HK1260200A1 - Targeted disruption of the mhc cell receptor - Google Patents

Description

Targeted disruption of MHC cell receptors

Cross Reference to Related Applications

This application claims us provisional application No. 62/269,410 filed on 12/18/2015; U.S. provisional application No. 62/305,097 filed on 8/3/2016; and 2016, 29, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure is in the field of human cell (including lymphocyte and stem cell) genome modification.

Background

Gene therapy holds tremendous potential for a new era of human therapeutics. These methods will enable the treatment of conditions that have not been addressed by standard medical practice. Gene therapy can involve many variations of gene editing techniques, such as disruption and modification of the locus, and insertion of expressible transgenes that can be controlled by specific exogenous promoters fused to the transgene, or by endogenous promoters present at the site of insertion into the locus.

For any real implementation of this technology, the delivery and insertion of transgenes are examples of obstacles that must be addressed. For example, while many gene delivery methods are potentially viable for therapeutic applications, they all involve a large number of tradeoffs between safety, persistence, and expression levels. Methods of providing transgenes in episomes (e.g., basal adenoviruses (Ad), adeno-associated viruses (AAV), and plasmid-based systems) are generally safe and can achieve high initial expression levels, however, these methods lack stable episomal replication, which can limit the duration of expression in mitotically active tissues. In contrast, delivery methods that result in random integration of the desired transgene (e.g., integrated Lentivirus (LV)) provide for more durable expression, but due to the nature of random insertion untargeted, may cause unregulated growth in the recipient cell, possibly leading to malignancy via activation of oncogenes in the vicinity of the randomly integrated transgene cassette. Furthermore, although transgene integration avoids loss of replication drive, it does not prevent eventual silencing of the exogenous promoter of the fusion transgene. Over time, such silencing results in reduced transgene expression for most non-specific insertion events. In addition, integration of the transgene rarely occurs in every target cell, which may make it difficult for the transgene of interest to achieve sufficiently high expression levels to achieve the desired therapeutic effect.

In recent years, new strategies for transgene integration have been developed that utilize site-specific nucleases (e.g., Zinc Finger Nucleases (ZFNs), transcription activator-like effector domain nucleases (TALENs), CRISP/Cas systems with engineered crRNA/tracr RNA ("single guide RNA") to guide specific cleavage, etc.) and cleavage of Cfp1/CRISPR systems to guide specific cleavage, etc.) to insert preferred (bias) at selected genomic loci. See, for example, U.S. patent nos. 9,255,250, 9,045,763, 9,005,973, 8,956,828, 8,945,868, 8,703,489, 8,586,526, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,067,317, 7,262,054, 7,888,121, 7,972,854, 7,914,796, 7,951,925, 8,110,379, 8,409,861; U.S. patent publication nos. 20030232410, 20050208489, 20050026157, 20050064474, 20060063231, 20080159996, 201000218264, 20120017290, 20110265198, 20130137104, 20130122591, 20130177983, and 20130177960 and 20150056705. Furthermore, targeted nucleases, developed based on the Archaeus (Argonaute) system (e.g., from Thermus thermophilus (T. thermophilus), known as 'TtAgo', see Swarts et al (2014) Nature 507(7491): 258-. This nuclease-mediated transgene integration pathway offers the prospect of improved transgene expression, increased safety, and expression persistence compared to classical integration pathways, as it allows for accurate transgene localization, thereby minimizing the risk of gene silencing or nearby oncogene activation.

T Cell Receptors (TCRs) are the major part of selective activation of T cells it bears some resemblance to antibodies, the antigen recognition part of the TCR usually consisting of two chains α and β, which aggregate to form heterodimers. it bears resemblance to antibodies in the way that the individual genes encoding the TCR β 0 and β 1 complexes are brought together TCR β (TCR β) and TCR β (TCR β) chains are each composed of two regions, the C-terminal constant region and the N-terminal variable region, the genomic loci encoding TCR β and β chains are similar to the antibody encoding loci in which TCR α comprises V and J segments, whereas the β chain locus comprises a D segment in addition to the V and J segments. for TCR β, there are additionally two different constant regions selected during the selection process during T cell rearrangement, the various segments thus allowing each T cell to comprise a unique variable part in TCR 29 and TCR 2, while this is called the complementary variable region (the T cell receptor is called the unique TCR 638 receptor epitope which is expressed by the first CDR 38023, which is expressed by the organism' T cell expressing the unique TCR 638, the antigen receptor itself expressed by the unique TCR 638, once the corresponding TCR 638 gene, the antibody is expressed by the unique TCR 638, the antibody expressing the antigen expressing the corresponding TCR 638, the antigen receptor.

