terms “individual,” “subject,” and “host” are used interchangeably herein and refer to any subject for which diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient. In some embodiments, the subject has or is suspected of having a FOXP3-related disorder or health condition. In some embodiments, the subject is a human who, at the time of diagnosis or thereafter, has been diagnosed with a risk of a FOXP3-related disorder or health condition. In some cases, the diagnosis of being at risk of a FOXP3-related disorder or health condition may be determined based on the presence of one or more mutations in the endogenous gene encoding FOXP3 or in a nearby genomic sequence that may affect FOXP3 expression. For example, in some embodiments, the subject has or is suspected of having an autoimmune disorder and / or has one or more symptoms of an autoimmune disorder. In some embodiments, the subject is a human who, at the time of diagnosis or thereafter, has been diagnosed with a risk of an autoimmune disorder. In some cases, a diagnosis of being at risk of autoimmune disorder can be determined based on the presence of one or more mutations in the endogenous FOXP3 gene or in nearby genomic sequences that may affect the expression of the FOXP3 gene.

[0067] When the term “treatment” is used to refer to a disease or condition, it means that the symptoms associated with the condition in which the individual is suffering are at least alleviated, where “alleviation” is used in a broad sense to mean that the degree of a parameter (e.g., symptoms) associated with the condition being treated (e.g., an autoimmune disease) is at least reduced. Treatment also includes a state in which the pathological condition or at least its associated symptoms are completely suppressed, for example, a state in which the onset of the pathological condition or its symptoms is prevented, or a state in which the pathological condition or its symptoms are completely eliminated so that the host no longer suffers from the pathological condition or at least the symptoms that characterize the pathological condition. Treatment thus includes (i) prevention, i.e., prevention of the onset of clinical symptoms, such as reducing the risk of developing clinical symptoms, for example, prevention of disease progression, and (ii) suppression, i.e., prevention of the onset or further onset of clinical symptoms, for example, reduction or complete suppression of active disease.

[0068] In this specification, “effective dose,” “pharmaceutically effective dose,” and “therapeutic effective dose” mean an amount of a composition sufficient to provide the desired utility when administered to a subject having a particular condition. As used in descriptions relating to ex vivo treatment of autoimmune disorders, the term “effective dose” refers to the amount of a population of therapeutic cells or their progeny cells required to prevent or alleviate at least one sign or symptom of an autoimmune disorder, and relates to an amount of a composition containing therapeutic cells or their progeny cells sufficient to provide the desired effect, for example, to treat the symptoms of an autoimmune disorder in a subject. Therefore, the term “therapeutic effective dose” refers to an amount of therapeutic cells or a composition containing therapeutic cells sufficient to promote a particular effect when administered to a subject in need of treatment, such as a subject having or at risk of having an autoimmune disorder. Furthermore, “effective dose” may include an amount sufficient to prevent or delay the onset of disease symptoms, an amount sufficient to alter the course of disease symptoms (for example, an amount sufficient to slow the progression of disease symptoms, but not limited to that), or an amount sufficient to improve disease symptoms. In descriptions of in vivo treatment of autoimmune disorders in subjects (e.g., patients) or genome editing in in vitro cultured cells, “effective dose” refers to the amount of components used for genome editing, such as gRNA, donor templates, and / or site-specific polypeptides (e.g., DNA endonucleases), required to edit the genome of cells in subjects or in vitro cultured cells. In any case, an appropriate “effective dose” can be determined by a person skilled in the art simply by performing routine experiments.

[0069] "Autoimmune diseases" include, but are not limited to, conditions in which the immune system is abnormally underactive or excessively activated. When the immune system is excessively activated, the body's own tissues are attacked and damaged (autoimmune disease). In immunodeficiency diseases, the body's ability to fight off invaders is weakened, resulting in reduced resistance to infection. Examples of autoimmune disorders or autoimmune diseases include, but are not limited to, systemic lupus erythematosus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, muscle diseases, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatous disease, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion disorders, NF-κB essential modulator (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX, immune dysregulation, polyglandular endocrine disorders, intestinal diseases, X-linked (IPEX) syndromes and / or ataxia telangiectasia. Immune disorders can be analyzed, for example, by examining the profiles of neuronal-specific autoantibodies or other biomarkers if they are detected in the serum or cerebrospinal fluid of the subject. In some embodiments of the methods provided herein, these methods are methods for treating, alleviating, or suppressing autoimmune disorders. In some embodiments, autoimmune disorders are systemic lupus erythematosus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, muscle diseases, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatous disease, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion disorders, NF-κB essential modulator (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX, immune dysregulation, polyglandular endocrine disorders, intestinal diseases, X-linked (IPEX) syndromes and / or ataxia telangiectasia.

[0070] IPEX syndrome refers to a rare disorder characterized by immune dysregulation, polyglandular endocrine disorders, intestinal diseases, and X-linking, associated with dysfunction of FOXP3, widely considered a master regulator of regulatory T cell lineages. Individuals with IPEX syndrome may exhibit symptoms such as autoimmune intestinal disease, psoriatic or eczematous dermatitis, nail dystrophy, autoimmune endocrine disorders, and / or autoimmune skin diseases (such as alopecia generalis and / or bullous pemphigoid). IPEX is an autoimmune disease in which the immune system attacks the body's own tissues and organs. IPEX syndrome leads to a deficiency of CD4+CD25+ T regulatory cells and a deficiency in the expression of the transcription factor FOXP3. The decrease in FOXP3 is thought to result from the activation of T cells without checks, following the deficiency of regulatory T cells.

[0071] "Organ transplantation" includes, but is not limited to, replacing a recipient's damaged organ or transferring an organ that is not present in the recipient's body, for example, by moving an organ from one body to another, or moving an organ from a donor site within the patient's own body to another site. The transplantation of organs and / or tissues within the same person's body is called autologous transplantation. Transplantation between two subjects belonging to the same species, as has been done in recent years, is called allogeneic transplantation. In allogeneic transplantation, organs or tissues obtained from a living or deceased person are transplanted. Some embodiments described herein provide methods for treating, suppressing, or mitigating side effects of organ transplantation, such as organ rejection in a subject.

[0072] Transplantable organs include, for example, the heart, kidneys, liver, lungs, pancreas, intestines, and / or thymus. Transplantable tissues include, for example, bone and tendons (called musculoskeletal grafts), cornea, skin, heart valves, nerves, and / or veins. The kidneys, liver, and heart are the most commonly transplanted organs. The cornea and musculoskeletal grafts are the most commonly transplanted tissues.

[0073] In some embodiments described herein, methods are provided for treating, suppressing, or mitigating organ transplant side effects, such as organ rejection, in subjects. In some embodiments, the subjects are selected or identified as subjects for administration of one or more anti-rejection agents. In some embodiments, the anti-rejection agents include prednisone, Imuran (azathioprine), CellCept (mycophenolate mofetil (MMF)), Myfortic (mycophenolate), Rapamune (sirolimus), Neoral (cyclosporine), and / or Prograf (tacrolimus).

[0074] In some embodiments, the subject is selected to be subjected to suppression, mitigation, or treatment using the genetically modified cells described in the embodiments of the present invention. In some embodiments, the subject is a subject who has experienced one or more side effects to an anti-inflammatory drug or an anti-rejection drug. Accordingly, representative cells or compositions provided herein are provided to the selected subject. Side effects of anti-rejection drugs include increased or decreased blood tacrolimus levels due to interaction with other drugs, nephrotoxicity, hypertension, neurotoxicity (tremor, headache, tingling, and insomnia), diabetes (hyperglycemia), diarrhea, nausea, alopecia, and / or hyperkalemia. Accordingly, the subject is selected by clinical or diagnostic evaluation to perform the treatment, suppression, or mitigation method described herein.

[0075] In this specification, “organ rejection” or “transplant rejection” includes, but is not limited to, the rejection and destruction of transplanted tissue by the recipient’s immune system.

[0076] Graft-versus-host disease (GVHD) includes, but is not limited to, medical complications that occur after tissue transplantation from a genetically different human. While GVHD often occurs in association with stem cell transplantation or bone marrow transplantation, the term GVHD is also used to refer to complications from other forms of transplanted tissue. Immune cells in donor-provided tissue recognize the recipient as a foreign body rather than "self." In some embodiments of the present invention, the methods of the present invention can be used to prevent or mitigate complications resulting from GVHD.

[0077] "Pharmaceutical additives" include, for example, inert substances used to obtain a composition by adding cells, but are not limited to these.

[0078] In this specification, “chimeric antigen receptor (CAR)” includes, but is not limited to, artificial T cell receptors or recombinant receptors, also known as chimeric T cell receptors, which can be used to transfer desired specificity to effector immune cells. A CAR may be a synthetically designed receptor comprising a ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder, wherein the ligand-binding domain is linked via a spacer domain to one or more intracellular signaling domains (e.g., costimulatory domains) derived from a T cell receptor or other receptor. In some embodiments, cells (such as mammalian cells) containing a chimeric antigen receptor are created, comprising a nucleic acid encoding a fusion protein. Using the chimeric antigen receptor, for example, the specificity of a monoclonal antibody or its binding portion can be transferred to T cells. In some embodiments of the present invention, the recombinant cell further comprises a sequence encoding a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is specific to a molecule on tumor cells. Using recombinant cells expressing a T cell receptor or a chimeric antigen receptor, it is possible to target specific tissues that require FOXP3 expression. Some embodiments of the present invention include a method for providing and delivering FOXP3 by targeting a specific tissue. In some embodiments, the tissue is a transplanted tissue. In some embodiments, the chimeric antigen receptor is specific to a target molecule on the transplanted tissue.

[0079] As described herein, the genetically modified cells of the present invention are cells genetically modified to express FOXP3, and therefore, in embodiments of the present invention, these cells are referred to as "T reg These are also called "phenotypic" cells.

[0080] In this specification, “protein sequence” includes, for example, a polypeptide sequence of amino acids that constitutes the primary structure of a protein, but is not limited thereto. Furthermore, “upstream” in this specification means the 5' position on a polynucleotide and the position toward the N-terminus on a polypeptide. Similarly, “downstream” in this specification means the 3' position on a nucleotide and the position toward the C-terminus on a polypeptide. Therefore, “N-terminus” refers to the position or specific position of an element on a polynucleotide toward the N-terminus on a polypeptide.

[0081] A "functional equivalent" or "fragment of a functional equivalent" relating to a protein description may have one or more conserved amino acid substitutions. A "conserved amino acid substitution" refers to an amino acid substitution to another amino acid that has equivalent properties to the original amino acid. The conserved amino acid group is shown below. [Table 1]

[0082] Conservative substitutions may be introduced at any position in a given peptide or its fragment. However, non-conservative substitutions may be preferable, and in particular, it may be preferable to introduce non-conservative substitutions at any one or more positions, but this is not limited to these cases. Non-conservative substitutions capable of forming a functional equivalent fragment of the peptide may, for example, substantially differ in polarity, charge, and / or steric bulk, while maintaining the functionality of the derivative or variant fragment.

[0083] Sequence identity (%) is determined by comparing two sequences at their optimal alignment on the comparison window. Parts of the polynucleotide or polypeptide sequences on the comparison window may have additions or deletions (such as gaps) compared to a reference sequence (without additions or deletions) for the optimal alignment of the two sequences. In some cases, sequence identity (%) can be calculated by measuring the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions on the comparison window, and multiplying the result by 100.

[0084] "Identity" or "identity (%)" of two or more nucleic acid sequences or polypeptide sequences refers to two or more sequences or subsequences that are determined to be identical when compared and aligned to obtain the greatest match within a comparison window or specified region, either using one of the sequence comparison algorithms described below or by manual alignment and visual determination, or two or more sequences or subsequences that have a specific percentage of identical amino acid residues or nucleotides (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity in a specific region, e.g., the entire polypeptide sequence or individual domains of the polypeptide). Such sequences are referred to as "substantially identical." This definition also applies to complementary strands of test sequences.

[0085] In this specification, the terms “complementary” or “substantially complementary” are used interchangeably and mean that a particular nucleic acid (e.g., DNA or RNA) has a nucleotide sequence capable of linking to another nucleic acid via non-covalent bonds in a sequence-specific and antiparallel manner (e.g., a particular nucleic acid specifically links to a complementary nucleic acid), for example, to form Watson-Crick base pairs and / or G / U base pairs. As is known in the Art, standard Watson-Crick base pairings include thymidine (T) and adenine (A) pairings, uracil (U) and adenine (A) pairings, and cytosine (C) and guanine (G) pairings.

[0086] A DNA sequence that "codes" a specific RNA is a DNA nucleic acid sequence that can be transcribed into RNA. DNA polynucleotides may code for RNA that is translated into proteins (mRNA), or for RNA that is not translated into proteins (e.g., tRNA, rRNA, or guide RNA; also referred to herein as “non-coding” RNA or “ncRNA”). A “protein-coding sequence” or “sequence that codes for a specific protein or polypeptide” is a nucleic acid sequence that, when controlled by an appropriate regulatory sequence in vitro or in vivo, is transcribed into mRNA (in the case of DNA) and translated into polypeptides (in the case of mRNA).

[0087] In this specification, “codon” refers to a single genetic coding unit in a DNA or RNA molecule, formed by a sequence of three nucleotides. In this specification, “codon degeneracy” refers to a genetic coding property that allows for changes in the nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide.

[0088] "Codon-optimized" or "codon optimization" refers to the modification of codons in the gene or coding region of a nucleic acid molecule to reflect codons typically used in the host organism without altering the polypeptide encoded by the DNA, for the purpose of transforming various hosts. Such optimizations include replacing at least one codon, multiple codons, or a very large number of codons with one or more codons that are frequently used in the organism's genes. Tables showing codon usage frequencies are readily available, for example, from the "Codon Usage Database" available at www.kazusa.or.jp / codon / (visited March 20, 2019). Those skilled in the art can use their knowledge of codon usage or codon preference in each organism to apply codon frequencies to a given polypeptide sequence, thereby encoding a polypeptide and producing nucleic acid fragments of a codon-optimized coding region that use codons optimally suited to a given species. Codon-optimized coding regions can be designed by various methods known to those skilled in the art.

[0089] For example, the terms “recombinant” or “engineered” used in reference to cells, nucleic acids, proteins, or vectors indicate that the cell, nucleic acid, protein, or vector has been modified by laboratory methods or is obtained as a result of laboratory methods. Therefore, for example, recombinant or engineered proteins include proteins produced by laboratory methods. Recombinant or engineered proteins may contain amino acid residues not found in natural (non-recombinant or wild-type) proteins, or may contain modified (e.g., labeled) amino acid residues. The terms “recombinant” or “engineered” may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include chemical modifications of a peptide, protein, or nucleic acid sequence (including one or more amino acids, deoxyribonucleotides, or ribonucleotides); addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; and addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence.

[0090] "Genome DNA" or "genome sequence" refers to the genomic DNA of an organism, including but not limited to the genomic DNA of bacteria, fungi, archaea, plants, or animals.

[0091] In this specification, "transgene," "exogenous gene," or "exogenous sequence" relating to nucleic acids refers to a nucleic acid sequence or gene that does not exist in the cell's genome but has been artificially introduced into the genome, for example, through genome editing.

[0092] In this specification, “endogenous gene” or “endogenous sequence” with respect to nucleic acids refers to a nucleic acid sequence or gene that is naturally present in the cell genome without being introduced through artificial means.

[0093] In this specification, the terms “expression” or “protein expression” refer to the translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological characteristics, and by quantitative or qualitative indicators. In some embodiments, the protein or the plurality of proteins are expressed in a configuration that allows for dimerization in the presence of a ligand.

[0094] In this specification, a “fusion protein” or “chimeric protein” is a protein created by ligating two or more genes that originally encoded separate proteins or parts of separate proteins. Fusion proteins can also be created from specific protein domains derived from two or more separate proteins. By translating such a fusion gene, a single polypeptide or a group of polypeptides with functional properties derived from each of the original proteins can be obtained. Recombinant fusion proteins can be artificially created by recombinant DNA technology used in biological research or therapy. Methods for creating such fusion proteins are known to those skilled in the art. Some fusion proteins are combinations of entire peptides and therefore may contain all domains of the original proteins, and in particular all functional domains. However, other fusion proteins, especially those not found in nature, are combinations of only parts of coding sequences and therefore do not retain the original function of the parent genes from which such proteins are derived.

[0095] A “vector,” “expression vector,” or “construct” is a nucleic acid used to introduce heterologous nucleic acids into cells, and can express heterologous nucleic acids in cells by containing various regulatory elements. Examples of vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some embodiments, the vector is a plasmid, minicircle, yeast, or viral genome. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a vector for protein expression in a bacterial system such as Escherichia coli. In this specification, “expression” or “protein expression” refers to the translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological characteristics, and by quantitative or qualitative indicators. In some embodiments, the protein or plurality of proteins are expressed in a configuration that allows for dimerization in the presence of a ligand. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is an adeno-associated virus (AAV) vector (for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, etc., and not limited to these).

[0096] In this specification, “fusion protein” or “chimeric protein” includes, but is not limited to, a protein created by ligating two or more genes that originally encoded separate proteins or portions of separate proteins. Fusion proteins can also be created from specific protein domains derived from two or more separate proteins. By translating such a fusion gene, a single polypeptide or a group of polypeptides with functional properties derived from each of the original proteins can be obtained. Recombinant fusion proteins can be artificially created by recombinant DNA technology used in biological research or therapy. Methods for creating such fusion proteins are known to those skilled in the art. Some fusion proteins are combinations of multiple whole peptides and therefore may contain all domains of the original proteins, and in particular all functional domains. However, other fusion proteins, especially those not found in nature, combine only portions of coding sequences and therefore do not retain the original function of the parental genes from which such proteins are derived. In some embodiments, we provide fusion proteins containing interferon and / or PD-1 protein or both.

[0097] A "conditional" or "inducible" promoter refers to a nucleic acid construct that contains a promoter that expresses a gene in the presence of an inducer, but substantially prevents gene expression in the absence of the inducer.

[0098] In this specification, "constitutive" refers to a nucleic acid construct that expresses a continuously produced polypeptide, as it contains a constitutive promoter.

[0099] In some embodiments, the inducible promoter exhibits low basal-level activity. In some embodiments, when using a lentiviral vector, the basal-level activity in cells where expression is not induced is a percentage within the range defined by 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less (but not 0%) of the activity when gene expression is induced in the cell, or any two of these values. Basal-level activity can be determined by measuring the expression level of the transgene (e.g., a marker gene) in the absence of an inducer (e.g., a drug) using flow cytometry. In some embodiments described herein, expression is measured using a marker protein such as Akt.

[0100] In some embodiments, when the inducible promoter is expressed, it can induce higher activity compared to when expression is not induced or at the basal level. In some embodiments, the activity level when expression is induced is 2, 4, 6, 8, 9, 10 or more times higher than when expression is not induced, or within a range defined by any two of these values. In some embodiments, the transgene under the control of the inducible promoter is off for a period defined by less than 10 days, less than 8 days, less than 6 days, less than 4 days, less than 2 days, or less than 1 day, or any two of these periods, in the absence of a transactivator, but is not off for 0 days.

[0101] In some embodiments, the inducible promoter is designed and / or modified to have low activity at the basal level, induce high levels of expression, and / or be switchable on and off for a short period of time.

[0102] In this specification, “dimeric chemical-induced signaling complex,” “dimeric CISC,” or “dimer” refers to two components that make up a CISC, which may or may not bind to each other to form a fusion protein complex. “Dimerization” refers to the process by which two separate entities bind to each other to form a single entity. In some embodiments, dimerization is stimulated by a ligand or drug. In some embodiments, “dimerization” refers to homodimerization, i.e., the binding and dimerization of two identical entities, for example, the binding and dimerization of two identical CISC components. In some embodiments, “dimerization” refers to heterodimerization, i.e., the binding and dimerization of two different entities, for example, the binding and dimerization of two different separate CISC components. In some embodiments, the dimerization of CISC components forms a cellular signaling pathway. In some embodiments, dimerization of CISC components enables selective expansion and proliferation of cells or cell populations. Further CISC systems may include CISC-gibberellin-based CISC dimerization systems or CISC-TMP-based CISC dimerization systems. Other chemically derivable dimerization (CID) systems and their components may also be used.

[0103] In this specification, “chemical-induced signaling complex” or “CISC” refers to a recombinant complex that initiates a signal within a cell and, as a direct result, undergoes dimerization upon ligand induction. A CISC may be a homodimer (a dimer of two identical components) or a heterodimer (a dimer of two different components). Therefore, in this specification, the term “homodimer” refers to a dimer consisting of two protein components described herein that have the same amino acid sequence. The term “heterodimer” refers to a dimer consisting of two protein components described herein that do not have the same amino acid sequence.

[0104] CISCs may be synthetic complexes, as described in further detail herein. "Synthetic" herein refers to complexes, proteins, dimers, or compositions described herein that are not natural and are not found in nature. In some embodiments, "IL2R-CISC" refers to a signaling complex comprising components of the interleukin-2 receptor. In some embodiments, "IL2 / 15-CISC" refers to a signaling complex comprising receptor signaling subunits shared by interleukin-2 and / or interleukin-15. In some embodiments, "IL7-CISC" refers to a signaling complex comprising components of the interleukin-7 receptor. Thus, CISCs may be named according to the constituent parts that constitute a particular CISC. Those skilled in the art will recognize that constituent parts of chemically induced signaling complexes may consist of natural or synthetic components useful for incorporation into CISCs. Therefore, these examples provided herein do not limit the invention.

[0105] CISC (chemically induced signaling complex) is a multi-component synthetic protein complex configured to be co-expressed as two chimeric proteins in host cells, as described in international patent application PCT / US2017 / 065746 (this document is incorporated herein by reference). The two chimeric protein components of CISC are one extracellular domain constituting half of the rapamycin-binding complex, which is fused to the intracellular signaling complex constituting the other half of the rapamycin-binding complex. By delivering the nucleic acid encoding CISC to a host cell, intracellular signaling can be regulated in the presence of rapamycin or rapamycin-related compounds.

[0106] In this specification, “cytokine receptor” refers to a receptor molecule that recognizes and binds to a cytokine. In some embodiments, cytokine receptors include modified cytokine receptor molecules (e.g., “cytokine receptor variants”) which involve substitutions, deletions, and / or additions to the amino acid sequence and / or nucleic acid sequence of the cytokine receptor. Thus, the term “cytokine receptor” is intended to include not only wild-type cytokine receptors but also recombinant cytokine receptors, synthetic cytokine receptors, and cytokine receptor variants. In some embodiments, the cytokine receptor is a fusion protein comprising an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain. In some embodiments, the components of the receptor (i.e., each domain of the receptor) are either native or synthetic. In some embodiments, each domain is of human origin.