During T cell activation, the TCR interacts with the antigen displayed as a peptide on the Major Histocompatibility Complex (MHC) of the antigen presenting cell. Recognition of the antigen-MHC complex by the TCR results in T cell stimulation which in turn leads to differentiation of T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+) in memory and effector lymphocytes. These cells can then be expanded in a clonal fashion to generate activated subpopulations capable of reacting with a particular antigen throughout the T cell population.

MHC proteins are heterodimers of two types — type I and type II. MHC type I proteins are the two proteins, α chain and β 2 microglobulin chain (sometimes also referred to as B2M), the α chain is a transmembrane protein encoded by a type I MHC gene, while β 2 microglobulin chain is a small extracellular protein encoded by a gene not located within the MHC gene cluster. α chain folds into 3 globular domains and, when associated with β 2 microglobulin, the globular structural complex is similar to an antibody complex.

Human HLA class I gene clusters include three major loci B, C and a, as well as several minor loci (including E, G and F, all present in the HLA region on chromosome 6.) HLA class II clusters also include three major loci DP, DQ and DR, and both class I and class II gene clusters are polymorphic, so that there are multiple different alleles of class I and class II within the population as well, some accessory proteins also play a role in HLA functionality β -2 microglobulin is used as a chaperone (encoded by B2M, located on chromosome 15) and stably expresses HLA a, B or C proteins on the cell surface, and also stabilizes antigen display grooves (display grooves) on class I structures, which are usually present in small amounts in serum and urine.

HLA plays a major role in transplant rejection. The acute phase of transplant rejection may occur within about 1-3 weeks and typically involves the action of host T lymphocytes on the donor tissue due to sensitization of the host system to donor HLA class I and II molecules. In most cases, the trigger antigen is HLA class I. For best success, donors are typed for HLA and matched as closely as possible to the patient recipient. But even donations between family members, even though they may possess a high percentage of HLA identity, are still not very successful. Therefore, to protect the transplanted tissue in the recipient, the patient often must undergo long-term immunograft therapy to avoid rejection. However, such treatment may lead to complications and significant morbidity as patients may have difficulty overcoming opportunistic infections. Modulation of a type I or II gene can be disrupted in the presence of some tumors, and such disruption can have an impact on the prognosis of the patient. For example, a reduction in B2M expression was found in metastatic colorectal cancer (Shrout et al (2008) Br J Canc 98: 1999). Because B2M has a key role in stabilizing MHC class I complexes, the reduction of B2M in certain solid cancers has been hypothesized to be a mechanism of immune escape from T cell-driven immune surveillance. Suppressed B2M expression has been shown to be the result of inhibiting the regulation of normal IFN γ B2M expression and/or specific mutations in the B2M coding sequence leading to gene knock-outs (Shrout et al, supra.) it is confusing that elevated B2M is also associated with some types of cancer. Elevated levels of B2M in urine are predictive of a variety of cancers, including prostate, Chronic Lymphocytic Leukemia (CLL) and non-hodgkin's lymphoma.

Adoptive Cell Therapy (ACT) is a developing form of cancer therapy that is based on the delivery of tumor-specific immune cells to a patient such that the delivered cells attack and clear the patient's cancer. ACT may involve the use of Tumor Infiltrating Lymphocytes (TILs), which are T cells isolated from a patient's own tumor mass and expanded ex vivo to be reperfused back into the patient. This approach has promise in treating metastatic melanoma, where in one study, long-term response rates of > 50% were observed (see, e.g., Rosenberg et al (2011) Clin cancer Res 17(13): 4550). TILs are a promising source of cells because they are a mixed pool of patients' own cells with T Cell Receptor (TCR) specificity for tumor associated antigens (TTA) presented on tumors (Wu et al (2012) Cancer J18 (2): 160). Other methods involve editing T cells isolated from the patient's blood so that they are engineered to be responsive to tumors in some way (Kalos et al (2011) scitranl Med 3(95):95ra 73).