[0107] In this specification, "FKBP" refers to the FK506-binding protein domain. FKBP refers to a family of proteins possessing prolyl isomerase activity, which are functionally related to cyclophyllin but not similar in terms of amino acid sequence. FKBP has been identified in many eukaryotes, from yeast to humans, and functions as a protein folding chaperone for proteins containing proline residues. FKBP belongs to the immunophilin family along with cyclophyllin. FKBP includes, for example, FKBP12, as well as proteins encoded by the AIP gene, AIPL1 gene, FKBP1A gene, FKBP1B gene, FKBP2 gene, FKBP3 gene, FKBP5 gene, FKBP6 gene, FKBP7 gene, FKBP8 gene, FKBP9 gene, FKBP9L gene, FKBP10 gene, FKBP11 gene, FKBP14 gene, FKBP15 gene, FKBP52 gene, and / or LOC541473 gene; including their homologs and functional protein fragments.

[0108] In this specification, “FRB” refers to the FKBP rapamycin-binding domain. The FRB domain is a polypeptide region (protein “domain”) configured to form a ternary complex with the FKBP protein and rapamycin or its rapalog. FRB domains are present in a variety of natural proteins, including mTOR proteins from humans and other species (also referred to herein as FRAP, RAPT1, or RAFT); yeast proteins containing Tor1 and / or Tor2; and / or FRAP homologs of the genus Candida. Both FKBP and FRB are major components of mammalian target of rapamycin (mTOR) signaling.

[0109] "Naked FKBP rapamycin-binding domain polypeptide" or "naked FRB domain polypeptide" refers to a polypeptide containing only the amino acid sequence of the FRB domain, or a protein in which 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the protein's amino acid sequence consists of the FRB domain's amino acid sequence. The FRB domain can be expressed as a 12kDa soluble protein (Chen, J. et al. (1995). Proc. Natl. Acad. Sci. USA, 92(11):4947-4951). The FRB domain forms a 4-helix bundle, which is a structural motif commonly found in globular proteins. The overall dimensions of the FRB domain are 30 Å × 45 Å × 30 Å, and the four helices are connected by short lower loops similar to the folding of cytochrome b562 (Choi, J. et al. (1996). Science, 273(5272):239-242). In some embodiments, the naked FRB domain contains the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71.

[0110] Cereblon interacts with damaged DNA-binding protein 1 and, together with Cullin4, forms an E3 ubiquitin ligase complex. This E3 ubiquitin ligase complex functions as a substrate receptor, and proteins recognized by cereblon can be ubiquitinated and degraded by the proteasome. Proteasome-mediated degradation of unnecessary or damaged proteins plays a crucial role in maintaining normal cellular function (e.g., cell survival, proliferation, and / or growth). Binding of immunomodulatory imides (IMIDs) (e.g., thalidomide) to cereblon is associated with teratogenicity, and cytotoxicity of IMIDs such as lenalidomide is also observed. Cereblon plays a central role in the binding, ubiquitination, and degradation of factors involved in maintaining myeloma cell function.

[0111] The “thalidomide-binding domain of cereblon” refers to an extracellular binding domain that interacts with IMIDs such as thalidomide, pomalidomide, lenalidomide, apremilast, or related analogues thereof. Some of the embodiments provided herein utilize analogues or variants of the thalidomide-binding domain of cereblon. In some embodiments, these extracellular binding domains are configured to bind IMID ligands simultaneously.

[0112] In some embodiments, the immunomodulatory imide drugs used in the methods described herein may include thalidomide (including its analogues, derivatives and / or pharmaceutically acceptable salts). Examples of thalidomide include Immunoprin, Salomid, Talidex, Talizer, Neurosedyn, α-(N-phthalimido)glutarimide, 2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione); or pomalidomide (including its analogues, derivatives and / or pharmaceutically acceptable salts). Examples of pomalidomide include pomalist, Imnovid, (RS)-4-amino-2-(2,6-dioxopiperidine-3-yl)isoindole-1,3-dione; or lenalidomide (including its analogues, derivatives and / or pharmaceutically acceptable salts). Examples of lenalidomides include revlimide, (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindole-2-yl)piperidine-2,6-dione; or apremilast (including its analogues, derivatives and / or pharmaceutically acceptable salts). Examples of apremilast include otezla, CC-10004, N-{2-[(1S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl}acetamide); or any combination thereof.

[0113] As used herein, the “extracellular binding domain” refers to one of the domains constituting a complex, configured to bind to a specific atom or molecule, and located outside the cell. In some embodiments, the extracellular binding domain of a CISC is the FKBP domain or a portion thereof. In some embodiments, the extracellular binding domain is the FRB domain or a portion thereof. In some embodiments, the extracellular binding domain is configured to stimulate the dimerization of two CISC components by binding to a ligand or drug. In some embodiments, the extracellular binding domain is configured to bind to a cytokine receptor modulator.

[0114] In this specification, “cytokine receptor modulator” refers to an agent that modulates the phosphorylation of downstream targets of cytokine receptors, the activation of signaling pathways associated with cytokine receptors, and / or the expression of specific proteins such as cytokines. Such agents may directly or indirectly modulate the phosphorylation of downstream targets of cytokine receptors, the activation of signaling pathways associated with cytokine receptors, and / or the expression of specific proteins such as cytokines. Examples of cytokine receptor modulators include, but are not limited to, cytokines; cytokine fragments; fusion proteins; and / or antibodies or their binding sites that immune-specifically bind to cytokine receptors or fragments thereof. Furthermore, examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides (e.g., soluble cytokine receptors), fusion proteins, and / or antibodies or their binding sites that immune-specifically bind to cytokines or fragments thereof.

[0115] In this specification, “activate” means the enhancement of at least one biological activity of the protein of interest. Similarly, “activation” means the state of the protein of interest in which the activity is enhanced. “Activatable” means that the protein of interest is activatable in the presence of a signal, drug, ligand, compound, or stimulus. In some embodiments, the dimers described herein are activated in the presence of a signal, drug, ligand, compound, or stimulus to become a signal-signaling dimer. In this specification, “signal-signaling” means the ability of a dimer or its configuration to initiate or sustain a downstream signaling pathway.

[0116] In this specification, “hinge domain” refers to a domain that may ligate an extracellular binding domain to a transmembrane domain, thereby conferring flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain closer to the cell membrane, minimizing the possibility of recognition by antibodies or their binding fragments. In some embodiments, the extracellular binding domain is located at the N-terminus of the hinge domain. In some embodiments, the hinge domain may be naturally occurring or synthetic.

[0117] In this specification, “transmembrane domain” or “TM domain” refers to a domain that is stable within a membrane, such as a cell membrane. The terms “transmembrane span,” “membrane endogenous protein,” and “membrane endogenous domain” are also used herein. In some embodiments, the hinge domain and extracellular domain are located at the N-terminus of the transmembrane domain. In some embodiments, the transmembrane domain is a native or synthetic domain. In some embodiments, the transmembrane domain is the IL-2 transmembrane domain.

[0118] In this specification, “signaling domain” refers to a domain of a fusion protein or a component of a CISC involved in a signaling cascade within a cell (such as a mammalian cell). “Signaling domain” refers to a signaling portion that provides a signal to a cell (such as a T cell) that mediates a cellular response (such as a T cell response), in addition to a primary signal provided by the CD3ζ chain of the TCR / CD3 complex, such as activation, proliferation, differentiation, and / or cytokine secretion. In some embodiments, the signaling domain is located at the N-terminal end of a transmembrane domain, hinge domain, and extracellular domain. In some embodiments, the signaling domain is a synthetic or native domain. In some embodiments, the signaling domain is a linked intracellular signaling domain. In some embodiments, the signaling domain is a cytokine signaling domain. In some embodiments, the signaling domain is an antigen signaling domain. In some embodiments, the signaling domain is an interleukin-2 receptor γ subunit (IL2Rγ or IL2Rg) domain. In some embodiments, the signaling domain is an interleukin-2 receptor β-subunit (IL2Rβ or IL2Rb) domain. In some embodiments, binding of a drug or ligand to the extracellular binding domain causes dimerization of the CISC components, resulting in activation of the signaling pathway and signaling via the signaling domain. In this specification, "signaling" refers to the activation of the signaling pathway by binding of a ligand or drug to the extracellular domain. Binding of the ligand or drug to the extracellular domain causes dimerization of the CISC components and activation of the signal.

[0119] In this specification, “IL2Rb” or “IL2Rβ” refers to the interleukin-2 receptor β subunit. Similarly, “IL2Rg” or “IL2Rγ” refers to the interleukin-2 receptor γ subunit, and “IL2Ra” or “IL2Rα” refers to the interleukin-2 receptor α subunit. The IL-2 receptor has three forms: α-chain, β-chain, and γ-chain, which are also subunits of other cytokine receptors. IL2Rβ and IL2Rγ are members of the type I cytokine receptor family. In this specification, “IL2R” refers to the interleukin-2 receptor, which is involved in T cell-mediated immune responses. IL2R is involved in receptor-dependent endocytosis and the transmission of pro-mitotic signals from interleukin-2. Similarly, "IL-2 / 15R" refers to the receptor signaling subunit shared by IL-2 and IL-15, which may include an α subunit (IL2 / 15Ra or IL2 / 15Rα), a β subunit (IL2 / 15Rb or IL2 / 15Rβ), or a γ subunit (IL2 / 15Rg or IL2 / 15Rγ).

[0120] In some embodiments, the chemically induced signaling complex is a heterodimerization-activated signaling complex comprising two components. In some embodiments, the first component comprises an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracellular signaling domains, which are one of the heterodimerization pairs. In some embodiments, the second component comprises an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracellular signaling domains, which are the other of the heterodimerization pairs. Thus, in some embodiments, two recombination events occur. In some embodiments, these two CISC components are expressed in cells such as mammalian cells. In some embodiments, cells such as mammalian cells, or a population of cells such as a mammalian cell population, are brought into contact with a ligand or factor that induces heterodimerization, thereby initiating signaling. In some embodiments, the homodimerization pair dimerizes, thereby expressing a single CISC component in cells such as mammalian cells, and the homodimerized CISC component initiates signaling.

[0121] In this specification, “ligand” or “drug” refers to a molecule having a desired biological effect. In some embodiments, an extracellular binding domain recognizes and binds to the ligand, forming a ternary complex comprising the ligand and two binding CISC components. Ligands include, but are not limited to, proteinaceous molecules such as peptides, polypeptides, proteins, post-translationally modified proteins, antibodies, and their binding moieties; small molecules (less than 1000 daltons), inorganic or organic compounds; and nucleic acid molecules such as, but are not limited to, double-stranded or single-stranded DNA, double-stranded or single-stranded RNA (e.g., antisense RNA, RNAi, etc.), aptamers, and triple-helical nucleic acid molecules. Ligands may originate from, be obtained from, or be derived from, or be obtained from, synthetic molecular libraries. In some embodiments, the ligand is a protein, an antibody or a part thereof, a small molecule, or a drug. In some embodiments, the ligand is rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog includes variants of rapamycin in which one or more modifications have been made to rapamycin, including demethylation, removal or substitution of methoxy at positions C7, C42 and / or C29; removal, derivatization or substitution of hydroxyl at positions C13, C43 and / or C28; reduction, removal or derivatization of ketone at positions C14, C24 and / or C30; substitution of a 6-membered pipecolate ring with a 5-membered prolyl ring; and another substitution on the cyclohexyl ring or substitution of the cyclohexyl ring with a substituted cyclopentyl ring.In some embodiments, the rapalog is everolimus, merilimus, novolimus, pimecrolimus, ridaflorimus, tacrolimus, temsirolimus, umilolimus, zotarolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, AP21967, sodium mycophenolate, benidipine hydrochloride, AP23573 or AP1903, or any metabolite, derivative and / or combination thereof. In some embodiments, the ligand is an IMID drug (e.g., thalidomide, pomalidomide, lenalidomide or related analogues).

[0122] In this specification, "simultaneous binding" refers to the simultaneous binding of two or more CISC components to a ligand, and in some cases, the binding of two or more CISC components to a ligand substantially simultaneously, forming a complex consisting of multiple components including CISC components and ligand components, resulting in signal activation. For simultaneous binding to occur, the CISC components must be configured to spatially bind to a single ligand, and both CISC components must be configured to bind to the same ligand (or different parts thereof).

[0123] In this specification, “selective expansion and proliferation” refers to the ability to expand and proliferate a desired cell, such as mammalian cells, or a desired cell population, such as a mammalian cell population. In some embodiments, selective expansion and proliferation refers to the development or expansion of a population of pure cells (such as mammalian cells) in which two genes have been recombined. One component of a dimerized CISC is involved in one gene recombination, and the other component is involved in the other gene recombination. Thus, each component of a heterodimerized CISC is associated with each gene recombination. By exposing cells to a ligand, it becomes possible to selectively expand and proliferate only cells (such as mammalian cells) that have both desired modifications. Thus, in some embodiments, the only cells (e.g., mammalian cells) that can respond to contact with the ligand are those that express both components of the heterodimerized CISC.

[0124] In some embodiments, rapamycin (including its analogues, derivatives and pharmaceutically acceptable salts) is used as a ligand or agent in the methods herein for the chemical induction of signaling complexes. Examples of rapamycin include sirolimus, rapamune, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[ Examples include (1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxazacyclohenthriacontin-1,5,11,28,29(4H,6H,31H)-pentone). Also, everolimus is an example, and everolimus includes its analogs, derivatives and pharmaceutically acceptable salts. Everolimus includes RAD001, Zortress, Certican, Afinitol, Votubia, 42-O-(2-hydroxyethyl)rapamycin, (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0 4 , 9Examples include hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone. Also, merilimus is mentioned, which includes its analogs, derivatives, and pharmaceutically acceptable salts. Examples of Merilimus include SAR943, 42-O-(tetrahydrofuran-3-yl)rapamycin (Merilimus-1); 42-O-(oxetan-3-yl)rapamycin (Merilimus-2); 42-O-(tetrahydropyran-3-yl)rapamycin (Merilimus-3); 42-O-(4-methyl,tetrahydrofuran-3-yl)rapamycin; 42-O-(2,5,5-trimethyl,tetrahydrofuran-3-yl)rapamycin; 42-O-(2,5-diethyl-2-methyl,tetrahydrofuran-3-yl)rapamycin; 42-O-(2H-pyran-3-yl,tetrahydro-6-methoxy-2-methyl)rapamycin; or 42-O-(2H-pyran-3-yl,tetrahydro-2,2-dimethyl-6-phenyl)rapamycin). Furthermore, nobolimus is mentioned, including its analogs, derivatives, and pharmaceutically acceptable salts. An example of nobolimus is 16-O-demethylrapamycin. Also mentioned is pimecrolimus, including its analogs, derivatives, and pharmaceutically acceptable salts. Examples of pimecrolimus include Elidel, (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-3-((E)-2-((1R,3R,4S)-4-chloro-3-methoxycyclohexyl)-1-methylvinyl)-8-ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24,26,26a-hexadecahydro-5,19-epoxy-3H-pyrido(2,1-c)(1,4)oxazacyclotricosin-1,17,20,21(4H,23H)-tetron, and 33-epi-chloro-33-desoxiascomycin. Also mentioned is lidahororimus, which includes its analogs, derivatives, and pharmaceutically acceptable salts.As for ridaforolimus, AP23573, MK-8669, dehorolimus, (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-12-((1R)-2-((1S,3R,4R)-4-((dimethylphosphinoyl)oxy)-3-methoxycyclohexyl)-1-methylethyl)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo(30.3.1.0. 4 , 9Examples of tacrolimus include hexatriaconta-16,24,26,28-tetraen-2,3,10,14,20-pentone. Also, tacrolimus is mentioned, and includes its analogues, derivatives, and pharmaceutically acceptable salts. Examples of tacrolimus include FK-506, fujimacin, Prograf, Advagraf, Protopic, 3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro Examples include -5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxazacyclotricosin-1,7,20,21(4H,23H)-tetron monohydrate. Also, temsirolimus is an example, including its analogs, derivatives, and pharmaceutically acceptable salts. Temsirolimus includes CCI-779, CCL-779, Torisel, (1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1, Examples include 5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentricontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate. Also, umilolimus is mentioned, including its analogs, derivatives and pharmaceutically acceptable salts.Examples of umilolimus include Biolimus, Biolimus A9, BA9, TRM-986, and 42-O-(2-ethoxyethyl)rapamycin. Zotarolimus is also included, encompassing its analogues, derivatives, and pharmaceutically acceptable salts. Examples of zotarolimus include ABT-578 and (42S)-42-deoxy-42-(1H-tetrazole-1-yl)-rapamycin. C20-metharylrapamycin is also included, encompassing its analogues, derivatives, and pharmaceutically acceptable salts. An example of C20-metharylrapamycin is C20-Marap. C16-(S)-3-methylindolerapamycin is also included, encompassing its analogues, derivatives, and pharmaceutically acceptable salts. Examples of C16-(S)-3-methylindolerapamycin include C16-iRap. Also, AP21967 is an example, and AP21967 includes its analogues, derivatives, and pharmaceutically acceptable salts. Examples of AP21967 include C-16-(S)-7-methylindolerapamycin. Also, sodium mycophenolate is an example, and sodium mycophenolate includes its analogues, derivatives, and pharmaceutically acceptable salts. Examples of sodium mycophenolate include CellCept, Myfortic, and (4E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhexa-4-enoic acid. Also, benidipine hydrochloride is an example, and benidipine hydrochloride includes its analogues, derivatives, and pharmaceutically acceptable salts. Examples of benidipine hydrochloride include Benidipinum and Coniel. Also mentioned is AP1903, which includes its analogs, derivatives, and pharmaceutically acceptable salts.Examples of AP1903 include rimiducid, [(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-[[2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-oxoethoxy]phenyl]propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate. Furthermore, any combination of these can be listed.

[0125] In this specification, "gibberellin" refers to the synthetic or natural form of a diterpenoid acid that has been synthesized in the terpenoid pathway in plastids and then modified in the endoplasmic reticulum and cytosol to become biologically active. Gibberellins may be natural gibberellins or their analogs, such as gibberellins derived from the ent-gibberellane skeleton or gibberellins synthesized via ent-kaurene, including gibberellin 1 (GA1), GA2, GA3, ..., GA136, and their analogs and derivatives. In some embodiments, gibberellins or their analogs or derivatives are used for the dimerization of CISCs.

[0126] In this specification, “SLF-TMP” or “synthetic FKBP ligand bound to trimethoprim” refers to a dimerizing agent used for CISC dimerization. In some embodiments, the SLF portion binds to a first CISC component and the TMP portion binds to a second CISC component, thereby causing CISC dimerization. In some embodiments, SLF can bind to, for example, FKBP, and TMP can bind to dihydrofolate reductase (eDHFR) derived from Escherichia coli.

[0127] In this specification, "simultaneous binding" refers to the simultaneous binding of two or more CISC components to a ligand, and in some cases, the binding of two or more CISC components to a ligand substantially simultaneously, forming a complex consisting of multiple components including CISC components and ligand components, resulting in signal activation. For simultaneous binding to occur, the CISC components must be configured to spatially bind to a single ligand, and both CISC components must be configured to bind to the same ligand (or different parts thereof).

[0128] In this specification, “selective expansion and proliferation” refers to the ability to expand and proliferate a desired cell, such as mammalian cells, or a desired cell population, such as a mammalian cell population. In some embodiments, selective expansion and proliferation refers to the development or expansion of a population of pure cells (such as mammalian cells) in which two genes have been recombined. One component of a dimerized CISC is involved in one gene recombination, and the other component is involved in the other gene recombination. Thus, each component of a heterodimerized CISC is associated with each gene recombination. By exposing cells to a ligand, it becomes possible to selectively expand and proliferate only cells (such as mammalian cells) that have both desired modifications. Thus, in some embodiments, the only cells (e.g., mammalian cells) that can respond to contact with the ligand are those that express both components of the heterodimerized CISC.

[0129] In this specification, “host cell” includes any type of cell (e.g., mammalian cell) that is sensitive to transformation, transfection, or transduction by a nucleic acid construct or vector. In some embodiments, the host cell, such as a mammalian cell, is a T cell or a regulatory T cell (T reg) In some embodiments, the host cell, such as a mammalian cell, is a hematopoietic stem cell. In some embodiments, the host cell is a CD34+ cell, a CD8+ cell, or a CD4+ cell. In some embodiments, the host cell is a CD8+ cytotoxic T lymphocyte selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the host cell is a CD4+ helper T lymphocyte selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In this specification, “cell population” refers to a group of cells (such as a group of mammalian cells) that includes two or more types of cells. In some embodiments, cells (such as mammalian cells) containing the protein sequence described herein or an expression vector encoding the protein sequence are produced.

[0130] In this specification, “transformed” or “transfected” means a cell (such as a mammalian cell), tissue, organ, or organism into which a foreign polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule may be incorporated into the genomic DNA of the recipient cell (such as a mammalian cell), tissue, organ, or organism, thereby allowing the introduced polynucleotide molecule to be passed on to subsequent offspring. Furthermore, “transgenic” cells (such as mammalian cells) or “transgenic” organisms, or “transfected” cells (such as mammalian cells) or “transfected” organisms include offspring of transgenic cells or organisms or transfected cells or organisms, including offspring produced by breeding programs that use such transgenic organisms as parents in mating, and which exhibit altered phenotypes resulting from the presence of the foreign polynucleotide molecule. “Transgenic” also means bacteria, fungi, or plants containing one or more heterologous polynucleotide molecules. “Transduction” means the transfer of genes into cells, such as mammalian cells, via viruses.

[0131] In this specification, “subject” refers to an animal that is the subject of treatment, observation, or experimentation. “Animals” include ectothermic vertebrates, warm-blooded vertebrates, and invertebrates such as fish, crustaceans, and reptiles, in particular mammals. “Mammals” include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates (such as monkeys, chimpanzees, and apes), in particular humans. In some embodiments, the subject is a human.

[0132] In some embodiments, the effective amount of ligand used to induce dimerization is 0.01nM, 0.02nM, 0.03nM, 0.04nM, 0.05nM, 0.06nM, 0.07nM, 0.08nM, 0.09nM, 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, 1.0nM, 1.5nM, 2.0nM, 2.5nM, 3.0nM, 3.5nM, 4.0nM, 4.5nM, 5.0nM. The concentration is within the range defined by 5.5nM, 6.0nM, 6.5nM, 7.0nM, 7.5nM, 8.0nM, 8.5nM, 9.0nM, 9.5nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, or 100nM, or any two of these values.

[0133] The “marker sequences” described herein encode cells containing the target protein (such as mammalian cells) or proteins used to select or track the target protein. Embodiments described herein provide fusion proteins which may contain marker sequences that can be selected in experiments such as flow cytometry.

[0134] In this specification, "cytotoxic T lymphocytes (CTLs)" refers to T lymphocytes that express CD8 on their cell surface (e.g., CD8 +This refers to T cells. In some embodiments, such cells are "memory" T cells (T cells) that have previously experienced an antigen. M Preferably, the cells are cytotoxic T lymphocytes. In some embodiments, cells for secreting fusion proteins are provided. In some embodiments, the cells are cytotoxic T lymphocytes. In this specification, "central memory" T cells (or "T") are used. CM A central memory T cell (T) is a cytotoxic T lymphocyte (CTL) that has experienced an antigen and, compared to naive cells, expresses CD62L, CCR-7, and / or CD45RO on its surface, but does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells are provided for secreting a fusion protein. In some embodiments, the cells are central memory T cells (T). CM In some embodiments, central memory cells may be positive for the expression of CD62L, CCR7, CD28, CD127, CD45RO, and / or CD95, but have reduced expression of CD54RA, compared to naive cells. In this specification, “effector memory” T cells (or “T EM A T cell is an antigen-experienced T cell that, compared to a central memory cell, does not express CD62L on its surface or has reduced CD62L expression, and compared to a naive cell, does not express CD45RA or has reduced CD45RA expression. In some embodiments, cells for secreting a fusion protein are provided. In some embodiments, the cells are effector memory T cells. In some embodiments, the effector memory cells may be negative for CD62L and / or CCR7 expression and positive or negative for CD28 and / or CD45RA expression compared to a naive cell or a central memory cell.

[0135] As described herein, “naive” T cells are T lymphocytes that have not experienced an antigen and, compared to central memory cells or effector memory cells, express CD62L and / or CD45RA and do not express CD45RO. In some embodiments, cells (such as mammalian cells) for secreting fusion proteins are provided. In some embodiments, the cells (such as mammalian cells) are naive T cells. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers, such as CD62L, CCR7, CD28, CD127, and / or CD45RA.

[0136] The “effector” T cells described herein are cytotoxic T lymphocytes that have experienced an antigen and, compared to central memory T cells or naive T cells, do not express CD62L, CCR7, and / or CD28, or have reduced expression of CD62L, CCR7, and / or CD28, and are granzyme B positive and / or perforin positive. In some embodiments, cells (such as mammalian cells) for secreting fusion proteins are provided. In some embodiments, the cells (such as mammalian cells) are effector T cells. In some embodiments, the cells (such as mammalian cells) do not express CD62L, CCR7, and / or CD28, or have reduced expression of CD62L, CCR7, and / or CD28, and are granzyme B positive and / or perforin positive, compared to central memory T cells or naive T cells.

[0137] As used herein, “epitope” refers to a portion of an antigen or molecule recognized by the immune system, including antibodies, T cells, and / or B cells. An epitope typically has at least seven amino acids and may be a linear epitope or a higher-order structural epitope. In some embodiments, cells expressing a fusion protein (such as mammalian cells) are provided, which further include a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor includes an scFv capable of recognizing an epitope on a cancer cell. As used in describing the various polypeptides or nucleic acids disclosed herein, “isolated” or “purified” refers to a polypeptide or nucleic acid that has been identified, separated, and / or recovered from other components in the environment in which it originally resides. It is preferable that isolated polypeptides or nucleic acids contain no other components to which they originally reside. Impurities in the environment in which isolated polypeptides or nucleic acids originally reside are typically substances that interfere with the diagnostic or therapeutic use of the polypeptide or nucleic acid, and include, for example, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, a method is provided comprising the steps of delivering a nucleic acid according to any one embodiment described herein or an expression vector according to any one embodiment described herein to a bacterial cell, a mammalian cell or an insect cell, growing the cell in a culture medium, inducing the expression of a fusion protein, and purifying and processing the fusion protein.

[0138] The “amino acid sequence identity (%)” for sequences described herein (e.g., CISC sequences) is defined as the percentage of amino acid residues in each of the extracellular binding domain, hinge domain, transmembrane domain, and / or signaling domain in the candidate sequence that match the amino acid residues in each domain in the reference sequence. This amino acid sequence identity is calculated after aligning the candidate sequence and the reference sequence and inserting gaps as necessary to calculate the maximum possible sequence identity (%), and conservative substitutions are not considered as part of the sequence identity. Alignment for determining amino acid sequence identity (%) can be performed in various ways that fall within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to maximize alignment over the full length of multiple sequences being compared. For example, calculating amino acid sequence identity (%) using the WU-BLAST-2 computer program (Altschul, SF et al. (1996). Methods Enzymol., 266:460-480) requires the use of several search parameters, most of which are set to their default values. Parameters not set to default values ​​(e.g., adjustable parameters) are set to overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. In some embodiments of CISC, the CISC includes an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, each of which may include a native or synthetic domain, or a mutant or cleaved form of the native domain.In some embodiments, a variant or cleavage of a given domain includes an amino acid sequence having sequence identity (%) within a range defined by 100%, 95%, 90%, or 85% sequence identity, or any two of the aforementioned percentages, with respect to the sequence shown in the sequence provided herein.

[0139] In this specification, “CISC variant polypeptide sequence” or “CISC variant amino acid sequence” means a protein sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity (or amino acid sequence identity (%) within the range defined by any two of these percentages) with the protein sequences provided herein, as defined below, or a specifically derived fragment thereof, such as a protein sequence of an extracellular binding domain, hinge domain, transmembrane domain, and / or signaling domain. Typically, a polypeptide or fragment of a CISC variant has at least 80% amino acid sequence identity with the amino acid sequence of CISC or a fragment derived therefrom, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, or more preferably at least 99% amino acid sequence identity with the amino acid sequence of CISC or a fragment derived therefrom. The variant does not include the native protein sequence.

[0140] In this specification, "T cells" or "T lymphocytes" may be obtained from any mammal, preferably from primates or other species, including monkeys, dogs, and humans. In some embodiments, the T cells are of the same species as the recipient (same species but from a different donor). In some embodiments, the T cells are autologous (the donor and recipient are the same). In some embodiments, the T cells are syngeneic (the donor and recipient are different, but identical twins).

[0141] In this specification, whether in the transitional clause or the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, these terms are to be interpreted as synonymous with the expressions “at least have” or “at least contain.” When the term “comprise(s)” is used in a context relating to a method, it means that the method includes at least the specified steps, and may include other steps. When the term “comprise(s)” is used in a context relating to a compound, composition, or apparatus, it means that the compound, composition, or apparatus includes at least the specified features or components, and may include other features or components.

[0142] genome editing system This disclosure provides a genome editing system for cells (e.g., lymphocytes) to modulate the expression, function, and / or activity of FOXP3 by targeting and incorporating nucleic acids encoding FOXP3 or functional derivatives into the cell genome. Furthermore, this disclosure provides a system for treating subjects with or suspected of having FOXP3-related disorders or health conditions, using genome editing ex vivo and / or in vivo. In some embodiments, the subjects have or are suspected of having autoimmune diseases (e.g., IPEX syndrome) or organ transplant-related disorders (e.g., graft-versus-host disease (GVHD)).

[0143] In some embodiments, (a) DNA endonucleases, or nucleic acids encoding said DNA endonucleases; (b) gRNA (e.g., sgRNA) or nucleic acid encoding such gRNA that can target the DNA endonuclease to a FOXP3 locus or a non-FOXP3 locus in the cell genome (e.g., AAVS1 (adeno-associated virus integration site)); and (c) Donor template including the code sequence of FOXP3 We provide a system that includes this. In some smooth ways, the DNA endonucleases include Cas1 endonuclease, Cas1B endonuclease, Cas2 endonuclease, Cas3 endonuclease, Cas4 endonuclease, Cas5 endonuclease, Cas6 endonuclease, Cas7 endonuclease, Cas8 endonuclease, Cas9 endonuclease (also known as Csn1 and Csx12), Cas100 endonuclease, Csy1 endonuclease, Csy2 endonuclease, Csy3 endonuclease, Cse1 endonuclease, Cse2 endonuclease, Csc1 endonuclease, Csc2 endonuclease, Csa5 endonuclease, Csn2 endonuclease, Csm2 endonuclease, Csm3 endonuclease, and Csm 4 endonucleases, Csm5 endonucleases, Csm6 endonucleases, Cmr1 endonucleases, Cmr3 endonucleases, Cmr4 endonucleases, Cmr5 endonucleases, Cmr6 endonucleases, Csb1 endonucleases, Csb2 endonucleases, Csb3 endonucleases, Csx17 endonucleases, Csx14 endonucleases, Csx The gRNA is selected from the group consisting of 10-endonuclease, Csx16-endonuclease, CsaX-endonuclease, Csx3-endonuclease, Csx1-endonuclease, Csx15-endonuclease, Csf1-endonuclease, Csf2-endonuclease, Csf3-endonuclease, Csf4-endonuclease, and Cpf1-endonuclease, as well as functional derivatives thereof. In some embodiments, the DNA endonucleases are Cas endonucleases, such as Cas9-endonuclease (e.g., Cas9-endonuclease derived from Streptococcus pyogenes). In some embodiments, the gRNA contains a spacer sequence complementary to the target sequence within the FOXP3 locus. In some embodiments, the gRNA contains a spacer sequence complementary to the target sequence within exon 1 of the FOXP3 locus. In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence in exon 1 of the FOXP3 locus.In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs. 2 or 5, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs. 2 or 5. In some embodiments, the gRNA includes a spacer sequence complementary to a target sequence in a non-FOXP3 locus (e.g., AAVS1 or TRAC). In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 15-20, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. 15-20. In some embodiments, the gRNA includes the spacer sequence shown in SEQ ID NO: 33 or 34, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NO: 33 or 34. In some embodiments, the coding sequence for FOXP3 encodes FOXP3 or a functional derivative thereof. In some embodiments, the coding sequence for FOXP3 is FOXP3 cDNA.In some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof has at least 70% or at least about 70% sequence identity with the sequence shown in SEQ ID NO: 68 or 69, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least more sequence identity, or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least more sequence identity. In some embodiments, the system comprises a Cas DNA endonuclease. In some embodiments, the system comprises a nucleic acid encoding a Cas DNA endonuclease. In some embodiments, the system comprises a gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the system includes a nucleic acid encoding a gRNA. In some embodiments, the system further includes one or more further gRNAs or nucleic acids encoding the one or more further gRNAs.

[0144] In some embodiments, according to any of the systems described herein, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7, 15-20, 27-29, 33, and 34, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7, 15-20, 27-29, 33, and 34. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 2, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 2. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 3, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 3. In some embodiments, the gRNA includes the spacer sequence shown in SEQ ID NO: 5, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NO: 5.

[0145] In some embodiments, according to any of the systems described herein, the Cas DNA endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease is a Cas9 endonuclease derived from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 is a Cas9 derived from Staphylococcus lugdunensis (SluCas9).

[0146] In some embodiments, according to any of the systems described herein, the nucleic acid sequence encoding FOXP3 or its functional derivative is codon-optimized for expression in host cells. In some embodiments, the nucleic acid sequence encoding FOXP3 or its functional derivative has at least 70% or at least about 70% sequence identity with the sequence shown in SEQ ID NO: 68 or 69, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least more sequence identity, or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least more sequence identity. In some embodiments, the nucleic acid sequence encoding FOXP3 or its functional derivative is codon-optimized for expression in human cells.

[0147] In some embodiments, according to any of the systems described herein, the system comprises a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in a host cell. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in a human cell. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA (such as a DNA plasmid). In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA (such as mRNA).

[0148] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, and a promoter configured to express FOXP3 or a functional derivative thereof. Examples of promoters include the MND promoter, the PGK promoter, and the EF1 promoter. In some embodiments, the promoter has the sequence shown in any of SEQ ID NOs: 113-115, or a variant having at least 85% identity with any one of SEQ ID NOs: 113-115. In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is the AAV6 vector.

[0149] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template being configured to incorporate the donor cassette into a genomic locus targeted by a gRNA included in the system by homologous recombination repair (HDR). In some embodiments, homologous arms corresponding to the sequence of the targeted genomic locus are positioned on either side of the donor cassette. In some embodiments, the length of the homologous arm is at least 0.2kb or at least about 0.2kb (for example, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb or at least more, or at least about 0.3kb, at least about 0.4kb, at least about 0.5kb, at least about 0.6kb, at least about 0.7kb, at least about 0.8kb, at least about 0.9kb, at least about 1kb or at least more). In some embodiments, the length of the homologous arm is at least 0.4kb or at least about 0.4kb, for example 0.45kb, 0.6kb, or 0.8kb. Typical homologous arms include 5' homologous arms having sequences shown in any of sequence numbers 90-97 and 106-107, and 3' homologous arms having sequences shown in any of sequence numbers 98-105 and 108-109. Further examples of typical homologous arms include homologous arms contained in a donor template having sequence number 37 or 38. Typical donor templates include donor templates having sequence number 37 or 38. In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV2 vector, an AAV5 vector, or an AAV6 vector. In some embodiments, the AAV vector is an AAV6 vector.

[0150] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template configured to be incorporated into a genomic locus targeted by a gRNA included in the system by non-homologous end joining (NHEJ). In some embodiments, gRNA target sites are located adjacent to one or both sides of the donor cassette. In some embodiments, gRNA target sites are located adjacent to both sides of the donor cassette. In some embodiments, the gRNA target sites are target sites of the gRNA included in the system. In some embodiments, the gRNA target sites of the donor template are the reverse complementary strand of the cellular genomic gRNA target site targeted by the gRNA included in the system. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV2 vector, an AAV5 vector, or an AAV6 vector. In some embodiments, the AAV vector is an AAV6 vector.

[0151] In some embodiments, any of the systems described herein includes a donor template comprising a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor cassette contains a post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus (WHP). In some embodiments, the WPRE is the full-length WPRE. In some embodiments, the WPRE is a truncated WPRE. Typical WPREs include those contained in a donor template having the sequence shown in any of SEQ ID NOs: 135-147. Typical donor templates having WPREs include those having the sequence shown in any of SEQ ID NOs: 135-147.

[0152] In some embodiments, any of the systems described herein includes a donor template comprising a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor cassette contains a ubiquitous chromatin opening element (UCOE). Typical UCOEs include those contained in a donor template having the sequence shown in any of SEQ ID NOs 158, 159, and 162. Typical donor templates having UCOEs include those having the sequence shown in any of SEQ ID NOs 158, 159, and 162.

[0153] In some embodiments, any of the systems described herein includes a donor template comprising a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor cassette containing a coding sequence for low affinity nerve growth factor receptor (LNGFR). In some embodiments, the coding sequence for LNGFR is upstream of the nucleic acid sequence encoding FOXP3 or a functional derivative thereof. In some embodiments, the coding sequence for LNGFR is downstream of the nucleic acid sequence encoding FOXP3 or a functional derivative thereof. Typical LNGFR coding sequences include those contained in a donor template having the sequence shown in any of sequence numbers 37, 38, 40, 42, 46, 47, 74, 76, 80, and 81. Typical LNGFR coding sequences include the sequence shown in sequence number 88 or 118, or variants having at least 85% identity with sequence number 88 or 118.

[0154] In some embodiments, any of the systems described herein includes a donor template comprising a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor cassette includes a 3' untranslated region (UTR) ligated to the 3' end of the nucleic acid sequence encoding FOXP3 or a functional derivative thereof. In some embodiments, the 3'UTR includes an SV40-polyA signal. A typical 3'UTR including an SV40-polyA signal includes a 3'UTR having the sequence shown in SEQ ID NO: 116. In some embodiments, the 3'UTR includes a 3'UTR derived from the human FOXP3 gene. A typical 3'UTR derived from the human FOXP3 gene is a 3'UTR having the sequence shown in SEQ ID NO: 117.

[0155] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template further comprising a nucleic acid encoding a 2A self-cleaving peptide between adjacent nucleic acids encoding the components of the system. In some embodiments, the donor template comprises a nucleic acid encoding a 2A self-cleaving peptide between each adjacent nucleic acid encoding the components of the system. In some embodiments, each 2A self-cleaving peptide is independently a T2A self-cleaving peptide or a P2A self-cleaving peptide. For example, in some embodiments, the donor template comprises, in this order from the 5' end to the 3' end, a promoter, a nucleic acid encoding the expression of FOXP3 or a functional variant thereof, a nucleic acid encoding a 2A self-cleaving peptide, and a nucleic acid encoding a selection marker. In some embodiments, the donor template comprises the nucleic acid shown in SEQ ID NO: 89, or a variant of the nucleic acid having at least 85% identity with SEQ ID NO: 89. In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0156] In some embodiments, according to any of the systems described herein, the DNA endonuclease or the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in liposomes or lipid nanoparticles. In some embodiments, the liposomes or lipid nanoparticles further comprise gRNA. In some embodiments, the liposomes or lipid nanoparticles are lipid nanoparticles. In some embodiments, the system comprises lipid nanoparticles containing a DNA endonuclease and a nucleic acid encoding gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA encoding the DNA endonuclease.

[0157] In some embodiments, according to any of the systems described herein, the DNA endonuclease complexes with gRNA to form a ribonucleoprotein (RNP) complex.

[0158] nucleic acid Genome-targeted nucleic acids or guide RNA This disclosure provides genome-targeted nucleic acids that can direct the activity of a relevant polypeptide (e.g., a site-directed polypeptide or DNA endonuclease) to a specific target sequence within a target nucleic acid. In some embodiments, the genome-targeted nucleic acid is RNA. Hereinafter, the genome-targeted RNA is referred to as “guide RNA” or “gRNA”. The guide RNA has at least a spacer sequence and a CRISPR repeat sequence that can hybridize to a target nucleic acid sequence of interest. In a type II system, the gRNA further has a second RNA called a tracrRNA sequence. In a type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize to form a double helix. In a type V guide RNA (gRNA), the crRNA forms a double helix. In either system, the double helix binds to a site-specific polypeptide to form a guide RNA-site-specific polypeptide complex. This genome-targeted nucleic acid confers target specificity to the complex formed by association with the site-specific polypeptide. Thus, this genome-targeted nucleic acid confers directionality to the activity of the site-specific polypeptide.

[0159] In some embodiments, the genome-targeting nucleic acid is a bimolecule guide RNA. In some embodiments, the genome-targeting nucleic acid is a single-molecule guide RNA. The bimolecule guide RNA has two RNA strands. The first strand has an optional spacer extension sequence, a spacer sequence, and a CRISPR minimal repeat sequence in the direction from the 5' end to the 3' end. The second strand has a tracrRNA minimal sequence (complementary to the CRISPR minimal repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The single-molecule guide RNA (sgRNA) of the type II system has an optional spacer extension sequence, a spacer sequence, a CRISPR minimal repeat sequence, a single-molecule guide linker, a tracrRNA minimal sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence in the direction from the 5' end to the 3' end. The optionally provided tracrRNA extension sequence may have elements that confer further functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the CRISPR minimal repeat sequence and the tracrRNA minimal sequence to form a hairpin structure. An optional tracrRNA elongation sequence has one or more hairpins. The single-molecule guide RNA (sgRNA) of the V-type system has the CRISPR minimal repeat sequence and a spacer sequence in the direction from the 5' end to the 3' end.

[0160] As an example, guide RNAs used in the CRISPR / Cas / Cpf1 system, or other smaller RNAs, can be easily synthesized by chemical means described below that are known in the art. Although chemical synthesis procedures are continuously expanding, due to the polynucleotide length significantly exceeding about 100 nucleotide lengths, purification of the RNA by procedures such as high-performance liquid chromatography (HPLC without using gels such as PAGE) tends to become difficult. As one approach used to produce long RNAs, there is a method of producing two or more molecules and then ligating them later. Very long RNAs, such as RNAs encoding Cas9 endonuclease or Cpf1 endonuclease, can be easily produced enzymatically. Various RNA modifications can be introduced during or after the process of chemical synthesis and / or enzymatic production of the RNA. For example, as reported in the art, modifications that enhance stability, modifications that reduce the possibility or degree of the innate immune response, and / or modifications that enhance other attributes can be mentioned.

[0161] In some embodiments, provided is a guide RNA (gRNA) comprising a spacer sequence complementary to a genomic sequence within the FOXP3 locus of a cell or a genomic sequence in the vicinity of the FOXP3 gene. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 1-7 and 27-29, or a variant of the spacer sequence having no more than 3 mismatches as compared to any of SEQ ID NOs: 1-7 and 27-29. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having no more than 3 mismatches as compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having no more than 3 mismatches as compared to any of SEQ ID NOs: 2, 3, and 5.

[0162] In some embodiments, a guide RNA (gRNA) is provided that includes a spacer sequence complementary to a genomic sequence within the AAVS1 gene of a cell or a genomic sequence in the vicinity within the AAVS1 gene. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs: 15 to 20, or a variant of the spacer sequence having three or fewer mismatches as compared to any of SEQ ID NOs: 15 to 20.

[0163] Guide RNAs produced by in vitro transcription may contain a mixture of the full-length guide RNA molecule and a portion of the guide RNA molecule. Chemically synthesized guide RNA molecules usually have a proportion of the full-length guide molecule exceeding 75%, and may further contain chemically modified bases such as chemically modified bases for enhancing the resistance of the guide RNA to cleavage by intracellular nucleases.

[0164] Spacer extension arrangement In some embodiments of genome-targeted nucleic acids, spacer extension sequences can modulate activity, confer stability, and / or provide sites for modifying genome-targeted nucleic acids. Spacer extension sequences can also modulate on-target or off-target activity or specificity. In some embodiments, spacer extension sequences are provided. The lengths of the spacer extension sequences are greater than 1 nucleotide, greater than 5 nucleotides, greater than 10 nucleotides, greater than 15 nucleotides, greater than 20 nucleotides, greater than 25 nucleotides, greater than 30 nucleotides, greater than 35 nucleotides, greater than 40 nucleotides, greater than 45 nucleotides, greater than 50 nucleotides, greater than 60 nucleotides, greater than 70 nucleotides, greater than 80 nucleotides, greater than 90 nucleotides, greater than 100 nucleotides, greater than 120 nucleotides, greater than 140 nucleotides, greater than 160 nucleotides, greater than 180 nucleotides, and greater than 200 nucleotides. The length may exceed the creotide length, exceed 220 nucleotides, exceed 240 nucleotides, exceed 260 nucleotides, exceed 280 nucleotides, exceed 300 nucleotides, exceed 320 nucleotides, exceed 340 nucleotides, exceed 360 nucleotides, exceed 380 nucleotides, exceed 400 nucleotides, exceed 1000 nucleotides, exceed 2000 nucleotides, exceed 3000 nucleotides, exceed 4000 nucleotides, exceed 5000 nucleotides, exceed 6000 nucleotides, or exceed 7000 nucleotides, or any other nucleotide length. The length of the spacer extension sequence may be 1 nucleotide or approximately 1 nucleotide, 5 nucleotides or approximately 5 nucleotides, 10 nucleotides or approximately 10 nucleotides, 15 nucleotides or approximately 15 nucleotides, 20 nucleotides or approximately 20 nucleotides,25 nucleotides or approximately 25 nucleotides, 30 nucleotides or approximately 30 nucleotides, 35 nucleotides or approximately 35 nucleotides, 40 nucleotides or approximately 40 nucleotides, 45 nucleotides or approximately 45 nucleotides, 50 nucleotides or approximately 50 nucleotides, 60 nucleotides or approximately 60 nucleotides, 70 nucleotides or approximately 70 nucleotides, 80 nucleotides or approximately 80 nucleotides, 90 nucleotides or approximately 90 nucleotides, 100 nucleotides or approximately 100 nucleotides, 120 nucleotides or approximately 120 nucleotides, 140 nucleotides or approximately 140 nucleotides, 160 nucleotides or approximately 160 nucleotides, 180 nucleotides or approximately 180 nucleotides, 200 nucleotides or approximately 200 nucleotides, 220 nucleotides or approximately 220 nucleotides, 240 nucleotides or approximately 24 The nucleotide length may be 0 nucleotides, 260 nucleotides or approximately 260 nucleotides, 280 nucleotides or approximately 280 nucleotides, 300 nucleotides or approximately 300 nucleotides, 320 nucleotides or approximately 320 nucleotides, 340 nucleotides or approximately 340 nucleotides, 360 nucleotides or approximately 360 nucleotides, 380 nucleotides or approximately 380 nucleotides, 400 nucleotides or approximately 400 nucleotides, 1000 nucleotides or approximately 1000 nucleotides, 2000 nucleotides or approximately 2000 nucleotides, 3000 nucleotides or approximately 3000 nucleotides, 4000 nucleotides or approximately 4000 nucleotides, 5000 nucleotides or approximately 5000 nucleotides, 6000 nucleotides or approximately 6000 nucleotides, or 7000 nucleotides or approximately 7000 nucleotides, or greater. The length of the spacer extension sequence is less than 1 nucleotide, less than 5 nucleotides, less than 10 nucleotides, less than 15 nucleotides, less than 20 nucleotides, less than 25 nucleotides, less than 30 nucleotides, less than 35 nucleotides, less than 40 nucleotides.The length may be less than 45 nucleotides, less than 50 nucleotides, less than 60 nucleotides, less than 70 nucleotides, less than 80 nucleotides, less than 90 nucleotides, less than 100 nucleotides, less than 120 nucleotides, less than 140 nucleotides, less than 160 nucleotides, less than 180 nucleotides, less than 200 nucleotides, less than 220 nucleotides, less than 240 nucleotides, less than 260 nucleotides, less than 280 nucleotides, less than 300 nucleotides, less than 320 nucleotides, less than 340 nucleotides, less than 360 nucleotides, less than 380 nucleotides, less than 400 nucleotides, less than 1000 nucleotides, less than 2000 nucleotides, less than 3000 nucleotides, less than 4000 nucleotides, less than 5000 nucleotides, less than 6000 nucleotides, less than 7000 nucleotides, or more. In some embodiments, the length of the spacer extension sequence is less than 10 nucleotides. In some embodiments, the length of the spacer extension sequence is 10 to 30 nucleotides. In some embodiments, the length of the spacer extension sequence is 30 to 70 nucleotides.