Chimeric Antigen Receptors (CARs) are molecules designed to target immune cells to specific molecular targets expressed on the cell surface in their most basic form, they are receptors that are introduced into cells that couple specific domains expressed extracellularly to signal transduction pathways within the cell, such that the cells are activated when the specific domains interact with their targets, CARs are typically prepared from functional domains that mimic T Cell Receptors (TCRs), where antigen-specific domains, such as scFv or some types of receptors, are fused to signal transduction domains, such as ITAM and other costimulatory domains, these constructs are then introduced into T cells to allow the T cells to be activated in the presence of cells expressing the target antigen, and when the T cells are reintroduced into patients, resulting in non-MHC dependent manner of attacking the target cells by activating T cells (see, Chicaybam et al (2011 Rev Immunol) 30:294, kas conspecific for treating various types of cancer, such as lymphoblastic lymphoma, CD 3982, or lymphoblastic lymphoma, ALL of which are promising for the development of clinical trials of HIV-mediated by engineered CAR, or HIV-T cells, including CD20, which is a clinically promising for the clinical trials for the treatment of lymphomatosis, including lymphomas.

ACTR (antibody-coupled T cell receptor) is an engineered T cell component that is capable of binding heterologously supplied antibodies. Binding of the antibody to the ACTR component assembles the T cell to interact with the antigen recognized by the antibody, and when the antigen is encountered, the ACTR, including the T cell, is triggered to interact with the antigen (see, U.S. patent publication No. 20150139943).

However, one drawback of adoptive cell therapy is that the source of the cellular product must be patient-specific (autologous) in order to avoid potential rejection of the transplanted cells. This has led researchers to develop methods to edit patient's own T cells to avoid such rejection. For example, a patient's T cells or hematopoietic stem cells can be manipulated in vitro by the addition of engineered CARs, ACTRs, and/or T Cell Receptors (TCRs), and then further treated with engineered nucleases to knock out T cell checkpoint inhibitors such as PD1 and/or CTLA4 (see PCT publication No. WO 2014/059173). In order to apply this technique to a larger patient population, it would be advantageous to develop a universal population of cells (allogeneic). In addition, TCR knockdown will result in cells that are unable to produce a Graft Versus Host Disease (GVHD) response once introduced into a patient.

Thus, there remains a need for compositions and methods that can be used to modify MHC gene expression (e.g., knock-out B2M) and/or knock-out TCR expression in T cells.

Disclosure of Invention

Disclosed herein are methods and compositions for partially or completely inactivating or disrupting the B2M gene, as well as methods and compositions for introducing and expressing a desired level of an exogenous TCR, CAR, or ACTR transgene in T lymphocytes after or simultaneously with disruption of the endogenous TCR and/or B2M. Further, provided herein are compositions and methods for deleting (inactivating) or inhibiting the B2M gene to produce HLA class I free T cells, stem cells, tissues, or whole organisms, e.g., cells that do not express one or more HLA receptors on their surface. In certain embodiments, the HLA-free cell or tissue is a human cell or tissue, which is advantageous for use in transplantation. In a preferred embodiment, T cells free of HLCs are prepared for adoptive T cell therapy.

In one aspect, described herein are isolated cells (e.g., eukaryotic cells such as mammalian cells including lymphocytes, stem cells (e.g., iPSC, embryonic stem cells, MSC or HSC) or progenitor cells) wherein expression of the β 2 microglobulin (B2M) gene is regulated by modification of exon 1 or exon 2 of the B2M gene in certain embodiments, the modification is within 1-5, 1-10 or 1-20 bases on either side of (flanking the genomic sequence of) SEQ ID NO:6-48 or 137-gene, or GGCCTTA, TCAAAT, TCAAATT, TTACTGA and/or AATTGAA. the modification may be by an exogenous fusion molecule comprising a functional domain (e.g., transcription regulatory domain, nuclease domain) and a DNA binding domain including, but not limited to (i) cells comprising an exogenous transcription factor, including a transcription factor that encodes a chimeric gene encoding the CTLA-137-48, or ACT-receptor gene, and/or a chimeric gene encoding an ACT receptor gene encoding the chimeric gene encoding the CTLA-137-48, or ACT-48, and/or ACT-receptor gene encoding the chimeric gene, wherein the chimeric gene is further expressed in the transgenic cells with an exogenous or/or chimeric gene encoding the CTTA-48-137-48, or/or the chimeric gene encoding the polypeptide or the polypeptide coding sequence of the polypeptide coding gene encoding the polypeptide coding for the polypeptide or the polypeptide, the.