[0165] In some embodiments, the spacer extension sequence has another portion (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme, etc.). In some embodiments, the other portion targets a nucleic acid and reduces or increases the stability of the nucleic acid. In some embodiments, the other portion is a transcriptional terminator segment (e.g., a transcription termination sequence). In some embodiments, the other portion functions in eukaryotic cells. In some embodiments, the other portion functions in prokaryotic cells. In some embodiments, the other portion functions in both eukaryotic and prokaryotic cells. Examples of suitable parts include, but are not limited to, 5' end caps (e.g., 7-methylguanylate caps (m7G)); riboswitch sequences (e.g., those that stabilize under control and / or allow access by proteins or protein complexes under control); sequences that form dsRNA double strands (e.g., hairpins); sequences that target RNA to subcellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that enable tracking (e.g., direct binding to fluorescent molecules, binding to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.); and / or modifications or sequences that provide binding sites for proteins (e.g., DNA-acting proteins such as transcription activators, transcription repressors, DNA methyltransferases, DNA methyl-degrading enzymes, histone acetyltransferases, histone deacetylases, etc.).

[0166] Spacer array The spacer sequence hybridizes to the sequence within the target nucleic acid. The spacer in the genome-targeted nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (e.g., base pairing). Therefore, the nucleotide sequence of the spacer varies depending on the sequence of the target nucleic acid.

[0167] In the CRISPR / Cas system described herein, the spacer sequence is designed to hybridize with the target nucleic acid located at the 5' end of the PAM in the Cas9 enzyme used in this system. The spacer may be a perfect match or a mismatch with the target sequence. Each Cas9 enzyme has a specific PAM sequence that recognizes the target DNA. For example, Streptococcus pyogenes recognizes a PAM in its target nucleic acid that has the sequence 5'-NRG-3' (where R represents A or G and N is any nucleotide adjacent to the 3' end of the target nucleic acid sequence targeted by the spacer sequence).

[0168] In some embodiments, the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has fewer than 20 nucleotides. In some embodiments, the target nucleic acid has more than 20 nucleotides. In some embodiments, the target nucleic acid has at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, or at least more nucleotides. In some embodiments, the target nucleic acid has at most 5, at most 10, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 30, or at most more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases adjacent to the 5' end of the first nucleotide of the PAM. In some embodiments, the PAM sequence used in the compositions and methods of this disclosure as a sequence recognized by Cas9 derived from Streptococcus pyogenes (Sp) is NGG.

[0169] In some embodiments, the length of the spacer sequence hybridizing to the target nucleic acid is at least 6 nucleotides (nt) or at least about 6 nucleotides (nt). The length of the spacer sequence is at least 6nt or at least about 6nt, at least 10nt or at least about 10nt, at least 15nt or at least about 15nt, at least 18nt or at least about 18nt, at least 19nt or at least about 19nt, at least 20nt or at least about 20nt, at least 25nt or at least about 25nt, at least 30nt or at least about 30nt, at least 35nt or at least about 35nt, or at least 40nt or at least about 40nt, about 6nt to about 80nt, about 6nt to about 50nt, about 6nt to about 45nt, about 6nt to about 40nt, about 6nt to about 35nt, about 6nt to Approximately 30nt, approximately 6nt to approximately 25nt, approximately 6nt to approximately 20nt, approximately 6nt to approximately 19nt, approximately 10nt to approximately 50nt, approximately 10nt to approximately 45nt, approximately 10nt to approximately 40nt, approximately 10 nt ~ about 35nt, about 10nt - about 30nt, about 10nt - about 25nt, about 10nt - about 20nt, about 10nt - about 19nt, about 19nt - about 25nt, about 19nt - about 30 The spacer sequence may be nt, approximately 19nt to approximately 35nt, approximately 19nt to approximately 40nt, approximately 19nt to approximately 45nt, approximately 19nt to approximately 50nt, approximately 19nt to approximately 60nt, approximately 20nt to approximately 25nt, approximately 20nt to approximately 30nt, approximately 20nt to approximately 35nt, approximately 20nt to approximately 40nt, approximately 20nt to approximately 45nt, approximately 20nt to approximately 50nt, or approximately 20nt to approximately 60nt. In some embodiments, the spacer sequence is 20 nucleotides long. In some embodiments, the spacer is 19 nucleotides long. In some embodiments, the spacer is 18 nucleotides long. In some embodiments, the spacer is 17 nucleotides long. In some embodiments, the spacer is 16 nucleotides long. In some embodiments, the spacer is 15 nucleotides long.

[0170] In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is 100% at the six consecutive 5' terminal nucleotides of the target sequence in the complementary strand of the target nucleic acid. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at least 60% at about 20 consecutive nucleotides. In some embodiments, the length of the spacer sequence and the length of the target nucleic acid may differ by 1 to 6 nucleotides, and this difference can be considered as one or more bulges.

[0171] In some embodiments, spacer sequences are designed or selected using a computer program. The computer program may use variables, such as, for example, predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC ratio (%), genomic frequency (e.g., changes at one or more locations caused by mismatches, insertions, or deletions in identical or similar sequences), methylation status, and the presence of SNPs.

[0172] CRISPR's minimum iteration array In some embodiments, the minimal repeat sequence of CRISPR is a sequence having at least 30% or at least about 30%, at least 40% or at least about 40%, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 65% or at least about 65%, at least 70% or at least about 70%, at least 75% or at least about 75%, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, or 100% sequence identity with a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

[0173] In some embodiments, the minimal repeat sequence of CRISPR has nucleotides that can hybridize to the minimal sequence of tracrRNA in cells. The minimal repeat sequence of CRISPR and the minimal sequence of tracrRNA form a double helix, for example, a base-paired double-stranded structure. Together, the minimal repeat sequence of CRISPR and the minimal sequence of tracrRNA bind to a site-directed polypeptide. At least a portion of the minimal repeat sequence of CRISPR hybridizes to the minimal sequence of tracrRNA. In some embodiments, at least a portion of the minimal repeat sequence of CRISPR has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 100% complementarity with the minimal sequence of tracrRNA. In some embodiments, at least a portion of the minimal repeat sequence of CRISPR has complementarity with the minimal sequence of tracrRNA of at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, or at most 100%.

[0174] The minimum repeat sequence length for CRISPR is approximately 7 nucleotides to 100 nucleotides. For example, the minimum repeat sequence length for CRISPR is 7 nucleotides (nt) to 50 nt or approximately 7 nt to 50 nt, 7 nt to 40 nt or approximately 7 nt to 40 nt, 7 nt to 30 nt or approximately 7 nt to 30 nt, 7 nt to 25 nt or approximately 7 nt to 25 nt, 7 nt to 20 nt or approximately 7 nt to 20 nt, 7 nt to 15 nt or approximately 7 nt to 15 nt, 8 nt to 40 nt or approximately 8 nt to 40 nt, 8 nt to 30 nt or approximately 8 nt to 30 nt, and 8 nt to 2 The minimum repeat length of CRISPR is approximately 5 nucleotides or 8 nucleotides to 25 nucleotides, 8 nucleotides to 20 nucleotides or 8 nucleotides to 20 nucleotides, 8 nucleotides to 15 nucleotides or 8 nucleotides to 15 nucleotides, 15 nucleotides to 100 nucleotides or 15 nucleotides to 100 nucleotides, 15 nucleotides to 80 nucleotides or 15 nucleotides to 80 nucleotides, 15 nucleotides to 50 nucleotides or 15 nucleotides to 50 nucleotides, 15 nucleotides to 40 nucleotides or 15 nucleotides to 40 nucleotides, 15 nucleotides to 30 nucleotides or 15 nucleotides to 30 nucleotides, or 15 nucleotides to 25 nucleotides or 15 nucleotides to 25 nucleotides. In some embodiments, the minimum repeat length of CRISPR is approximately 9 nucleotides. In some embodiments, the minimum repeat length of CRISPR is approximately 12 nucleotides.

[0175] In some embodiments, the CRISPR minimal repeat sequence has at least about 60% identity with a reference CRISPR minimal repeat sequence (e.g., wild-type crRNA from S. pyogenes) in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides. For example, the CRISPR minimal repeat sequence has at least 65% or at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, or at least 100% identity with a reference CRISPR minimal repeat sequence in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides.

[0176] Minimal sequence of tracrRNA In some embodiments, the minimum tracrRNA sequence is a sequence having at least 30% or at least about 30%, at least 40% or at least about 40%, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 65% or at least about 65%, at least 70% or at least about 70%, at least 75% or at least about 75%, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, or at least 100% sequence identity with a reference tracrRNA sequence (e.g., wild-type tracrRNA from S. pyogenes).

[0177] In some embodiments, the minimal tracrRNA sequence has nucleotides that can hybridize to the minimal CRISPR repeat sequence in a cell. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence form a double helix, for example, a base-paired double-stranded structure. Together, the minimal tracrRNA sequence and the minimal CRISPR repeat sequence bind to a site-directed polypeptide. At least a portion of the minimal tracrRNA sequence can hybridize to the minimal CRISPR repeat sequence. In some embodiments, the minimal tracrRNA sequence has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 100% complementarity with the minimal CRISPR repeat sequence.

[0178] The minimum sequence length of tracrRNA is approximately 7 nucleotides to approximately 100 nucleotides. For example, the minimum sequence length of tracrRNA may be approximately 7 nucleotides (nt) to approximately 50 nt, approximately 7 nt to approximately 40 nt, approximately 7 nt to approximately 30 nt, approximately 7 nt to approximately 25 nt, approximately 7 nt to approximately 20 nt, approximately 7 nt to approximately 15 nt, approximately 8 nt to approximately 40 nt, approximately 8 nt to approximately 30 nt, approximately 8 nt to approximately 25 nt, approximately 8 nt to approximately 20 nt, approximately 8 nt to approximately 15 nt, approximately 15 nt to approximately 100 nt, approximately 15 nt to approximately 80 nt, approximately 15 nt to approximately 50 nt, approximately 15 nt to approximately 40 nt, approximately 15 nt to approximately 30 nt, or approximately 15 nt to approximately 25 nt. In some embodiments, the minimum sequence length of tracrRNA is approximately 9 nucleotides. In some embodiments, the minimum sequence length of tracrRNA is approximately 12 nucleotides. In some embodiments, the minimum tracrRNA sequence consists of a 23-48 nt tracrRNA as described in Jinek, M. et al. (2012). Science, 337(6096):816-821.

[0179] In some embodiments, the minimal tracrRNA sequence has at least about 60% identity with the reference tracrRNA minimal sequence (e.g., wild-type tracrRNA from S. pyogenes) in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides. For example, the minimal tracrRNA sequence has at least 65% or at least about 65% identity with the reference tracrRNA minimal sequence in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides, at least 70% or at least about 70% identity, at least 75% or at least about 75% identity, at least 80% or at least about 80% identity, at least 85% or at least about 85% identity, at least 90% or at least about 90% identity, at least 95% or at least about 95% identity, at least 98% or at least about 98% identity, at least 99% or at least about 99% identity, or at least 100% identity.

[0180] In some embodiments, the double-strand formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has a double helix structure. In some embodiments, the double-strand formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or at least more nucleotides. In some embodiments, the double-strand formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has at most about 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or at most more nucleotides.

[0181] In some embodiments, the double-strand has mismatches (e.g., the two strands included in the double-strand do not have 100% complementarity). In some embodiments, the double-strand has at least about 1, at least about 2, at least about 3, at least about 4 or at least about 5 mismatches. In some embodiments, the double-strand has at most about 1, at most about 2, at most about 3, at most about 4, or at most about 5 mismatches. In some embodiments, the double-strand has 2 or fewer mismatches.

[0182] Bulge In some embodiments, a "bulge" exists in the double helix formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence. The bulge is a nucleotide unpaired region within the double helix. In some embodiments, the bulge contributes to the binding of the double helix to a site-specific polypeptide. The bulge has an unpaired sequence 5'-XXXY-3' on one strand of the double helix (where X is any purine base and Y is a nucleotide that can form a fluctuating base pair with a nucleotide on the opposite strand) and an unpaired nucleotide region on the other strand. The number of unpaired nucleotides on each strand of the double helix may vary.

[0183] In one example, the bulge has an unpaired purine base (e.g., adenine) on the CRISPR minimal repeat strand that forms the bulge. In some embodiments, the bulge has an unpaired sequence 5'-AAGY-3' on the tracrRNA minimal sequence strand that forms the bulge (where Y is a nucleotide that can form a fluctuating base pair with a nucleotide on the CRISPR minimal repeat strand).

[0184] In some embodiments, the bulge on the CRISPR minimal repeat chain side forming the double helix has at least one, at least two, at least three, at least four, at least five, or at least more unpaired nucleotides. The bulge on the CRISPR minimal repeat chain side forming the double helix has at most one, at most two, at most three, at most four, at most five, or at most more unpaired nucleotides. In some embodiments, the bulge on the CRISPR minimal repeat chain side forming the double helix has one unpaired nucleotide.

[0185] In some embodiments, the bulge on the tracrRNA minimal sequence side forming the double helix has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least more unpaired nucleotides. In some embodiments, the bulge on the tracrRNA minimal sequence side forming the double helix has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10, or at most more unpaired nucleotides. In some embodiments, the bulge on the second strand forming the double helix (for example, on the tracrRNA minimal sequence side forming the double helix) has 4 unpaired nucleotides.

[0186] In some embodiments, the bulge has at least one fluctuating base pair. In some embodiments, the bulge has one or fewer fluctuating base pairs. In some embodiments, the bulge has at least one purine nucleotide. In some embodiments, the bulge has at least three purine nucleotides. In some embodiments, the bulge sequence has at least five purine nucleotides. In some embodiments, the bulge sequence has at least one guanine nucleotide. In some embodiments, the bulge sequence has at least one adenine nucleotide.

[0187] hairpin In various embodiments, one or more hairpins are present at the 3' end of the minimal tracrRNA sequence within the 3' tracrRNA sequence.

[0188] In some embodiments, the hairpin begins at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, or at least more nucleotides from the 3' end of the last paired nucleotide of the double helix consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence. In some embodiments, the hairpin may begin at a maximum of about 1, at a maximum of about 2, at a maximum of about 3, at a maximum of about 4, at a maximum of about 5, at a maximum of about 6, at a maximum of about 7, at a maximum of about 8, at a maximum of about 9, at a maximum of about 10, or at a maximum of more nucleotides from the 3' end of the last paired nucleotide of the double helix consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence.

[0189] In some embodiments, the hairpin has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more consecutive nucleotides, or at least about one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more consecutive nucleotides. In some embodiments, the hairpin has at most about one, at most about two, at most about three, at most about four, at most about five, at most about six, at most about seven, at most about eight, at most about nine, at most about ten, at most about fifteen or more consecutive nucleotides.

[0190] In some embodiments, the hairpin has a CC dinucleotide (for example, two consecutive cytosine nucleotides).

[0191] In some embodiments, the hairpin has a double-stranded nucleotide (for example, a hairpin nucleotide in which nucleotides hybridize with each other). For example, the hairpin has a CC dinucleotide that hybridizes to a GG dinucleotide in the hairpin double-stranded 3' tracrRNA sequence.

[0192] One or more hairpins can interact with the guide RNA interaction region of a site-specific polypeptide.

[0193] In some embodiments, there are two or more hairpins, and in some embodiments, there are three or more hairpins.

[0194] 3' tracrRNA sequence In some embodiments, the 3' tracrRNA sequence has a sequence that has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 100% sequence identity with a reference tracrRNA sequence (e.g., tracrRNA from S. pyogenes).

[0195] In some embodiments, the length of the 3' tracrRNA sequence is 6 nucleotides to 100 nucleotides or approximately 6 nucleotides to approximately 100 nucleotides. For example, the length of the 3' tracrRNA sequence may be approximately 6 nucleotides (nt) to approximately 50 nt, approximately 6 nt to approximately 40 nt, approximately 6 nt to approximately 30 nt, approximately 6 nt to approximately 25 nt, approximately 6 nt to approximately 20 nt, approximately 6 nt to approximately 15 nt, approximately 8 nt to approximately 40 nt, approximately 8 nt to approximately 30 nt, approximately 8 nt to approximately 25 nt, approximately 8 nt to approximately 20 nt, approximately 8 nt to approximately 15 nt, approximately 15 nt to approximately 100 nt, approximately 15 nt to approximately 80 nt, approximately 15 nt to approximately 50 nt, approximately 15 nt to approximately 40 nt, approximately 15 nt to approximately 30 nt, or approximately 15 nt to approximately 25 nt. In some embodiments, the length of the 3' tracrRNA sequence is approximately 14 nucleotides.

[0196] In some embodiments, the 3'tracrRNA sequence has at least about 60% identity with a reference 3'tracrRNA sequence (e.g., a wild-type 3'tracrRNA sequence from S. pyogenes) in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides. For example, the 3'tracrRNA sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least 100% identity with a reference 3'tracrRNA sequence (e.g., a wild-type 3'tracrRNA sequence from S. pyogenes) in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides.

[0197] In some embodiments, the 3' tracrRNA sequence has two or more double-stranded regions (e.g., hairpins, hybridized regions). In some embodiments, the 3' tracrRNA sequence has two double-stranded regions.

[0198] In some embodiments, the 3' tracrRNA sequence has a stem-loop structure. In some embodiments, the stem-loop structure of the 3' tracrRNA has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or at least more nucleotides. In some embodiments, the stem-loop structure of the 3' tracrRNA has at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten, or at most more nucleotides. In some embodiments, the stem-loop structure has a functional moiety. For example, the stem-loop structure may have an aptamer, a ribozyme, a hairpin that interacts with a protein, a CRISPR array, an intron, or an exon. In some embodiments, the stem-loop structure has at least about one, at least about two, at least about three, at least about four, at least about five, or at least more functional parts. In some embodiments, the stem-loop structure has at most about one, at most about two, at most about three, at most about four, at most about five, or at most more functional parts.

[0199] In some embodiments, the hairpin of the 3' tracrRNA sequence has a P domain. In some embodiments, the P domain on the hairpin has a double-stranded region.

[0200] tracrRNA elongation sequence In some embodiments, the tracrRNA elongation sequence is provided when the guide RNA is a single-molecule guide or a bi-molecule guide. In some embodiments, the length of the tracrRNA elongation sequence is approximately 1 nucleotide to approximately 400 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is greater than 1 nucleotide, greater than 5 nucleotides, greater than 10 nucleotides, greater than 15 nucleotides, greater than 20 nucleotides, greater than 25 nucleotides, greater than 30 nucleotides, greater than 35 nucleotides, greater than 40 nucleotides, greater than 45 nucleotides, greater than 50 nucleotides, greater than 60 nucleotides, greater than 70 nucleotides, greater than 80 nucleotides, greater than 90 nucleotides, greater than 100 nucleotides, greater than 120 nucleotides, greater than 140 nucleotides, greater than 160 nucleotides, greater than 180 nucleotides, greater than 200 nucleotides, greater than 220 nucleotides, greater than 240 nucleotides, greater than 260 nucleotides, greater than 280 nucleotides, greater than 300 nucleotides, greater than 320 nucleotides, greater than 340 nucleotides, greater than 360 nucleotides, greater than 380 nucleotides, or greater than 400 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is approximately 20 nucleotides to approximately 5000 nucleotides or more. In some embodiments, the length of the tracrRNA elongation sequence exceeds 1000 nucleotides.In some embodiments, the length of the tracrRNA elongation sequence is less than 1 nucleotide, less than 5 nucleotides, less than 10 nucleotides, less than 15 nucleotides, less than 20 nucleotides, less than 25 nucleotides, less than 30 nucleotides, less than 35 nucleotides, less than 40 nucleotides, less than 45 nucleotides, less than 50 nucleotides, less than 60 nucleotides, less than 70 nucleotides, less than 80 nucleotides, less than 90 nucleotides, less than 100 nucleotides, less than 120 nucleotides, less than 140 nucleotides, less than 160 nucleotides, less than 180 nucleotides, less than 200 nucleotides, less than 220 nucleotides, less than 240 nucleotides, less than 260 nucleotides, less than 280 nucleotides, less than 300 nucleotides, less than 320 nucleotides, less than 340 nucleotides, less than 360 nucleotides, less than 380 nucleotides, less than 400 nucleotides, or more. In some embodiments, the length of the tracrRNA elongation sequence may be less than 1000 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is less than 10 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 10 to 30 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 30 to 70 nucleotides.

[0201] In some embodiments, the tracrRNA elongation sequence has a functional moiety (e.g., a stability control sequence, a ribozyme, or an endoribonuclease binding sequence). In some embodiments, the functional moiety is a transcriptional terminator segment (e.g., a transcription termination sequence). In some embodiments, the full length of the functional moiety is approximately 10 nucleotides (nt) to approximately 100 nucleotides, approximately 10 nt to approximately 20 nt, approximately 20 nt to approximately 30 nt, approximately 30 nt to approximately 40 nt, approximately 40 nt to approximately 50 nt, approximately 50 nt to approximately 60 nt, approximately 60 nt to approximately 70 nt, approximately 70 nt to approximately 80 nt, approximately 80 nt to approximately 90 nt, approximately 90 nt to approximately 100 nt, approximately 15 nt to approximately 80 nt, approximately 15 nt to approximately 50 nt, approximately 15 nt to approximately 40 nt, approximately 15 nt to approximately 30 nt, or approximately 15 nt to approximately 25 nt. In some embodiments, the functional moiety functions in eukaryotic cells. In some embodiments, the functional portion functions in prokaryotic cells. In some embodiments, the functional portion functions in both eukaryotic and prokaryotic cells.

[0202] Examples of suitable functional portions of a tracrRNA elongation sequence include, but are not limited to, a 3' polyadenylated tail; riboswitch sequences (e.g., those that stabilize under control and / or allow access by proteins or protein complexes under control); sequences that form a dsRNA double helix (e.g., hairpins); sequences that target RNA to subcellular locations (e.g., the nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that enable tracking (e.g., direct binding to fluorescent molecules, binding to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.); and / or modifications or sequences that provide a binding site for proteins (e.g., DNA-acting proteins such as transcription activators, transcription repressors, DNA methyltransferases, DNA methyl-degrading enzymes, histone acetyltransferases, histone deacetylases, etc.). In some embodiments, the tracrRNA elongation sequence has a primer binding site or molecular index (e.g., a barcode sequence). In some embodiments, the tracrRNA elongation sequence has one or more affinity tags.

[0203] Linker sequence of single molecule guide In some embodiments, the length of the linker sequence of a single-molecule guide nucleic acid is approximately 3 nucleotides to approximately 100 nucleotides. For example, in Jinek, M. et al. (2012). Science, 337(6096):816-821, a simple "tetraloop" (-GAAA-) consisting of four nucleotides was used. The linker lengths were approximately 3 nucleotides (nt) to approximately 90 nt, approximately 3 nt to approximately 80 nt, approximately 3 nt to approximately 70 nt, approximately 3 nt to approximately 60 nt, approximately 3 nt to approximately 50 nt, approximately 3 nt to approximately 40 nt, approximately 3 nt to approximately 30 nt, approximately 3 nt to approximately 20 nt, or approximately 3 nt to approximately 10 nt. For example, the linker length may be approximately 3nt to 5nt, 5nt to 10nt, 10nt to 15nt, 15nt to 20nt, 20nt to 25nt, 25nt to 30nt, 30nt to 35nt, 35nt to 40nt, 40nt to 50nt, 50nt to 60nt, 60nt to 70nt, 70nt to 80nt, 80nt to 90nt, or 90nt to 100nt. In some embodiments, the linker length of the single-molecule guide nucleic acid is 4 to 40 nucleotides long. In some embodiments, the linker length is at least about 100 nucleotides, at least about 500 nucleotides, at least about 1000 nucleotides, at least about 1500 nucleotides, at least about 2000 nucleotides, at least about 2500 nucleotides, at least about 3000 nucleotides, at least about 3500 nucleotides, at least about 4000 nucleotides, at least about 4500 nucleotides, at least about 5000 nucleotides, at least about 5500 nucleotides, at least about 6000 nucleotides, at least about 6500 nucleotides, or at least about 7000 nucleotides, or at least more.In some embodiments, the length of the linker is at most about 100 nucleotides, at most about 500 nucleotides, at most about 1000 nucleotides, at most about 1500 nucleotides, at most about 2000 nucleotides, at most about 2500 nucleotides, at most about 3000 nucleotides, at most about 3500 nucleotides, at most about 4000 nucleotides, at most about 4500 nucleotides, at most about 5000 nucleotides, at most about 5500 nucleotides, at most about 6000 nucleotides, at most about 6500 nucleotides, or at most about 7000 nucleotides, or at most more.

[0204] Linkers can have a variety of sequences, but in some embodiments, the linker does not have a sequence with a broad region homologous to other parts of the guide RNA, because the presence of such a homologous broad region can lead to intramolecular binding that may interfere with other functional regions of the guide RNA. In Jinek, M. et al. (2012). Science, 337(6096):816-821, a simple sequence of four nucleotides—GAAA—was used, but various other sequences, such as longer sequences, can be used as well.

[0205] In some embodiments, the linker sequence has functional moieties. For example, the linker sequence may have one or more features such as an aptamer, a ribozyme, a hairpin that interacts with a protein, a protein binding site, a CRISPR array, an intron, or an exon. In some embodiments, the linker sequence has at least about one, at least about two, at least about three, at least about four, at least about five, or at least more functional moieties. In some embodiments, the linker sequence has at most about one, at most about two, at most about three, at most about four, at most about five, or at most more functional moieties.

[0206] In some embodiments, the genomic sites targeted by gRNAs according to this disclosure may be located within the FOXP3 gene in the genome (e.g., the human genome), within the FOXP3 gene in the genome, or in the vicinity of the FOXP3 locus in the genome. Typical guide RNAs targeting such sites include the spacer sequences shown in SEQ ID NOs: 1-7, 15-20, and 27-29. For example, a gRNA containing the spacer sequence shown in SEQ ID NO: 1 may have a spacer sequence including i) the sequence of SEQ ID NO: 1, ii) sequences from positions 2-20 of SEQ ID NO: 1, iii) sequences from positions 3-20 of SEQ ID NO: 1, iv) sequences from positions 4-20 of SEQ ID NO: 1, and so on. As will be understood by those skilled in the art, each guide RNA is designed to contain a spacer sequence complementary to its genomic target sequence. For example, each spacer sequence shown in SEQ ID NOs: 1-7, 15-20, and 27-29 can be incorporated into a single RNA chimera or (together with the corresponding tracrRNA) into a crRNA. See Jinek, M. et al. (2012). Science, 337(6096):816-821 and Deltcheva, E. et al. (2011). Nature, 471:602-607.

[0207] Donor DNA or donor template Site-directed polypeptides, such as DNA endonucleases, can introduce double-strand or single-strand breaks into nucleic acids (e.g., genomic DNA). Double-strand breaks can stimulate intrinsic DNA repair pathways in cells (e.g., homology-dependent repair (HDR), non-homologous end joining, alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining (MMEJ)). NHEJ can repair the cleaved target nucleic acid without requiring a homologous template. This can result in small deletions or insertions (indels) in the target nucleic acid at the cleavage site, potentially leading to disruption or alteration of gene expression. Homologous-dependent repair (HDR), also known as homologous recombination (HR), can occur when a homologous repair template or donor is available.

[0208] Homologous donor templates have sequences homologous to the sequences adjacent to the cleavage site of the target nucleic acid. Generally, sister chromatids are used by cells as repair templates. On the other hand, repair templates for genome editing are often provided as exogenous nucleic acids such as plasmids, double-stranded oligonucleotides, single-stranded oligonucleotides, double-stranded oligonucleotides, or viral nucleic acids. In exogenous donor templates, an additional nucleic acid sequence (such as an introduced gene) or modification (such as a change or deletion of one or more bases) is generally introduced between adjacent homologous regions so that the additional nucleic acid sequence or modified nucleic acid sequence is incorporated into the target gene locus. MMEJ yields genetic results similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ achieves the desired end-join DNA repair result by utilizing homologous sequences consisting of several base pairs adjacent to the cleavage site. In some cases, the expected repair result can be predicted by analyzing the short homologous sequences (microhomology) predicted in the target region of the nuclease.

[0209] Therefore, in some cases, homologous recombination is used to insert an exogenous polynucleotide sequence into the cleavage site of the target nucleic acid. Hereinafter, the exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor, donor sequence, or polynucleotide donor template). In some embodiments, a donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide is inserted into the cleavage site of the target nucleic acid. In some embodiments, the donor polynucleotide is an exogenous polynucleotide sequence, for example, a sequence that is not naturally present at the cleavage site of the target nucleic acid.

[0210] If a sufficient concentration of exogenous DNA molecules is supplied to the nucleus of a cell where a double-strand break occurs, the exogenous DNA can be inserted into the double-strand break site during the NHEJ repair process, potentially leading to permanent addition to the genome. Such exogenous DNA molecules are referred to as donor templates in some embodiments. If the donor template contains the coding sequence of a target gene, such as the FOXP3 gene (also referred to herein as a “donor cassette”), along with relevant regulatory sequences such as promoters, enhancers, polyA sequences, and / or splice acceptor sequences as needed, the target gene can be expressed from the integrated copy in the genome and may be permanently expressed for the duration of the cell’s life. Furthermore, the integrated copy from the donor DNA template can be transmitted to daughter cells when the cell divides.

[0211] If a donor DNA template containing adjacent DNA sequences homologous to the DNA sequences on both sides of a double-strand break site (called homologous arms) is present in sufficient concentration, this donor DNA template can be incorporated via the HDR pathway. The homologous arms act as substrates for homologous recombination between the donor template and the sequences on both sides of the double-strand break site. This allows for error-free insertion of a donor template in which the sequences on both sides of the double-strand break site have not been altered from the sequences of the unmodified genome.

[0212] Donors provided for HDR editing are highly diverse, but generally, they contain the intended editing sequence and short or long homologous arms on either side, enabling annealing to genomic DNA. The homologous region adjacent to the introduced gene alteration site may be less than 30 bp, or it may be as large as a few kilobase cassette, which may also contain promoters or cDNA. Both single-stranded and double-stranded oligonucleotide donors can be used. The size of these oligonucleotides ranges from less than 100 nt to several kilobases or more, but longer ssDNA can also be generated and used. Double-stranded donors such as PCR amplicons, plasmids, and minicircles are commonly used. Generally, AAV vectors have been shown to be a very effective means of delivering donor templates, but the limit for packaging to individual donors is less than 5 kb. Active transcription of the donor has been shown to triple the HDR rate, indicating that conversion can be increased by including a promoter. Conversely, CpG methylation of the donor may decrease gene expression and HDR rate.

[0213] In some embodiments, donor DNA can be introduced alone or together with a nuclease by various methods, such as transfection, nanoparticles, microinjection, or viral transduction. In some embodiments, the availability of donor in HDR can be enhanced by using various methods for linking donor DNA and nuclease. Examples of such methods include linking the donor to a nuclease, linking it to a DNA-binding protein that binds to the vicinity of the donor and nuclease, or linking it to a protein involved in DNA end joining or DNA repair.

[0214] In addition to genome editing using NHEJ or HDR, site-directed gene insertion can be performed using both the NHEJ pathway and HR. Such combined techniques are applicable in specific situations that may involve intron / exon boundaries. NHEJ has been demonstrated to be effective for ligation in introns, while error-free HDR may be more suitable for coding regions.

[0215] In some embodiments, the exogenous sequence intended for insertion into the genome is a nucleotide sequence encoding FOXP3 or a functional derivative thereof. Examples of functional derivatives of FOXP3 include FOXP3 derivatives having substantially equivalent activity to wild-type FOXP3 (e.g., wild-type human FOXP3), such as FOXP3 derivatives exhibiting at least 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, 95% or about 95%, or at least 100% or about 100% of the activity of wild-type FOXP3. In some embodiments, functional derivatives of FOXP3 may have at least 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 85% or about 85%, 90% or at least 90%, 95% or at least about 95%, 96% or at least about 96%, 97% or at least about 97%, 98% or at least about 98%, or 99% or at least about 99% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, those skilled in the art can test the functionality or activity of the compound (e.g., peptides or proteins) using various methods known in the art. Further examples of functional derivatives of FOXP3 include fragments of modified FOXP3 or fragments of wild-type FOXP3 having conservative modifications to one or more amino acid residues of the full length of wild-type FOXP3.Therefore, in some embodiments, the nucleic acid sequence encoding a functional derivative of FOXP3 may have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 is human wild-type FOXP3.

[0216] In some embodiments in which nucleic acids encoding FOXP3 or its functional derivatives are inserted, the cDNA of the FOXP3 gene or its functional derivative can be inserted into the genome of a subject having an abnormal FOXP3 gene or its regulatory sequence. In such cases, the donor DNA or donor template may be an expression cassette or vector construct containing a sequence (e.g., a cDNA sequence) encoding FOXP3 or its functional derivative.

[0217] In some embodiments of the donor template described herein, which includes a donor cassette, the donor cassette is either adjacent to a gRNA target site at one end or adjacent to gRNA target sites at both ends. For example, such a donor template may include a donor cassette having a gRNA target site at the 5' end and / or a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' end. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' end and a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' end and a gRNA target site at the 3' end, wherein these two gRNA target sites contain the same sequence. In some embodiments, the donor template includes at least one gRNA target site, the at least one gRNA target site in the donor template includes the same sequence as the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. In some embodiments, the donor template includes at least one gRNA target site, the at least one gRNA target site in the donor template includes the reverse complementary strand of the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' end and a gRNA target site at the 3' end, the two gRNA target sites in the donor template include the same sequence as the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. In some embodiments, the donor template comprises a donor cassette having a gRNA target site at its 5' end and a gRNA target site at its 3' end, wherein these two gRNA target sites in the donor template comprise the reverse complementary strands of the gRNA target sites in the target gene locus into which the donor cassette of the donor template is incorporated.

[0218] In some embodiments, a donor template is provided for targeted incorporation into the FOXP3 locus, comprising a nucleotide sequence encoding FOXP3 or a functional derivative thereof, wherein the donor template comprises, from 5' to 3', i) a first gRNA target site; ii) a splice acceptor; iii) a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and iv) a polyadenylation signal. In some embodiments, the donor template further comprises a second gRNA target site downstream of the polyadenylation signal. In some embodiments, the first gRNA target site and the second gRNA target site are the same. In some embodiments, the donor template further comprises a polynucleotide spacer between i) the first gRNA target site and ii) the splice acceptor. In some embodiments, the polynucleotide spacer is 18 nucleotides long. In some embodiments, the donor template has one end adjacent to a first AAV ITR and / or the other end adjacent to a second AAV ITR. In some embodiments, the first AAV ITR is AAV2 ITR and / or the second AAV ITR is AAV2 ITR. In some embodiments, FOXP3 is human wild-type FOXP3.

[0219] Nucleic acids encoding site-directed polypeptides or DNA endonucleases In some embodiments, based on the foregoing, nucleic acid sequences (or oligonucleotides) encoding site-directed polypeptides or DNA endonucleases can be used in the genome editing method and the composition. The nucleic acid sequence encoding the site-directed polypeptide may be DNA or RNA. If the nucleic acid sequence encoding the site-directed polypeptide is RNA, this RNA can be covalently bound to a gRNA sequence or exist as a separate sequence. In some embodiments, peptide sequences of site-directed polypeptides or DNA endonucleases can be used instead of these nucleic acid sequences.

[0220] vector In another embodiment, the Disclosure provides nucleic acids having a nucleotide sequence encoding the genome-targeted nucleic acid of the Disclosure, site-directed polypeptides of the Disclosure, and / or any nucleic acid or protein molecule necessary to carry out embodiments of the methods of the Disclosure. In some embodiments, such nucleic acids are vectors (e.g., recombinant expression vectors).

[0221] Examples of expression vectors envisioned in the present invention include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, Simian virus 40 (SV40), herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., vectors derived from retroviruses such as mouse leukemia virus; splenic necrosis virus; and Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary cancer virus), and other recombinant vectors. Other vectors envisioned for use in eukaryotic target cells include, but are not limited to, pXT1 vector, pSG5 vector, pSVK3 vector, pBPV vector, pMSG vector, and pSVLSV40 vector (Pharmacia). Further vectors envisioned for use in eukaryotic target cells include, but are not limited to, pCTx-1 vector, pCTx-2 vector, and pCTx-3 vector. Other vectors may also be used as long as they are compatible with the host cell.

[0222] In some embodiments, the vector has one or more transcriptional and / or translational regulatory elements. Depending on the host / vector system used, various suitable transcriptional and translational regulatory elements, such as constitutive promoters, inducible promoters, transcriptional enhancer elements, and transcriptional terminators, can be used in the expression vector. In some embodiments, the vector is a self-inactivating vector that inactivates a viral sequence or components of the CRISPR mechanism or other elements.

[0223] Examples of suitable eukaryotic promoters (e.g., promoters that function in eukaryotic cells) include, but are not limited to, the cytomegalovirus (CMV) promoter, the earliest thymidine kinase promoter of herpes simplex virus (HSV), early SV40, late SV40, retroviral long terminal repeats (LTRs), human elongation factor 1 promoter (EF1), a hybrid construct fused with the cytomegalovirus (CMV) enhancer to the chicken β-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase 1 locus promoter (PGK), and mouse metallothionein I.

[0224] Various promoters, such as RNA polymerase III promoters like U6 and H1, can be useful for expressing small RNAs, including guide RNAs used with Cas endonucleases. Descriptions and parameters that facilitate the use of such promoters are well-known in the art, and new information and approaches are constantly being reported. See, for example, Ma, H. et al. (2014). Molecular Therapy - Nucleic Acids 3:e161, doi:10.1038 / mtna.2014.12.

[0225] The expression vector may further contain ribosome binding sites for translation initiation and transcription termination. It may also contain appropriate sequences for amplification of expression. Furthermore, because the expression vector is fused to a site-directed polypeptide, it may contain nucleotide sequences encoding non-natural tags (e.g., histidine tags, hemagglutinin tags, green fluorescent protein, etc.) that are expressed as part of the fusion protein.

[0226] In some embodiments, the promoter is an inductive promoter (e.g., a heat shock promoter, a tetracycline-regulating promoter, a steroid-regulating promoter, a metal-regulating promoter, an estrogen receptor-regulating promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., a CMV promoter, or a UBC promoter). In some embodiments, the promoter is a spatially restricted promoter and / or a temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). In some embodiments, if at least one gene expressed in a host cell is inserted into the genome and then expressed under the control of an endogenous promoter present in that genome, the vector does not contain a promoter for this gene.

[0227] In some embodiments, the first vector may encode a first CISC component comprising a first extracellular binding domain or a portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or a portion thereof, and the second vector may encode a second CISC component comprising a second extracellular binding domain or a portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or a portion thereof.

[0228] In some embodiments, the expression vector includes a nucleic acid encoding a protein sequence shown in any of SEQ ID NOs: 48-61. In some embodiments, the expression vector includes a nucleic acid sequence shown in SEQ ID NO: 67. SEQ ID NO: 67 encodes a protein sequence shown in SEQ ID NO: 54.

[0229] In some embodiments, the expression vector is a variant of SEQ ID NO: 67, shown in SEQ ID NO: 65. SEQ ID NO: 65 encodes the protein sequences shown in SEQ ID NOs: 50 and 51.

[0230] In some embodiments, the expression vector is a variant of SEQ ID NO: 67, shown in SEQ ID NO: 66. SEQ ID NO: 66 encodes the protein sequences shown in SEQ ID NOs: 52 and 53.

[0231] In some embodiments, the expression vector comprises a nucleic acid having at least 80%, 85%, 90%, 95%, 98%, or 99% nucleic acid sequence identity (or nucleic acid sequence identity (%) within the range defined by any two of these percentages) with the nucleotide sequence provided herein, or a specifically induced fragment thereof. In some embodiments, the expression vector comprises a promoter. In some embodiments, the expression vector comprises the nucleic acid encoding a fusion protein. In some embodiments, the vector is RNA or DNA.

[0232] Site-directed polypeptide or DNA endonuclease Modification of target DNA by NHEJ and / or HDR can result in, for example, mutations, deletions, alterations, integrations, gene modifications, gene substitutions, gene tagging, transgene insertions, nucleotide deletions, gene disruption, translocations, and / or gene mutations. The integration of non-native nucleic acids into genomic DNA is an example of genome editing.

[0233] Site-directed polypeptides are nucleases used in genome editing to cleave DNA. Site-directed polypeptides can be administered to cells or subjects as one or more polypeptides, or as one or more mRNAs encoding such polypeptides.

[0234] In relation to the CRISPR / Cas system or the CRISPR / Cpf1 system, the site-directed polypeptide binds to a guide RNA, thereby allowing the guide RNA to identify the site on the target DNA to which the site-directed polypeptide is directed. In the embodiments of the CRISPR / Cas system or the CRISPR / Cpf1 system described herein, the site-directed polypeptide is an endonuclease, such as a DNA endonuclease.

[0235] In some embodiments, the site-directed polypeptide has multiple nucleic acid cleavage domains (e.g., nuclease sites). Two or more nucleic acid cleavage domains can be linked via a linker. In some embodiments, the linker is flexible. The length of the linker may be 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, 9 amino acid length, 10 amino acid length, 11 amino acid length, 12 amino acid length, 13 amino acid length, 14 amino acid length, 15 amino acid length, 16 amino acid length, 17 amino acid length, 18 amino acid length, 19 amino acid length, 20 amino acid length, 21 amino acid length, 22 amino acid length, 23 amino acid length, 24 amino acid length, 25 amino acid length, 30 amino acid length, 35 amino acid length, 40 amino acid length, or longer.

[0236] The naturally occurring wild-type Cas9 enzyme has two nuclease domains: an HNH nuclease domain and a RuvC domain. The Cas9 enzyme as envisioned herein has an HNH nuclease domain or an HNH-like nuclease domain, and / or a RuvC nuclease domain or a RuvC-like nuclease domain.

[0237] The HNH domain or HNH-like domain has McrA-like folding. The HNH domain or HNH-like domain has two antiparallel β-chains and one α-helix. The HNH domain or HNH-like domain has a metal-binding site (e.g., a divalent cation-binding site). The HNH domain or HNH-like domain can cleave a single strand of the target nucleic acid (e.g., the complementary strand of the target strand of crRNA).

[0238] RuvC domains or RuvC-like domains possess RNaseH folding or RNaseH-like folding. RuvC domains or RNaseH domains are involved in a diverse range of nucleic acid-based functions, including actions on both RNA and DNA. RNaseH domains have five β-chains surrounded by multiple α-helices. RuvC domains or RNaseH domains or RuvC-like domains or RNaseH-like domains possess metal-binding sites (e.g., divalent cation-binding sites). RuvC domains or RNaseH domains or RuvC-like domains or RNaseH-like domains can cleave a single strand of target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0239] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity with a typical wild-type site-directed polypeptide ([e.g., Cas9 from S. pyogenes, SEQ ID NO: 8 described in US2014 / 0068797, or Cas9 described in Sapranauskas, R. et al. (2011). Nucleic Acids Res, 39(21): 9275-9282], and various other site-directed polypeptides).

[0240] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity with the nuclease domain of a typical wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes (shown above)).

[0241] In some embodiments, the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. In some embodiments, the site-directed polypeptide has at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. In some embodiments, the HNH nuclease domain of the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. In some embodiments, the RuvC nuclease domain of the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids.