Thus, in one aspect, described herein are cells in which the expression of the B2M gene is regulated (e.g., activated, suppressed or inactivated) — in preferred embodiments, exon 1 or exon 2 of B2M is regulated. regulation may be by an exogenous molecule that binds to the B2M gene and modulates the expression of B2M (e.g., an engineered transcription factor including a DNA binding domain and a transcriptional activation or suppression domain) and/or by sequence modification of the B2M gene (e.g., using a nuclease that cleaves the B2M gene and modifies the gene sequence by insertion and/or deletion) in certain embodiments, the expression of one or more other genes is also regulated (e.g., a TCR gene, such as a TCRA gene) in certain embodiments, a cell that includes an engineered nuclease that induces the knockout of the B2M gene, such that the type I TCR I instability is achieved in preferred embodiments, the type I instability of the TCR I instability of the modified cell surface instability, the instability of the type I TCR I-modified cell, wherein the exogenous TCR cd 2M gene is further includes a transgene expression of a transgene coding region encoding a TCR cd-T2 expressing a T-cd, or a-cd.

In certain embodiments, the cells described herein comprise modifications (e.g., deletions and/or insertions, in combination with engineered TF to inhibit B2M) of the B2M gene (e.g., modifications of exon 1 or exon 2). In certain embodiments, the modification is to SEQ ID NO 6-48 and/or 137-205, which includes modifications by binding, cleavage, insertion and/or deletion of one or more nucleotides located within any of these sequences and/or within 1-50 base pairs (including any value such as between 1-5, 1-10 or 1-20 base pairs) of the gene (genomic) sequence flanking these sequences in the B2M gene. In certain embodiments, the cell comprises one or more modifications (binding, cleavage, insertion, and/or deletion) within the following sequences: GGCCTTA, TCAAATT, TCAAAT, TTACTGA and/or AATTGAA within the B2M gene (e.g., exon 1 and/or exon 2, see fig. 1). In certain embodiments, the modification comprises binding to an engineered TF described herein such that B2M gene expression is modulated, e.g., inhibited or activated. In other embodiments, the modification is a genetic modification (change in nucleotide sequence) located at or adjacent to one or more nuclease binding (target) and/or one or more cleavage sites, including, but not limited to, modifying sequences within 1-300 base pairs (or any number of base pairs therebetween) upstream, downstream of (and/or including 1 or more base pairs of) one or more cleavage sites and/or binding sites; modifications include and/or are located within 1-100 base pairs (or any number of base pairs therebetween) on either side of one or more binding and/or cleavage sites; modifications include and/or are located on either side of one or more binding and/or cleavage sites (e.g., 1-5, 1-10, 1-20 or more base pairs) within 1-50 base pairs (or any number of base pairs therebetween); and/or modifying one or more base pairs within the nuclease binding site and/or cleavage site. In certain embodiments, the modification is at or adjacent (e.g., 1-300, 1-50, 1-20, 1-10, or 1-5 or more base pairs or any number of base pairs therebetween) to the B2M gene sequence surrounding any one of SEQ ID NOS 6-48 or 137-205. In certain embodiments, the modification comprises modifying the B2M gene within one or more of the sequences set forth in SEQ ID NO 6-48 or 137-205 or within GGCCTTA, TCAAATT, TCAAAT, TTACTGA, and/or AATTGAA (e.g., exon 1 and/or exon 2) of the B2M gene, e.g., modifying one or more of these sequences by 1 or more base pairs. In certain embodiments, the nuclease-mediated genetic modification is located between paired target sites (when the target is cleaved using a dimer). Nuclease-mediated genetic modifications can include insertions and/or deletions of any number of base pairs, including insertions of non-coding sequences of any length and/or transgenes of any length and/or deletions of 1 base pair to over 1000kb (or any value therebetween, but not limited to 1-100 base pairs, 1-50 base pairs, 1-30 base pairs, 1-20 base pairs, 1-10 base pairs, or 1-5 base pairs).