[0242] In some embodiments, the site-directed polypeptide has a modified form of a typical wild-type site-directed polypeptide. The modified form of a typical wild-type site-directed polypeptide has mutations that reduce the nucleic acid cleavage activity of the site-directed polypeptide. In some embodiments, the modified form of a typical wild-type site-directed polypeptide has nucleic acid cleavage activity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of a typical wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes mentioned above). In some embodiments, the modified form of the site-directed polypeptide may have substantially no nucleic acid cleavage activity. When the site-directed polypeptide has a modified form that has substantially no nucleic acid cleavage activity, such a modified form is referred to herein as "enzymatically inactive."

[0243] In some embodiments, the modification of the site-directed polypeptide has a mutation that can induce a single-strand break (SSB) on the target nucleic acid (for example, by cleaving only one of the sugar-phosphate backbones of the double-stranded target nucleic acid). In some embodiments, this mutation results in a nucleic acid cleavage activity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of one or more of the multiple nucleic acid cleavage domains of the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes as described above). In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave the non-complementary strand of the target nucleic acid while retaining the ability to cleave the complementary strand. In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave the complementary strand of the target nucleic acid while retaining the ability to cleave the non-complementary strand. For example, mutations occur in which one or more nucleic acid cleavage domains (e.g., nuclease domains) are inactivated at typical wild-type S. pyogenes Cas9 polypeptide residues such as Asp10, His840, Asn854, and Asn856. In some embodiments, the residues to which mutations are induced correspond to the Asp10, His840, Asn854, and Asn856 residues of a typical wild-type S. pyogenes Cas9 polypeptide (as determined, for example, by sequence and / or structural alignment). Examples of such mutations include, but are not limited to, D10A, H840A, N854A, or N856A. Those skilled in the art will understand that mutations other than alanine substitutions are also appropriate.

[0244] In some embodiments, the D10A mutation, when combined with one or more of the H840A, N854A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the H840A mutation, when combined with one or more of the D10A, N854A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N854A mutation, when combined with one or more of the H840A, D10A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N856A mutation, when combined with one or more of the H840A, N854A, and D10A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. Site-directed polypeptides having a substantially inactive single nuclease domain are called "nickases".

[0245] In some embodiments, variants of RNA-inducible endonucleases (e.g., Cas9) can be used to enhance the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is typically led by a single-strand guide RNA designed to hybridize with a specific sequence of about 20 nucleotides in the target sequence (such as an endogenous genomic locus). However, since a few mismatches can be tolerated between the guide RNA and the target locus, the required homologous sequence length at the target site may be efficiently shortened to, for example, about 13 nt, thereby increasing the likelihood that the CRISPR / Cas9 complex will bind to another location in the target genome and cleave a double-strand nucleic acid. This is also known as an off-target cleavage. On the other hand, since Cas9 nickase variants each cleave only one strand, a pair of nickases must bind in close proximity on opposite strands of the target nucleic acid to produce a double-strand break, thereby creating a pair of nicks and resulting in a double-strand break. This requires two separate guide RNAs (one for each nickase) to bind in close proximity on opposite strands of the target nucleic acid. Adhering to this requirement effectively doubles the minimum length of homologous sequence needed to produce a double-strand break, thus reducing the likelihood of the double-strand break occurring elsewhere in the genome, as the two guide RNA sites (if present) are less likely to be close enough to each other to produce a double-strand break. As reported in the art, nickases can also be used to facilitate HDR rather than NHEJ. By performing HDR using a specific donor sequence that effectively mediates the desired modification, the selected modification can be introduced into a target site in the genome. Descriptions of various CRISPR / Cas systems for use in gene editing are found, for example, in WO2013 / 176772 and Sander, JD et al. (2014). Nature Biotechnology, 32(4):347-355, as well as the references cited in these publications.

[0246] In some embodiments, site-directed polypeptides (e.g., variants of site-directed polypeptides, variants of site-directed polypeptides, enzymatically inactive site-directed polypeptides, and / or site-directed polypeptides enzymatically inactive under specific conditions) target nucleic acids. In some embodiments, site-directed polypeptides (e.g., variants of endoribonucleases, variants of endoribonucleases, enzymatically inactive endoribonucleases, and / or enzymatically inactive endoribonucleases under specific conditions) target DNA. In some embodiments, site-directed polypeptides (e.g., variants of endoribonucleases, variants of endoribonucleases, enzymatically inactive endoribonucleases, and / or enzymatically inactive endoribonucleases under specific conditions) target RNA.

[0247] In some embodiments, the site-directed polypeptide has one or more non-natural sequences (for example, the site-directed polypeptide is a fusion protein).

[0248] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), a nucleic acid-binding domain, and two nucleic acid-cleaving domains (such as an HNH domain and a RuvC domain).

[0249] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains (such as an HNH domain and a RuvC domain).

[0250] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains, one or both of which have at least 50% amino acid identity with the nuclease domain of Cas9 derived from bacteria (e.g., S. pyogenes).

[0251] In some embodiments, the site-directed polypeptide comprises an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), and a linker that anneals a non-natural sequence (e.g., a nuclear localization signal) or the site-directed polypeptide to the non-natural sequence.

[0252] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), with one or both of the nucleic acid cleavage domains having a mutation that reduces the cleavage activity of these nuclease domains by at least 50%.

[0253] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), with a mutation at the 10th aspartic acid position of one of the nuclease domains and / or a mutation at the 840th histidine position of one of the nuclease domains, the mutation reducing the cleavage activity of the nuclease domain by at least 50%.

[0254] In some embodiments, one or more site-directed polypeptides (e.g., DNA endonucleases) comprise two nickases that cooperate to produce one double-strand break at a specific locus in the genome, or one or more site-directed polypeptides comprise four nickases that cooperate to produce two double-strand breaks at a specific locus in the genome, or one site-directed polypeptide (e.g., DNA endonuclease) acts to produce one double-strand break at a specific locus in the genome.

[0255] In some embodiments, polynucleotides encoding site-directed polypeptides can be used for genome editing. In some such embodiments, the polynucleotide encoding the site-directed polypeptide is codon-optimized for expression in cells containing the target DNA of interest, according to methods known in the art. For example, if the target nucleic acid of interest is present in human cells, it is conceivable that a polynucleotide encoding Cas9 could be optimized for human codons and used to construct the Cas9 polypeptide.

[0256] The following provides some examples of site-directed polypeptides that can be used in various embodiments of this disclosure.

[0257] CRISPR Endonuclease System CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (such as bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a type of immune system useful in defending prokaryotes from foreign invaders such as viruses and phages. The function of a CRISPR locus consists of three stages: incorporation of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invading nucleic acids. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

[0258] The CRISPR locus contains numerous short repeat sequences called "repetitive sequences." When expressed, these repeat sequences can form secondary hairpin structures (e.g., hairpins) and / or single-stranded sequences without a fixed structure. Repetitive sequences usually occur as clusters and vary considerably between species. The repeat sequences are spaced at regular intervals by unique intervening sequences called "spacers," forming a locus with a repeat sequence-spacer-repetitive sequence structure. The spacers are either identical to or highly homologous to known invading sequences. The unit consisting of spacers and repeat sequences codes for crisprRNA (crRNA), which is processed to obtain the mature form of the unit consisting of spacers and repeat sequences. crRNA has a "seed" sequence or spacer sequence involved in targeting the target nucleic acid (in its natural form in prokaryotes, the spacer sequence targets the nucleic acid of the invader). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0259] The CRISPR locus further contains polynucleotide sequences encoding CRISPR-related (Cas) genes. Cas genes encode endonucleases involved in the biosynthetic and interference stages of crRNA function in prokaryotes. Some Cas genes have homologous secondary and / or tertiary structures.

[0260] Type II CRISPR System In the biosynthesis of crRNA in the naturally occurring type II CRISPR system, trans-activated CRISPR RNA (tracrRNA) is required. The tracrRNA is modified by endogenous RNase III and then hybridizes to the crRNA repeat sequence in the pre-crRNA array. Endogenous RNase III is recruited to cleave the pre-crRNA. The cleaved crRNA is trimmed by exoribonuclease (e.g., 5' end trimming) to produce the mature form of crRNA. The tracrRNA remains hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-directed polypeptide (e.g., Cas9). In the crRNA-tracrRNA-Cas9 complex, the complex is led by the crRNA to a target nucleic acid that the crRNA can hybridize to. Hybridization of the crRNA to the target nucleic acid activates Cas9, which then cleaves the target nucleic acid. In the type II CRISPR system, the target nucleic acid is called a protospacer adjacent motif (PAM). In fact, PAM is essential for facilitating the binding of site-directed polypeptides (e.g., Cas9) to target nucleic acids. Type II systems (also known as Nmeni or CASS4) are further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek, M. et al. (2012). Science, 337(6096):816-821 demonstrates the usefulness of the CRISPR / Cas9 system for RNA-programmable genome editing, and WO 2013 / 176772 further describes numerous examples and applications of the CRISPR / Cas endonuclease system for site-directed gene editing.

[0261] V-type CRISPR system The type V CRISPR system has several key differences from the type II system. For example, Cpf1 is a single-chain RNA-inducible endonuclease, but unlike the type II system, it lacks tracrRNA. In fact, a CRISPR array associated with Cpf1 processes into mature crRNA without requiring further transactivated tracrRNA. The type V CRISPR array processes into short mature crRNAs of 42–44 nucleotides in length, each mature crRNA beginning with a 19-nucleotide direct repeat sequence followed by a 23–25 nucleotide spacer sequence. In contrast, mature crRNAs in the type II system begin with a 20–24 nucleotide spacer sequence followed by a 22-nucleotide or approximately 22-nucleotide direct repeat sequence. Furthermore, Cpf1 utilizes a T-rich protospacer flanking motif, allowing the target DNA following this short T-rich PAM to be efficiently cleaved by the Cpf1-crRNA complex. This is in contrast to the type II system, where a G-rich PAM following the target DNA is utilized. Therefore, the type V system cleaves the target at a position away from the PAM, while the type II system cleaves the target adjacent to the PAM. Furthermore, in contrast to the type II system, Cpf1 cleaves the DNA double strand at a shifted position, resulting in a 4-nucleotide or 5-nucleotide 5' end overhang. In contrast, double-strand breaks by the type II system result in blunt ends. Cpf1 is predicted to contain a RuvC-like endonuclease domain, similar to the type II system, but unlike the type II system, it lacks a second HNH endonuclease domain.

[0262] Cas gene / polypeptide and protospacer adjacent motif A typical CRISPR / Cas polypeptide is the Cas9 polypeptide, shown in Figure 1 of Fonfara, I. et al. (2014). Nucleic Acids Research, 42(4):2577-2590. The CRISPR / Cas gene naming system has undergone significant revisions since the discovery of the Cas gene. Figure 5 by Fonfara et al. (2014) shows PAM sequences for Cas9 polypeptides derived from various species.

[0263] A complex of genome-targeted nucleic acids and site-directed polypeptides Genome-targeted nucleic acids form complexes by interacting with site-directed polypeptides (e.g., nucleic acid-inducible nucleases such as Cas9). The genome-targeted nucleic acid (e.g., gRNA) leads the site-directed polypeptide to the target nucleic acid.

[0264] As described above, in some embodiments, site-directed polypeptides and genome-targeted nucleic acids can be administered to cells or subjects separately. On the other hand, in some other embodiments, site-directed polypeptides can be pre-complexed with one or more guide RNAs, or site-directed polypeptides can be pre-complexed with tracrRNA and one or more crRNAs. The pre-complexed material can be administered to cells or subjects. Such pre-complexed material is known as ribonucleoprotein particles (RNPs).

[0265] CISC component As described herein, in some embodiments, one or more protein sequences are provided that encode dimerizable CISC components. The one or more protein sequences may have a first sequence and a second sequence. In some embodiments, the first sequence encodes a first CISC component which may include a first extracellular binding domain or part thereof, a hinge domain, a transmembrane domain, and a signaling domain or part thereof. In some embodiments, the second sequence encodes a second CISC component which may include a second extracellular binding domain or part thereof, a hinge domain, a transmembrane domain, and a signaling domain or part thereof. In some embodiments, the first and second CISC components may be configured to dimerize, preferably simultaneously, in the presence of a ligand when expressed.

[0266] In some embodiments, we provide protein sequences of two heterodimerizing CISC components, or sequences encoding two heterodimerizing CISC components. In some embodiments, the first CISC component is the IL2Rγ-CISC complex.

[0267] In some embodiments, the IL2Rγ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 48. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 48.

[0268] In some embodiments, the IL2Rγ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 50. The embodiments also include a nucleic acid sequence encoding the protein sequence of SEQ ID NO: 50.

[0269] In some embodiments, the IL2Rγ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 52. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 52.

[0270] In some embodiments, the IL2Rγ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 54. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 54.

[0271] In some embodiments, the protein sequence of the first CISC component includes a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Nucleic acid sequences encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain are also included in the embodiments. In some embodiments, the protein sequence of the first CISC component, including the first extracellular binding domain, a hinge domain, a transmembrane domain, and / or a signaling domain, includes an amino acid sequence having sequence identity within a range defined by 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity, or any two of these percentages, with respect to the sequence shown in SEQ ID NOs.

[0272] In some embodiments, the second CISC component is a complex containing IL2Rβ. In some embodiments, the IL2Rβ-CISC complex contains the amino acid sequence shown in SEQ ID NO: 49. The nucleic acid sequence encoding the protein sequence of SEQ ID NO: 49 is also included in the embodiments.

[0273] In some embodiments, the IL2Rβ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 51. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 51.

[0274] In some embodiments, the IL2Rβ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 53. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 53.

[0275] In some embodiments, the IL2Rβ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 55. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 55.

[0276] In some embodiments, the second CISC component is a complex containing IL7Rα. In some embodiments, the IL7Rα-CISC complex includes the amino acid sequence shown in SEQ ID NO: 56. The embodiments also include a nucleic acid sequence encoding the protein sequence of SEQ ID NO: 56.

[0277] In some embodiments, the protein sequence of the second CISC component includes a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Nucleic acid sequences encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain of the second CISC component are also included in the embodiments. In some embodiments, the protein sequence of the second CISC component, including the second extracellular binding domain, a hinge domain, a transmembrane domain, and / or a signaling domain, includes an amino acid sequence having sequence identity within a range defined by 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity, or any two of these percentages, with respect to the sequence shown in SEQ ID NOs.

[0278] In some embodiments, the protein sequence may include a linker. In some embodiments, the linker contains one, two, three, four, five, six, seven, eight, nine, or ten amino acids (such as glycine), or a large number of amino acids (such as glycine), or a number of amino acids within a range defined by any two of these numbers. In some embodiments, the glycine spacer contains at least three glycine molecules. In some embodiments, the glycine spacer contains the sequence shown in SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. Nucleic acid sequences encoding SEQ ID NOs: 62-64 are also included in the embodiments. In some embodiments, the transmembrane domain is located at the N-terminus of the signaling domain, the hinge domain is located at the N-terminus of the transmembrane domain, the linker is located at the N-terminus of the hinge domain, and the extracellular binding domain is located at the N-terminus of the linker.

[0279] In some embodiments, the present invention provides protein sequences of two homodimerizing CISC components, or sequences encoding two homodimerizing CISC components. In some embodiments, the first CISC component is an IL2Rγ-CISC complex. In some embodiments, the IL2Rγ-CISC complex includes the amino acid sequence shown in SEQ ID NO: 58. The embodiments also include nucleic acid sequences encoding the protein sequence of SEQ ID NO: 58.

[0280] In some embodiments, the protein sequence of the first CISC component includes a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Nucleic acid sequences encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain are also included in the embodiments. In some embodiments, the protein sequence of the first CISC component, including the first extracellular binding domain, a hinge domain, a transmembrane domain, and / or a signaling domain, includes an amino acid sequence having sequence identity within a range defined by 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity, or any two of these percentages, with respect to the sequence shown in Sequence ID No. 58.

[0281] In some embodiments, the second CISC component is a complex containing IL2Rβ or a complex containing IL2Rα. In some embodiments, the IL2Rβ-CISC complex contains the amino acid sequence shown in SEQ ID NO: 57. The nucleic acid sequence encoding the protein sequence of SEQ ID NO: 57 is also included in the embodiments.

[0282] In some embodiments, the IL2Rα-CISC complex includes the amino acid sequence shown in SEQ ID NO: 59. The embodiments also include the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 59.

[0283] In some embodiments, the protein sequence of the second CISC component includes a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Nucleic acid sequences encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain of the second CISC component are also included in the embodiments. In some embodiments, the protein sequence of the second CISC component, including the second extracellular binding domain, a hinge domain, a transmembrane domain, and / or a signaling domain, includes an amino acid sequence having sequence identity within a range defined by 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity, or any two of these percentages, with respect to the sequence shown in SEQ ID NO: 57 or SEQ ID NO: 59.

[0284] In some embodiments, the protein sequence may include a linker. In some embodiments, the linker contains one, two, three, four, five, six, seven, eight, nine, or ten amino acids (such as glycine), or a large number of amino acids (such as glycine), or a number of amino acids within a range defined by any two of these numbers. In some embodiments, the glycine spacer contains at least three glycine molecules. In some embodiments, the glycine spacer contains the sequence shown in SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. Nucleic acid sequences encoding SEQ ID NOs: 62-64 are also included in the embodiments. In some embodiments, the transmembrane domain is located at the N-terminus of the signaling domain, the hinge domain is located at the N-terminus of the transmembrane domain, the linker is located at the N-terminus of the hinge domain, and the extracellular binding domain is located at the N-terminus of the linker.

[0285] In some embodiments, the sequences encoding the two homodimerizing CISC components incorporate an FKBP F36V domain that homodimerizes with the ligand AP1903.

[0286] In some embodiments, a protein sequence of a single homodimerizable CISC component or a sequence encoding a single homodimerizable CISC component is provided. In some embodiments, the single CISC component is an IL7Rα-CISC complex. In some embodiments, the IL7Rα-CISC complex includes the amino acid sequence shown in SEQ ID NO: 60. The embodiments also include a nucleic acid sequence encoding the protein sequence of SEQ ID NO: 60.

[0287] In some embodiments, the single CISC component is an MPL-CISC complex. In some embodiments, the MPL-CISC complex includes the amino acid sequence shown in SEQ ID NO: 61. The embodiments also include a nucleic acid sequence encoding the protein sequence of SEQ ID NO: 61.

[0288] In some embodiments, the protein sequence of the single CISC component includes a protein sequence encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain. Nucleic acid sequences encoding an extracellular binding domain, a hinge domain, a transmembrane domain, or a signaling domain are also included in the embodiments. In some embodiments, the protein sequence of the first CISC component, comprising a first extracellular binding domain, a hinge domain, a transmembrane domain, and / or a signaling domain, includes an amino acid sequence having sequence identity within a range defined by 100%, 99%, 98%, 95%, 90%, 85%, or 80% sequence identity, or any two of these percentages, with respect to the sequence shown in SEQ ID NO: 60 or SEQ ID NO: 61.

[0289] In some embodiments, the protein sequence may include a linker. In some embodiments, the linker contains one, two, three, four, five, six, seven, eight, nine, or ten amino acids (such as glycine), or a large number of amino acids (such as glycine), or a number of amino acids within a range defined by any two of these numbers. In some embodiments, the glycine spacer contains at least three glycine molecules. In some embodiments, the glycine spacer contains the sequence shown in SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. Nucleic acid sequences encoding SEQ ID NOs: 62-64 are also included in the embodiments. In some embodiments, the transmembrane domain is located at the N-terminus of the signaling domain, the hinge domain is located at the N-terminus of the transmembrane domain, the linker is located at the N-terminus of the hinge domain, and the extracellular binding domain is located at the N-terminus of the linker.

[0290] In some embodiments, the sequence encoding a single homodimerizable CISC component incorporates an FKBP F36V domain that homodimerizes with the ligand AP1903.

[0291] Genome editing methods In organisms requiring the expression of the FOXP3 protein or its functional derivatives, one method for expressing the FOXP3 protein or its functional derivatives involves targeting an endogenous FOXP3 gene or a non-FOXP3 gene that is sufficiently expressed in an associated cell type (e.g., T cells) using genome editing, such that a nucleic acid containing a coding sequence encoding the FOXP3 protein is incorporated into the non-FOXP3 gene. The expression of the incorporated coding sequence is then induced by the endogenous promoter of the endogenous FOXP3 gene or the non-FOXP3 gene. In some embodiments in which the non-FOXP3 gene is targeted, the expression of the non-FOXP3 gene is controlled to prevent expression in unrelated cell types, such as targeting the cell type (e.g., lymphocytes, CD4+ cells such as CD4+ T cells, or cells derived therefrom, e.g., T cells). regIt is desirable that it be specific to the cell.

[0292] In some embodiments, the knock-in method involves knocking in a sequence encoding FOXP3 or a functional derivative of FOXP3 into a genomic sequence, such as a wild-type FOXP3 gene (e.g., a wild-type human FOXP3 gene), FOXP3 cDNA, or a FOXP3 minigene (having a natural or synthetic enhancer and promoter, one or more exons, a natural or synthetic intron, and a natural or synthetic 3'UTR and polyadenylation signal). In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located at, within, or near the FOXP3 locus. In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located at, within, or near exon 1 of the FOXP3 locus.

[0293] In some embodiments provided herein, a method for knocking in a sequence encoding FOXP3 or a functional derivative thereof into a genome is provided. In one embodiment, the disclosure provides insertion of a nucleic acid comprising a sequence encoding FOXP3 or a functional derivative thereof into a cellular genome. In some embodiments, the sequence encoding FOXP3 encodes wild-type FOXP3. Functional derivatives of FOXP3 include FOXP3 derivatives having substantially equivalent activity to wild-type FOXP3 (e.g., wild-type human FOXP3), such as FOXP3 derivatives exhibiting at least 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, 95% or about 95%, or at least 100% or about 100% of the activity of wild-type FOXP3. In some embodiments, the functional derivatives of FOXP3 have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 is encoded by an intron-deleting nucleotide sequence (e.g., FOXP3 cDNA). Those skilled in the art can test the functionality or activity of derivatives of FOXP using methods known in the art. Further examples of functional derivatives of FOXP3 include wild-type FOXP3 fragments having modifications to one or more amino acid residues of the full length of wild-type FOXP3.Therefore, in some embodiments, the nucleic acid sequence encoding a functional derivative of FOXP3 may have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 or its functional variant is human wild-type FOXP3.

[0294] In some embodiments, the genome editing method of the present invention utilizes a DNA endonuclease such as CRISPR / Cas endonuclease to genetically introduce (knock in) a sequence encoding FOXP3 or a functional derivative thereof. In some manner, the DNA endonucleases include Cas1 endonuclease, Cas1B endonuclease, Cas2 endonuclease, Cas3 endonuclease, Cas4 endonuclease, Cas5 endonuclease, Cas6 endonuclease, Cas7 endonuclease, Cas8 endonuclease, Cas9 endonuclease (also known as Csn1 and Csx12), Cas100 endonuclease, Csy1 endonuclease, Csy2 endonuclease, Csy3 endonuclease, Cse1 endonuclease, Cse2 endonuclease, Csc1 endonuclease, Csc2 endonuclease, Csa5 endonuclease, Csn2 endonuclease, Csm2 endonuclease, Csm3 endonuclease, Csm4 endonuclease, and Csm5 endonuclease. Endonuclease, Csm6 endonuclease, Cmr1 endonuclease, Cmr3 endonuclease, Cmr4 endonuclease, Cmr5 endonuclease, Cmr6 endonuclease, Csb1 endonuclease, Csb2 endonuclease, Csb3 endonuclease, Csx17 endonuclease, Csx14 endonuclease, Csx10 endonuclease, Csx16 endonuclease The DNA endonucleases are CsaX endonucleases, Csx3 endonucleases, Csx1 endonucleases, Csx15 endonucleases, Csf1 endonucleases, Csf2 endonucleases, Csf3 endonucleases, Csf4 endonucleases, or Cpf1 endonucleases, their homologs, recombinants of natural molecules, codon-optimized or modified versions thereof, or any combination thereof. In some embodiments, the DNA endonucleases are Cas9. In some embodiments, the Cas9 are Cas9 derived from Streptococcus pyogenes (spCas9).In some embodiments, the Cas9 is Cas9 derived from Staphylococcus lugdunensis (SluCas9).

[0295] In some embodiments, the cells targeted for genome editing have one or more mutations in their genome that reduce the expression of the endogenous FOXP3 gene compared to its expression in normal cells that do not have the mutation. Normal cells may be healthy cells or control cells derived from (or isolated from) another subject that does not have the FOXP3 gene abnormality. In some embodiments, the cells targeted for genome editing may be cells derived from (or isolated from) a subject that requires treatment for a FOXP3 gene-related condition or FOXP3 gene-related disorder (e.g., a subject suffering from an autoimmune disorder (e.g., IPEX syndrome)). Thus, in some embodiments, the expression of the endogenous FOXP3 gene in such cells is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the expression of the endogenous FOXP3 gene in normal cells.

[0296] In some embodiments, a method for editing the genome of lymphocyte cells, (a) Cas DNA endonucleases (e.g., Cas9 endonucleases) or nucleic acids encoding said Cas DNA endonucleases; (b) a gRNA (e.g., sgRNA) or nucleic acid encoding the gRNA that can target the Cas DNA endonuclease to a FOXP3 locus or a non-FOXP3 locus (e.g., AAVS1) in the genome of the lymphocyte cell; and (c) Donor template including the code sequence of FOXP3 The present invention provides a method comprising the step of providing a substance to lymphocyte cells. In some embodiments, the Cas DNA endonuclease is a Cas9 endonuclease (for example, a Cas9 endonuclease derived from Streptococcus pyogenes). In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence within the FOXP3 locus. In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence within exon 1 of the FOXP3 locus. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7 and 27-29. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence in a non-FOXP3 locus (e.g., AAVS1). In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 15-20, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 15-20. In some embodiments, the FOXP3 coding sequence encodes FOXP3 or a functional derivative thereof. In some embodiments, the FOXP3 coding sequence is FOXP3 cDNA. A typical FOXP3 cDNA sequence may be contained in an AAV donor template having the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the method includes the step of providing the lymphocyte cells with the Cas DNA endonuclease. In some embodiments, the method includes the step of providing the lymphocyte cells with the nucleic acid encoding the Cas DNA endonuclease. In some embodiments, the method includes the step of providing the lymphocyte cells with the gRNA.In some embodiments, the gRNA is sgRNA. In some embodiments, the method includes the step of providing the lymphocyte cells with the nucleic acid encoding the gRNA. In some embodiments, the method further includes the step of providing the lymphocyte cells with one or more further gRNAs or the nucleic acids encoding the one or more further gRNAs.

[0297] In some embodiments, according to any of the methods for editing the cell genome described herein, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is Cas9 derived from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 is Cas9 derived from Staphylococcus lugdunensis (SluCas9).

[0298] In some embodiments, according to any of the methods for editing the cell genome described herein, the nucleic acid sequence encoding FOXP3 or a functional derivative is codon-optimized for expression in the lymphocyte. In some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative has at least about 70% sequence identity with the sequence shown in SEQ ID NO: 68, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least more sequence identity. In some embodiments, the cell is a human cell.

[0299] In some embodiments, according to any of the methods for editing the genome of cells described herein, the method uses a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in the lymphocyte. In some embodiments, the cells are human cells, for example, human CD4+ T cells. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA (such as a DNA plasmid). In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA (such as mRNA).

[0300] In some embodiments, according to any of the methods for editing the cell genome described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor template is configured such that the donor cassette is incorporated by homologous recombination repair (HDR) into the genomic locus targeted by the gRNA of (b) above. In some embodiments, homologous arms corresponding to the sequence of the targeted genomic locus are positioned on both sides of the donor cassette. In some embodiments, the length of the homologous arms is at least about 0.2 kb (e.g., at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb or more). In some embodiments, the length of the homologous arms is at least about 0.4 kb. Typical homologous arms include 5' homologous arms having the arrangement shown in any of sequence numbers 90-97 and 106-107, and 3' homologous arms having the arrangement shown in any of sequence numbers 98-105 and 108-109. In some embodiments, the homologous arms at the 5' end and 3' end of the donor mold are the same. In some embodiments, the homologous arms at the 5' end and 3' end of the donor mold are different.

[0301] In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0302] In some embodiments, according to any of the methods for editing the cell genome described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor template is configured to be incorporated by non-homologous end joining (NHEJ) into the genomic locus targeted by the gRNA of (b) above. In some embodiments, gRNA target sites are located adjacent to one or both sides of the donor cassette. In some embodiments, gRNA target sites are located adjacent to both sides of the donor cassette. In some embodiments, the gRNA target sites are target sites of the gRNA included in the system. In some embodiments, the gRNA target sites of the donor template are the reverse complementary strand of the cellular genome gRNA target site targeted by the gRNA included in the system. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0303] In some embodiments, according to any of the methods for editing the cell genome described herein, the DNA endonuclease or the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in liposomes or lipid nanoparticles. In some embodiments, the liposomes or lipid nanoparticles further comprise gRNA. In some embodiments, the liposomes or lipid nanoparticles are lipid nanoparticles. In some embodiments, the method uses lipid nanoparticles containing a DNA endonuclease and a nucleic acid encoding gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA encoding the DNA endonuclease.

[0304] In some embodiments, according to any of the methods for editing the cell genome described herein, the DNA endonuclease is pre-complexed with gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex is delivered to the lymphocytes by electroporation. In some embodiments, the donor template is an AAV donor template encoded in an AAV vector (e.g., an AAV6 vector). In some embodiments, the AAV donor template is delivered to the cells at the same time as, or approximately at the same time as, the RNP complex. For example, in some embodiments, the RNP complex is electroporated into the cells, and the AAV donor template is transduced on the same day. In some embodiments, the RNP complex is electroporated into the cells and the AAV donor template is transduced, with a time difference of approximately 12 hours or less between electroporation and transduction (e.g., 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, or less). In some embodiments, after electroporating the RNP complex into the cells, the cells are seeded and transduced with the AAV donor template. In some embodiments, before providing the cells with RNP and the AAV donor template, the cells are pre-stimulated in the presence of a factor that can activate and proliferate the cells (e.g., anti-CD3 antibody and / or anti-CD28 antibody, e.g., anti-CD3 / anti-CD28 beads). In some embodiments, pre-stimulation is performed over at least about 12 hours (for example, over at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours or more). In some embodiments, pre-stimulation is performed over at least about 72 hours. In some embodiments, pre-stimulation is performed over about 1 × 10 5 pieces / ml~1×10 7This is carried out in a cell composition with a density of cells / ml (approximately 2.5 × 10⁻⁶). 5 pieces / ml, approximately 5×10 5 pieces / ml, approximately 7.5×10 5 pieces / ml, approximately 1×10 6 pieces / ml, approximately 2.5×10 6 pieces / ml, approximately 5×10 6 pieces / ml, or approximately 7.5 x 10 6 This is carried out in cell compositions with a density of cells / ml, including any range between these values. In some embodiments, the concentration of cells in the cell composition is about 5 × 10⁻⁶ 5 The concentration is per ml.

[0305] In some embodiments, the frequency of targeted incorporation of the donor template into the FOXP3 locus within the cell genome is about 0.1% to about 99% according to any of the cell genome editing methods described herein. In some embodiments, the frequency of targeted incorporation is about 2% to about 70% (e.g., about 2% to about 65%, about 2% to about 55%, about 3% to about 70%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, or about 10% to about 50%). In some embodiments, the cells are cells of a subject (such as a human subject).

[0306] In some embodiments, according to the method for editing the genome of cells described herein, the cells are cryopreserved after editing.

[0307] Selection of target sequence In some embodiments, the position of the 5' terminal boundary and / or the 3' terminal boundary can be shifted relative to a specific reference locus to facilitate or enhance a particular application of gene editing, and this depends in part on the endonuclease system selected to perform the gene editing, as will be further described and illustrated herein.

[0308] In a first embodiment (but not limited to) of such target sequence selection, many endonuclease systems have rules or criteria for initially selecting a cleavable target site, for example, CRISPR type II or V endonucleases require specific requirements for PAM sequence motifs at particular positions adjacent to the DNA cleavage site.

[0309] In another embodiment (but not limited to) relating to the selection or optimization of target sequences, the frequency of “off-target” activity (e.g., the frequency of double-strand breaks occurring at sites other than the selected target sequence) with a particular combination of target sequence and gene-editing endonuclease is evaluated relative to the frequency of on-target activity. In some cases, cells precisely edited at a desired locus may have a selective advantage compared to other cells. Specific examples of selective advantages include, but are not limited to, the acquisition of various attributes such as improved replication rate, persistence, tolerance to specific conditions, improved engraftment success or persistence after in vivo introduction into a target, and other attributes related to maintaining or improving the number or viability of such cells. In another case, cells precisely edited at a desired locus may be selected as positive cells by one or more screening methods used for the identification, sorting, or other selection purposes of precisely edited cells. Selective advantages and targeted selection methods can utilize phenotypes associated with the modification. In some embodiments, cells may be edited two or more times to produce a second modification for creating a novel phenotype used for the selection or purification of an intended cell population. Such a second modification can be made by adding a second gRNA for a selection marker or screening marker. In some cases, a DNA fragment containing cDNA and a selection marker can be used to precisely edit cells at the desired locus.

[0310] In embodiments, regardless of whether selective advantages or targeted selection are applicable in particular cases, target sequences are selected considering off-target frequency to improve the effectiveness of applying selective advantages or targeted selection and / or reduce the possibility of undesirable modifications at sites other than the desired target. As further described and illustrated herein and in the Art, off-target activity arises under the influence of various factors, such as the similarities and differences between the target site and various off-target sites, and the specific endonuclease used. Bioinformatics tools are available to assist in predicting off-target activity, and in many cases, such tools can be used to identify the sites most likely to occur, and such predictions and identifications can be experimentally evaluated to estimate the relative frequency of off-target activity to on-target activity, thereby enabling the selection of sequences with high relative on-target activity. Specific examples of such techniques are provided herein, and others are known in the Art.

[0311] Another aspect of target sequence selection relates to homologous recombination events. Sequences sharing homologous regions can play a central role in homologous recombination events that delete intervening sequences. Such recombination events occur during the normal replication process of chromosomes and other DNA sequences, as well as at other points in DNA sequence synthesis, such as during double-strand break repair, which occurs regularly in the normal cell replication cycle. However, they can also be enhanced by various events (such as UV light and other DNA break inducers) or the presence of certain drugs (such as various chemical inducers). Many of these inducers indiscriminately generate double-strand breaks (DSBs) in the genome, and DSBs are regularly induced and repaired even in normal cells. During double-strand break repair, the original sequence can be reconstructed with complete fidelity, but in some cases, short insertions or deletions (called "indels") may be introduced at the DSB site.

[0312] Furthermore, as can be seen with the endonuclease systems described herein, double-strand breaks (DSBs) can be specifically induced at particular locations, which can then be used to trigger directed or selective recombination events at selected chromosomal locations. The tendency for homologous sequences to be recombined in DNA repair (and replication) can be utilized in a variety of situations and forms one basis for the application of gene editing systems such as CRISPR, in which the target sequence provided by a "donor" polynucleotide is inserted into a specific location on the desired chromosome by homologous recombination repair.

[0313] Desired deletions can be created using homologous regions between specific sequences, and these homologous regions can be short "microhomology" regions, sometimes as short as 10 base pairs or less. For example, a single double-strand break (DSB) is introduced at a site that exhibits microhomology with a neighboring sequence. In the normal repair process of such DSBs, deletion of the intervening sequence occurs frequently, which results from recombination facilitated by the DSB and its associated cellular repair processes.

[0314] However, in some situations, selecting a target sequence within a homologous region may result in much larger deletions, such as gene fusion (deletion within the coding region), and such results may or may not be desirable in certain circumstances.

[0315] The examples provided herein further illustrate the selection of various target regions for constructing double-stroke bonds (DSBs) designed to insert genes encoding FOXP3, and the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events. In some embodiments, target loci are selected from the FOXP3 locus, the AAVS1 locus, and the TCRa(TRAC) locus.

[0316] Nucleic acid modification In some embodiments, as described in detail herein and as known in the art, the polynucleotides introduced into cells have one or more modifications that can be used individually or in combination for, for example, enhancing activity, stability or specificity, altering delivery, reducing or otherwise enhancing the innate immune response of the host cell.

[0317] In certain embodiments, modified polynucleotides are used in a CRISPR / Cas9 system, in which case the guide RNA (single-molecule guide or bi-molecule guide) and / or the DNA or RNA encoding the Cas endonuclease introduced into the cell can be modified as described and illustrated below. Such modified polynucleotides can be used in a CRISPR / Cas9 system to edit one or more genomic loci.

[0318] When using the CRISPR / Cas9 system for such an example (but not limited to) applications, modifying the guide RNA can improve the formation or stability of the CRISPR / Cas9 genome editing complex containing the guide RNA, which may be formed from a single-molecule guide or a bi-molecule guide and a Cas endonuclease. Alternatively, modifying the guide RNA can improve the initiation, stability, or dynamics of the interaction between the genome editing complex and the target sequence in the genome, which can be used, for example, to enhance on-target activity. Alternatively, modifying the guide RNA can improve specificity, for example, by improving the relative genome editing rate at the on-target site compared to the effect at other sites (off-target).

[0319] Alternatively, or in addition to the above, the stability of guide RNA can be improved by modifying its resistance to degradation by ribonucleases (RNases) present in cells, thereby extending the half-life of the guide RNA in cells. Modifications that extend the half-life of guide RNA may be particularly useful in embodiments in which Cas endonucleases are introduced into cells and edited using RNA that needs to be translated to produce endonucleases, because the extended half-life of the guide RNA introduced simultaneously with the endonuclease-encoding RNA allows for an extension of the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist in the cell.

[0320] Alternatively, or in addition to the foregoing, modifications can be utilized to reduce the likelihood or extent to which RNA introduced into cells induces an innate immune response. As will be discussed later herein and as is known in the art, such immune responses have been well evaluated with respect to RNA interference (RNAi), such as small interfering RNAs (siRNAs), and tend to be associated with shortening of the RNA half-life and / or induction of cytokines or other factors related to the immune response.

[0321] It is also possible to modify the RNA encoding the endonuclease introduced into the cell with one or more modifications, including, but not limited to, modifications that improve the stability of the RNA (such as modifications that increase degradation by RNAse present in the cell), modifications that enhance the translation of the product (e.g., endonuclease), and / or modifications that reduce the likelihood or degree to which the RNA introduced into the cell induces an innate immune response.

[0322] Various modifications, such as those mentioned above and other modifications, can be used in combination. For example, in the case of CRISPR / Cas9, one or more modifications can be added to the guide RNA (including those exemplified above), and / or one or more modifications can be added to the RNA encoding the Cas endonuclease (including those exemplified above).

[0323] delivery In some embodiments, nucleic acid molecules used in the methods provided herein, for example, nucleic acids encoding genome-targeted nucleic acids and / or site-directed polypeptides of this disclosure, are packaged inside or on the surface of a delivery carrier for delivery to cells. Possible delivery carriers include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. Various targeting moieties can be used to enhance the selective interaction between such carriers and desired cell types or locations, as reported in the Art.

[0324] The introduction of the complexes, polypeptides, or nucleic acids of this disclosure into cells can be carried out by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, or nucleic acid delivery via nanoparticles.

[0325] In some embodiments, guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) can be delivered by a delivery carrier using a virus known in the art or a non-virus delivery carrier. Alternatively, one or more endonuclease polypeptides can be delivered by a delivery carrier using a virus known in the art or a non-virus delivery carrier, such as electroporation or lipid nanoparticles. In some embodiments, DNA endonucleases can be delivered as one or more polypeptides alone, or pre-complexed with one or more guide RNAs, or pre-complexed with tracrRNA and one or more crRNAs.

[0326] In embodiments, polynucleotides can be delivered by non-viral delivery carriers, including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. Several specific examples of non-viral delivery carriers are described in Peer, D. et al. (2011). Gene Therapy, 18: 1127-1133 (this document focuses on non-viral delivery carriers for siRNA, which are also useful for the delivery of other polynucleotides).

[0327] In embodiments, polynucleotides such as guide RNA, sgRNA, and mRNA encoding endonucleases can be delivered to cells or targets by lipid nanoparticles (LNPs).

[0328] While non-viral nucleic acid delivery methods have been tested in both animal and human models, the most well-developed system is lipid nanoparticles. Lipid nanoparticles (LNPs) generally consist of an ionizable cationic lipid and three or more additional components, which are typically cholesterol, DOPE, and lipid-containing polyethylene glycol (PEG) (see, for example, Example 2). The cationic lipid can bind to positively charged nucleic acids to form a dense complex that protects the nucleic acid from degradation. As they pass through a microfluidic system, the components self-assemble to form particles 50–150 nM in size, with the nucleic acid encapsulated in a core complexed with the cationic lipid and surrounded by a lipid bilayer-like structure. After injection into the target circulatory system, these particles can bind to apolipoprotein E (apoE), a ligand for the LDL receptor, which mediates uptake into hepatocytes of the liver via receptor-mediated endocytosis. This type of LNP has been shown to efficiently deliver mRNA and siRNA to hepatocytes of the liver in rodents, primates, and humans. After endocytosis, LNPs reside in endosomes. The encapsulated nucleic acid escapes the endosome due to the ionizable properties of cationic lipids. This allows the nucleic acid to be delivered to the cytoplasm, where the mRNA is translated into the encoded protein. After escaping from the endosome, Cas9 mRNA can be translated into Cas9 protein and form a complex with gRNA. In some embodiments, nuclear translocation of the Cas9 protein / gRNA complex is facilitated by including a nuclear localization signal in the Cas9 protein sequence. Alternatively, a small gRNA can pass through the nuclear pore complex and form a complex with the Cas9 protein in the nucleus. Once the gRNA / Cas9 complex enters the nucleus, it scans the genome to find homologous target sites and selectively creates double-strand breaks at the desired target sites in the genome. The half-life of RNA molecules in vivo is generally short, ranging from a few hours to a few days. Similarly, the half-life of proteins also tends to be short, ranging from a few hours to a few days.Therefore, in some embodiments, delivery of gRNA and Cas9 mRNA using LNPs can result in only transient expression and activity of the gRNA / Cas9 complex. This can offer the advantage of reducing the frequency of off-target cleavage and, therefore, minimizing the risk of genotoxicity in some embodiments. LNPs are generally less immunogenic than viral particles. While many humans already have immunity to AAV, they do not have pre-existing immunity to LNPs. Furthermore, the likelihood of an adaptive immune response to LNPs is low, allowing for repeated administration of LNPs.

[0329] Several types of ionizable cationic lipids have been developed for use in LNPs. These include C12-200 (Love, KT et al. (2010). Proc. Natl. Acad. Sci. USA, 107(5):1864-1869), MC3, LN16, and MD1. In one type of LNP, the GalNac moiety is attached to the outside of the LNP and functions as a ligand for liver uptake via the asialoglycoprotein receptor. LNPs are formulated using one of these cationic lipids to deliver gRNA and Cas9 mRNA to the liver.

[0330] In some embodiments, LNPs refer to particles having a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. Alternatively, the size of the nanoparticles may be in the range of 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0331] LNPs can be prepared from cationic lipids, anionic lipids, or neutral lipids. Neutral lipids such as the fusion phospholipid DOPE and the membrane component cholesterol can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to low stability and rapid clearance, and the possibility of inducing inflammatory or anti-inflammatory reactions. LNPs can also contain hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0332] Any lipid or combination known in the art can be used to prepare LNPs. Examples of lipids used to prepare LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0333] In this embodiment, LNPs can be prepared by combining multiple types of lipids in any molar ratio. Furthermore, LNPs can also be prepared by combining single or multiple polynucleotides with single or multiple lipids in various molar ratios.

[0334] In embodiments, site-directed polypeptides and genome-targeted nucleic acids can be administered separately to cells or targets. Alternatively, site-directed polypeptides can be pre-complexed with one or more guide RNAs, or with tracrRNA and one or more crRNAs. The pre-complexed material can then be administered to cells or targets. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs).

[0335] RNA can form specific interactions with other RNA or DNA. While this property is utilized in many biological processes, it also carries the risk of indiscriminate interactions in nucleic acid-rich cellular environments. One solution to this problem is to form ribonucleoprotein particles (RNPs) in which RNA is pre-complexed with endonucleases. Another advantage of RNPs is the protection of RNA from degradation.

[0336] In some embodiments, the endonuclease contained in the RNP may or may not be modified. Similarly, the gRNA, crRNA, tracrRNA, or sgRNA may or may not be modified. Various modifications are known in the art and can be used.

[0337] Endonucleases and sgRNAs can typically be combined in a 1:1 molar ratio. Alternatively, endonucleases, crRNAs, and tracrRNAs can typically be combined in a 1:1:1 molar ratio. However, RNPs can be prepared using various molar ratios.

[0338] In some embodiments, delivery can be carried out using recombinant adeno-associated virus (AAV) vectors. Techniques for producing rAAV particles to provide cells with the functions of an AAV genome and a helper virus, packaged to contain the polynucleotides, rep gene, and cap gene to be delivered, are known in the art. The production of rAAV requires the presence of the functions of the rAAV genome, the AAV rep gene and cap gene isolated from this rAAV genome (e.g., not contained internally), and the helper virus within a single cell (referred to herein as a packaging cell). The AAV rep and cap genes may be derived from a serotype of AAV that allows for the creation of recombinant viruses, or from a serotype of AAV different from the ITR in the rAAV genome. Examples of AAV serotypes include, but are not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The creation of pseudotyped rAAVs is disclosed, for example, in international patent application WO01 / 83692. See Table 1. Table 1 shows the AAV serotypes and Genbank accession numbers of several selected AAVs. [Table 2]

[0339] In some embodiments, the method for producing packaging cells involves creating a cell line that stably expresses all the components necessary for producing AAV particles. For example, a single plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, and AAV rep and cap genes separate from this rAAV genome, along with a selection marker such as a neomycin resistance gene, is incorporated into the cell genome. The AAV genome is introduced into a bacterial plasmid by methods such as GC tailing (Samulski, RJ et al. (1982). Proc. Natl. Acad. Sci. USA, 79(6):2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin, CA et al. (1983). Gene, 23(1):65-73), or direct blunt-end ligation (Senapathy, P. et al. (1984). J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. Advantages of this method include cell selection and the suitability of the cells for mass production of rAAV. Another preferred method involves introducing the rAAV genome, as well as the rep and cap genes, into packaging cells using adenovirus or baculovirus instead of plasmids.

[0340] General principles for the production of rAAV are reviewed, for example, in Carter, BJ (1992). Curr. Opin. Biotechnol., 3(5):533-539; and Muzyczka, M. (1992). Curr. Top. Microbiol. Immunol., 158:97-129.Various approaches are described in Tratschin, JD et al. (1984). Mol. Cell. Biol., 4(10):2072-2081; Hermonat, PL et al. (1984). Proc. Natl. Acad. Sci. USA, 81(20):6466-6470; Tratschin, JD et al. (1985). Mo1. Cell. Biol. 5(11):3251-3260; McLaughlin, SK et al. (1988). J. Virol. , 62(6):1963-1973; Lebkowski, JS et al. (1988). Mol. Cell. Biol., 8(10):3988-3996.; Samulski, RJ et al. (1989), J. Virol., 63(9):3822-3828; U.S. Patent No. 5,173,414; WO 95 / 13365 and its corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin, P. et al. (1995). Vaccine, 13(13):1244-1250; Paul, RW et al. (1993). Hum. Gene Ther., 4(5):609-615; Clark, KR et al. (1996). Gene Ther. 3(12):1124-1132; as described in U.S. Patent Nos. 5,786,211; 5,871,982; and 6,258,595.

[0341] The serotype of the AAV vector can be matched to the target cell type. For example, typical cell types described later can be transduced with AAV of the serotypes described herein. For example, AAV2 and AAV6 are suitable serotypes of AAV vectors for hematopoietic stem cells, but are not limited to these. In some embodiments, the serotype of the AAV vector is AAV6.

[0342] In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity with any one of sequence numbers 33-36 and 161. In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity with sequence number 33. In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 34 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 35 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 36 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 161 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more).

[0343] In addition to adeno-associated virus vectors, other viral vectors can also be used. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpesviruses, Epstein-Barr virus, papovaviruses, poxviruses, vaccinia viruses, and herpes simplex viruses.

[0344] In some embodiments, Cas9 mRNA, sgRNA targeting one or two loci of the FOXP3 gene, and donor DNA are formulated by encapsulating each separately in lipid nanoparticles, or all of them together in a single lipid nanoparticle, or all of them together in two or more lipid nanoparticles.

[0345] In some embodiments, Cas9 mRNA is formulated by encapsulation in lipid nanoparticles, while sgRNA and donor DNA are delivered by integration into an AAV vector.

[0346] Cas9 nuclease can be delivered as a DNA plasmid, mRNA, or protein. Guide RNA can be expressed from the same DNA or delivered as RNA. This RNA can be chemically modified to alter or increase its half-life and / or reduce the likelihood or extent of an immune response. Endonuclease proteins can also be complexed with gRNA before delivery. Viral vectors enable efficient delivery. Split Cas9 and smaller orthologs of Cas9 can be packaged in AAVs, as well as HDR donors. Various nonviral delivery methods exist for delivering each of these components, and nonviral and viral delivery methods can be used in combination. For example, nanoparticles can be used to deliver the protein and guide RNA, and AAVs can be used to deliver the donor DNA.

[0347] In some embodiments relating to the delivery of genome editing components for therapeutic procedures, at least two components, namely a sequence-specific nuclease and a DNA donor template, are delivered into the nucleus of the cells to be transformed (e.g., lymphocytes). In some embodiments, the AAV is selected from serotypes AAV2 and AAV6. In some embodiments, the DNA donor template packaged in the AAV is first administered to a subject (e.g., a human subject) by peripheral intravenous injection, followed by the administration of the sequence-specific nuclease. The advantage of first delivering the donor DNA template packaged in the AAV is that the delivered donor DNA template is stably maintained in the nucleus of the transduced lymphocyte, thereby enabling the subsequent administration of the sequence-specific nuclease, which creates a double-strand break in the genome and enables the incorporation of the DNA donor by HDR or NHEJ. In some embodiments, it is desirable that the sequence-specific nuclease remain active in the target cells for only the time required to promote targeted incorporation of the transgene, at a level sufficient to exert the desired therapeutic effect. When sequence-specific nucleases maintain activity in cells for extended periods, this increases the frequency of off-target double-strand breaks. Specifically, the frequency of off-target breaks is a function of the off-target cleavage efficiency multiplied by the duration of nuclease activity. Because mRNA and the proteins translated from it are short-lived in cells, delivery of sequence-specific nucleases in mRNA form results in a shorter duration of nuclease activity, ranging from a few hours to several days. Therefore, delivery of sequence-specific nucleases to cells already containing donor templates is expected to maximize the ratio of targeted integration to off-target integration.

[0348] In some embodiments, the sequence-specific nuclease is CRISPR-Cas9, which consists of a sgRNA and Cas9 nuclease directed to the FOXP3 locus. In some embodiments, the Cas9 nuclease is delivered as mRNA encoding the Cas9 protein, operably fused to one or more nuclear localization signals (NLS). In some embodiments, the sgRNA and Cas9 mRNA are delivered to lymphocyte cells (e.g., CD4+ T cells) by being packaged in lipid nanoparticles.

[0349] In some embodiments, to promote the nuclear localization of the donor template, a 366 bp region consisting of the origin of replication and initial promoter of Simian virus 40 (SV40), which can promote plasmid nuclear localization, can be added to the donor template. Other DNA sequences that bind to cellular proteins can also be used to improve DNA nuclear translocation.

[0350] Genetically modified cells and genetically modified cell populations In one embodiment, the disclosure herein provides a method for producing genetically modified cells by editing the genome of cells. In several embodiments, a population of genetically modified cells is provided. Thus, genetically modified cells refer to cells having at least one genetic modification introduced by genome editing (e.g., genome editing using the CRISPR / Cas9 system). In some embodiments, the genetically modified cells are genetically modified lymphocytes, such as T cells, including human CD4+ T cells. In some embodiments, the T cells are human T cells obtained from subjects suffering from IPEX syndrome. Genetically modified cells having an incorporated FOXP3 coding sequence are assumed herein.

[0351] The compositions described herein provide genetically modified cells (e.g., mammalian cells) comprising the protein sequence or expression vector described herein. Thus, cells (such as mammalian cells) for secreting dimerized CISCs are provided, which comprise the protein sequence according to any one of the embodiments described herein or the expression vector according to any one of the embodiments described herein. In some embodiments, the cells are mammalian cells such as lymphocytes. In some embodiments, the cells are lymphocytes such as lymphocytes.

[0352] In some embodiments, the cells are progenitor T cells or regulatory T cells. In some embodiments, the cells are stem cells, such as hematopoietic stem cells. In some embodiments, the cells are NK cells. In some embodiments, the cells are CD34+ T lymphocytes, CD8+ T lymphocytes, and / or CD4+ T lymphocytes. In some embodiments, the cells are B cells. In some embodiments, the cells are neural stem cells.

[0353] In some embodiments, the cells are CD8+ T-cytotoxic lymphocytes and may include naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, or bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes and may include naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, or bulk CD4+ T cells.

[0354] The lymphocytes (T lymphocytes) can be recovered by known techniques and enriched or removed by known techniques such as flow cytometry and / or immunomagnetic selection, or affinity binding with antibodies. After the enrichment and / or removal steps, the desired T lymphocytes can be expanded and proliferated in vitro by known techniques or variations thereof, which are readily understood by those skilled in the art. In some embodiments, the T cells are autologous T cells obtained from a patient.

[0355] For example, a desired T cell population or subpopulation can be expanded by adding a T lymphocyte population before proliferation to an in vitro culture medium, then adding feeder cells such as non-dividing peripheral blood mononuclear cells (PBMCs) to the medium (for example, adding feeder cells in such a ratio that the post-addition cell population contains at least 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the initial population at the start of expansion culture), and incubating the medium (for example, for a time sufficient to sufficiently expand the T cell number). The non-dividing feeder cells may include PBMC feeder cells irradiated with gamma rays. In some embodiments, the PBMCs are irradiated with gamma rays at 3000-3600 rads to prevent cell division. In some embodiments, to prevent cell division of the PBMCs, the PBMCs are irradiated with gamma rays at 3000 rad, 3100 rad, 3200 rad, 3300 rad, 3400 rad, 3500 rad, or 3600 rad, or any other radiation value between two endpoints of these values. The order in which T cells or feeder cells are added to the culture medium may be changed as needed. Typically, the culture can be incubated under conditions suitable for T lymphocyte proliferation, such as a temperature. The temperature for proliferation of human T lymphocytes is typically at least 25°C, preferably at least 30°C, and more preferably 37°C. In some embodiments, the temperature for proliferation of human T lymphocytes is 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, or 37°C, or any other temperature between two endpoints of these values.

[0356] T lymphocytes can be isolated and sorted into naive T cell subpopulations, memory T cell subpopulations, and effector T cell subpopulations, respectively, before or after expansion and proliferation.

[0357] CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive CD8+ cells, central memory CD8+ cells, and effector memory CD8+ cells by identifying the cell surface antigens associated with each of these cells. In some embodiments, memory T cells are present in both the CD62L+ subset and the CD62L- subset derived from CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ fractions and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, central memory T CM The expression of phenotypic markers includes CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and granzyme B is negative or shows low expression. In some embodiments, central memory T cells are CD45RO+, CD62L+, and / or CD8+ T cells. In some embodiments, effector T cells are expressed as CD45RO+, CD62L+, and / or CD8+. E These cells are negative for CD62L, CCR7, CD28, and / or CD127, and positive for granzyme B and / or perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers, such as CD62L, CCR7, CD28, CD3, CD127, and / or CD45RA.

[0358] CD4+ helper T cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, and / or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and / or CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and / or CD45RO-.

[0359] Whether it be mammalian cells or mammalian cell populations, these cells or cell populations are selected for expansion and proliferation based on whether or not they have undergone two different genetic recombination events. If mammalian cells or cell populations undergo one or fewer genetic recombination events, dimerization does not occur even when a ligand is added. However, if mammalian cells or cell populations undergo two genetic recombination events, the addition of a ligand causes dimerization of CISC components, followed by the generation of a signaling cascade. Therefore, mammalian cells or cell populations may be selected based on their responsiveness to contact with a ligand. In some embodiments, the amount of ligand added is 0.01nM, 0.02nM, 0.03nM, 0.04nM, 0.05nM, 0.06nM, 0.07nM, 0.08nM, 0.09nM, 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, 1.0nM, 1.5nM, 2.0nM, 2.5nM, 3.0nM, 3.5nM, 4.0nM, 4.5nM, 5.0nM, 5.5nM, 6.0 The concentration may be within the range defined by nM, 6.5nM, 7.0nM, 7.5nM, 8.0nM, 8.5nM, 9.0nM, 9.5nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, or 100nM, or any two of these values.

[0360] In some embodiments, cells such as mammalian cells or cell populations such as mammalian cell populations may be positive for dimerized CISCs based on markers expressed as a result of signaling pathways. Therefore, whether a cell population is positive for dimerized CISCs may be determined by flow cytometry using staining with surface marker-specific antibodies and isotype-matched control antibodies.

[0361] In some embodiments, a recombinant cell comprising a protein sequence according to any one of the embodiments described herein or an expression vector according to any one of the embodiments described herein has a phenotype similar to that of natural thymic T reg (tT reg ). As used herein, such a recombinant cell is also referred to as "edT reg ". In some embodiments, edT reg is characterized by i) high expression of any one or more (2, 3, 4 or 5) of FOXP3, CD25, CTLA4, ICOS and LAG3 and / or ii) low expression of CD127. In some embodiments, edT reg is characterized by high expression of FOXP3, CD25, CTLA4, ICOS and LAG3 and low expression of CD127. In some embodiments, edT reg has a memory phenotype. In some embodiments, edT reg is characterized by high expression of CD45RO. In some embodiments, edT reg is characterized by low expression of Helios. In some embodiments, edT reg is characterized by a reduced response to stimulation of inflammatory cytokines compared to non-recombinant control cells. In some embodiments, edT reg is characterized by a reduced response to stimulation of IL-2, IFNγ and / or TNFα compared to non-recombinant control cells. In some embodiments, edT reg is characterized by a reduced response to stimulation of IL-2, IFNγ and TNFα compared to non-recombinant control cells.

[0362] In some embodiments, a genetically modified cell comprising a protein sequence according to any one of the embodiments described herein or an expression vector according to any one of the embodiments described herein can be enriched by known techniques such as affinity binding. For example, a genetically modified cell expressing LNGFR can be enriched by affinity binding to an LNGFR-selective material such as beads conjugated with an anti-LNGFR antibody or a binding fragment thereof.

[0363] In some embodiments, the genetically modified cell is edT reg and when edT reg is administered to a mouse model of graft-versus-host disease (GVHD), the onset of GVHD in the mouse model is delayed and / or the survival rate of the mouse model is increased as compared to a control mouse model administered non-genetically modified control cells. In some embodiments, edT reg is administered to the mouse model by an intraperitoneal route or an intravenous route. In some embodiments, a cell composition comprising at least 60% or at least about 60% (at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least more than that, or at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least more than that) of edT reg is administered to the mouse model. In some embodiments, a cell composition comprising 70% or about 70% of edT reg is administered to the mouse model. In some embodiments, a cell composition comprising 90% or about 90% of edT reg is administered to the mouse model.

[0364] In some embodiments, the cells are not germ cells.

[0365] Manufacturing method This provides a method for creating genetically modified cells. A step of providing a cell containing a first nucleic acid that includes at least one target gene locus; A step of providing a CAS9 protein, or a second nucleic acid sequence encoding the CAS9 protein; A step of introducing the CAS9 protein or a second nucleic acid into the cells; The steps include introducing a third nucleic acid encoding at least one CRISPR guide sequence configured to hybridize to at least one target gene locus, or a set of nucleic acids encoding said at least one CRISPR guide sequence; and A step of introducing a fourth nucleic acid, including a gene delivery cassette, into the cells. Includes.

[0366] In some embodiments, the method further includes a step of activating the cells, which is performed before introducing a second nucleic acid into the cells. The activation may be performed by contacting the cells with CD3 and / or CD28. The CD3 and / or CD28 may be supported on a solid carrier such as beads.

[0367] In some embodiments, the at least one target gene locus is the FOXP3 locus, the AAVS1 locus, or the TCR(TRAC) locus. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are provided incorporated into one or more vectors.

[0368] In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vectors are adeno-associated virus (AAV) vectors. In some embodiments, the AAV vectors are self-complementary vectors. In some embodiments, the AAV vectors are single-stranded vectors. In some embodiments, the AAV vectors are a combination of self-complementary vectors and single-stranded vectors.

[0369] In some embodiments, the second nucleic acid encoding the CAS9 protein is mRNA. In some embodiments, the at least one guide sequence includes any of the sequences shown in SEQ ID NOs: 1-7, 15-20, 27-29, 33, and / or 34. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids, and / or the fourth nucleic acid are codon-optimized for expression in eukaryotic cells (such as human cells). Those skilled in the art will understand codon optimization, and the nucleic acids may be optimized by computer-aided methods.

[0370] In some embodiments, the fourth nucleic acid includes a sequence encoding a FOXP3 cDNA sequence optimized for human codons.

[0371] In some embodiments, the fourth nucleic acid sequence includes the sequence shown in SEQ ID NO: 68 or 69. In some embodiments, the fourth nucleic acid further includes a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter, or an E2F promoter.

[0372] In some embodiments, the fourth nucleic acid further comprises sequences encoding low-affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB, and / or LNGFRe (a sequence encoding the LNGFR epitope). LNGFR may be used as a marker for cell enrichment.

[0373] Cells possessing μCISC, CISCγ, or FRB may be used in the composition and method, thereby enabling the utilization of intracellular signaling of CISCs via rapamycin and mitigating the adverse effects of rapamycin or rapamycin-related compounds on the proliferation and viability of host cells possessing the FOXP3 gene.

[0374] In some embodiments, the method further includes the step of introducing a fifth nucleic acid comprising a second gene delivery cassette into the cells. In some embodiments, the fifth nucleic acid is provided incorporated into a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid comprises sequences encoding CISC, FRB, marker proteins, μCISC, and / or βCISC.

[0375] In some embodiments, the fourth and / or fifth sequence further comprises a sequence encoding a P2A self-cleaving peptide. In some embodiments, the fourth and / or fifth sequence further comprises a sequence encoding a poly-A sequence. In some embodiments, the poly-A sequence comprises the 3'UTR of SV40 poly-A or FOXP3. In some embodiments, the fourth sequence comprises the sequence shown in any of SEQ ID NOs. 37-42. In some embodiments, the fourth and fifth sequences are introduced into the cells, wherein the fourth sequence includes the sequence shown in SEQ ID NO: 37 and the fifth sequence includes the sequence shown in SEQ ID NO: 43; the fourth sequence includes the sequence shown in SEQ ID NO: 37 and the fifth sequence includes the sequence shown in SEQ ID NO: 44; the fourth sequence includes the sequence shown in SEQ ID NO: 38 and the fifth sequence includes the sequence shown in SEQ ID NO: 43; the fourth sequence includes the sequence shown in SEQ ID NO: 38 and the fifth sequence includes the sequence shown in SEQ ID NO: 44; the fourth sequence includes the sequence shown in SEQ ID NO: 45 and the fifth sequence includes the sequence shown in SEQ ID NO: 46; or the fourth sequence includes the sequence shown in SEQ ID NO: 45 and the fifth sequence includes the sequence shown in SEQ ID NO: 47.

[0376] In some embodiments, the cells are primary human lymphocytes.

[0377] In some embodiments, the fourth nucleic acid includes at least one homologous arm having a locus-specific sequence, the length of which is configured to achieve efficient packaging into an AAV vector. The homologous arm may be configured to add another gene to the construct.

[0378] In some embodiments, the length of the at least one homologous arm is within the range defined by 0.25kb, 0.3kb, 0.45kb, 0.6kb, or 0.8kb, or any two of these values. In some embodiments, the marker is LNGF, RQR8, or EGFRt.

[0379] In some embodiments, the method further includes introducing a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3 into the cells. In some embodiments, the method further includes selecting the cells by increasing the concentration of the marker.

[0380] In some embodiments, the method is performed on an input cell population to generate an output cell population in which one or more cells are modified. In some embodiments, the modified cells in the output cell population express a surface marker (e.g., LNGFR) that is not expressed in the unmodified cells in the output cell population. In some embodiments, the method further includes a step of enriching the output cell population to obtain modified cells. Modified cells can be enriched by known techniques, such as affinity binding. For example, modified cells expressing LNGFR can be enriched by affinity binding to an LNGFR-selective material, such as beads conjugated with an anti-LNGFR antibody. By enriching the modified cells, the yield and purity of the modified cells to be subsequently expanded and proliferated can be increased. In some embodiments, the output cell population is enriched to obtain modified cells, so that the proportion of modified cells (e.g., LNGFR+ modified cells) in the enriched cell population is at least 90% or at least about 90% (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least more, or at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least more).

[0381] Furthermore, cells for FOXP3 expression, prepared by a method according to any one of the embodiments described herein, are provided. In some embodiments, the cells are primary human lymphocytes. In some embodiments, the FOXP3 is constitutively expressed or under controlled expression.

[0382] In some embodiments, cells for FOXP3 expression are provided, comprising nucleic acid encoding a gene encoding FOXP3. In some embodiments, the gene encoding FOXP3 is incorporated into a FOXP3 locus or a non-FOXP3 locus. In some embodiments, the non-FOXP3 locus is the AAVS1 locus or the TCRa(TRAC) locus. In some embodiments, the cells are primary human lymphocytes. In some embodiments, the cells express CISCβ:FRB-IL2Rβ, DISC, CISC-FRB, μDISC, μCISC-FRB, FRB, LNGFR and / or LNGFRe. In some embodiments, the cells express T reg Includes phenotype.

[0383] In some embodiments, a composition comprising cells according to any one of the embodiments described herein is provided. In some embodiments, the composition comprises a pharmaceutical additive.

[0384] In some embodiments, methods are provided for treating, alleviating, and / or suppressing a disease and / or condition in a subject, comprising the step of providing a subject having the disease and / or condition with cells or a composition according to any one of the embodiments described herein. In some embodiments, by providing the subject with the cells, the immune response of the subject is inhibited or suppressed. In some embodiments, the immune response inhibited or suppressed is a T cell-mediated inflammatory response.

[0385] In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an X-linked (IPEX) syndrome. In some embodiments, the condition is a graft-versus-host disease (GVHD). In some embodiments, the condition is a condition related to solid organ transplantation.

[0386] In some embodiments, a method for producing genetically modified cells, A step of providing a cell containing a first nucleic acid that includes at least one target gene locus; A step of providing a CAS9 protein, or a second nucleic acid sequence encoding the CAS9 protein; A step of introducing the CAS9 protein or a second nucleic acid into the cells; The steps include introducing a third nucleic acid encoding at least one CRISPR guide sequence configured to hybridize to at least one target gene locus, or a set of nucleic acids encoding said at least one CRISPR guide sequence; and A step of introducing a fourth nucleic acid, including a gene delivery cassette, into the cells. This provides a method that includes [something]. In some embodiments, the method further includes a step of activating the cells, which is performed before introducing the second nucleic acid into the cells. In some embodiments, the activation is performed by contacting the cells with CD3 and / or CD28. In some embodiments, the at least one target locus is the FOXP3 locus, the AAVS1 locus, or the TCR (TRAC) locus. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids, and / or the fourth nucleic acid are provided incorporated into one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is a self-complementary vector. In some embodiments, the AAV vector is a single-stranded vector. In some embodiments, the AAV vector is a combination of a self-complementary vector and a single-stranded vector. In some embodiments, the second nucleic acid encoding the CAS9 protein is mRNA. In some embodiments, the at least one guide sequence includes a sequence shown in any of SEQ ID NOs: 1-7, 15-20, 27-29, 33, and / or 34. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids, and / or the fourth nucleic acid are codon-optimized for expression in eukaryotic cells (such as human cells). In some embodiments, the fourth nucleic acid includes a sequence encoding a FOXP3 cDNA sequence optimized for human codons. In some embodiments, the fourth nucleic acid sequence includes a sequence shown in SEQ ID NO: 68 or 69. In some embodiments, the fourth nucleic acid further includes a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter, or an E2F promoter. In some embodiments, the fourth nucleic acid further includes a sequence encoding a low-affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB, and / or LNGFRe (a sequence encoding an LNGFR epitope).In some embodiments, the method further includes the step of introducing a fifth nucleic acid comprising a second gene delivery cassette into the cells. In some embodiments, the fifth nucleic acid is provided incorporated into a vector. In some embodiments, the ve...

Claims

[Claim 1] The inventions described herein.

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