The modified cells of the invention may be lymphocytes (e.g., T cells), stem/progenitor cells (e.g., induced pluripotent stem cells (ipscs), embryonic stem cells (e.g., human ES), Mesenchymal Stem Cells (MSCs), or Hematopoietic Stem Cells (HSCs)). The stem cells may be totipotent or pluripotent (e.g., partially differentiated, such as HSCs that are pluripotent bone marrow or lymphoid stem cells). In other embodiments, the invention provides methods for producing a cell having a deletion genotype for HLA expression (nullgeotype). Any of the modified stem cells described herein (modifying the B2M locus) can then be differentiated to produce differentiated (in vivo or in vitro) cells derived from the stem cells described herein. Any of the modified stem cells described herein can include further modifications in other genes of interest (e.g., TCRA, TCRB, PD1, CTLA4, etc.).

In another aspect, the compositions (modified cells) and methods described herein can be used, for example, to treat or prevent or ameliorate a disorder. The methods generally include (a) cleaving or down-regulating an endogenous B2M gene in an isolated cell (e.g., a T cell or lymphocyte) using a nuclease (e.g., ZFN or TALEN) or a nuclease system such as CRISPR/Cas or Cfp1/CRISPR with an engineered crRNA/tracr RNA, or using an engineered transcription factor (e.g., ZFN-TF, TALE-TF, Cfp1-TF, or Cas9-TF), such that the B2M gene is inactivated or down-regulated; and (b) introducing the cell into a subject, thereby treating or preventing the disorder.

In some embodiments, the gene encoding TCR α (TCRA) and/or TCR β (TCRB) is also inactivated or down-regulated.

The transcription factor and/or one or more nucleases can be introduced into the cell as mRNA, in protein form, and/or as a DNA sequence encoding one or more nucleases. In certain embodiments, introducing the isolated cell into a subject further comprises other genetic modifications such as integration of exogenous sequences (to the cleaved B2M, TCR gene, or other genes, e.g., safe harbor gene or locus) and/or inactivation (e.g., nuclease-mediated) of other genes, e.g., one or more HLA and/or TAP genes. The exogenous sequence can be introduced via a vector (e.g., Ad, AAV, LV), or by using techniques such as electroporation. In some embodiments, the protein is introduced into the cells by cell extrusion (cell sizing) (see Kollmann seger et al (2016) Nat Comm 7,10372doi:10.1038/ncomms 10372). In some aspects, the compositions include isolated cell fragments and/or (partially or fully) differentiated cells.

In some aspects, the mature cells may be used in cell therapy, e.g., for adoptive cell transfer. In other embodiments, the cells used for T cell transplantation comprise other genetic modifications of interest. In one aspect, the T cell comprises an inserted Chimeric Antigen Receptor (CAR) specific for a cancer marker. In another aspect, the inserted CAR is specific for a CD19 marker signature of a B cell malignancy. Such cells can be used in therapeutic compositions for treating patients without the need for matching HLA, and can therefore be used as "off-the-shelf" therapeutic agents for any patient in need thereof.

In another aspect, a B2M regulated (modified) T cell comprises an inserted antibody-coupled T cell receptor (ACTR) donor sequence in some embodiments, the ACTR donor sequence is inserted into a B2M or TCR gene to disrupt the gene expression following nuclease-induced cleavage in embodiments, the donor sequence is inserted into a "safe harbor" locus, such as the AAVS1, HPRT, albumin, and CCR5 genes in some embodiments, the ACTR sequence is inserted via targeted integration, wherein the ACTR donor sequence comprises flanking homology arms having homology with sequences flanking the cleavage site of the engineered nuclease in some embodiments, the ACTR donor sequence further comprises a promoter and/or other transcriptional regulatory sequences in other embodiments, the ACTR donor sequence lacks a promoter in some embodiments, the ACTR donor is inserted into a TCR β encoding gene (TCRB) in some embodiments, the insertion is via targeted cleavage of a constant region of the gene (TCR β) or in other embodiments, the ACTR donor gene is inserted into a TCR 48363, preferably a TCR coding region of the TCR ra, the TCR coding region is inserted into a TCR ra 5, or TCR coding region in other embodiments, the TCR 6778, the TCR coding for additional TCR gene is inserted into a TCR ra coding region in some embodiments, preferably TCR ra coding for TCR ra, TCR ra 5, TCR coding for TCR.

Also provided are pharmaceutical compositions comprising modified cells described herein (e.g., T cells or stem cells having an inactivated B2M gene), or pharmaceutical compositions comprising one or more B2M gene binding molecules (e.g., engineered transcription factors and/or nucleases) as described herein. In certain embodiments, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients. The modified cells, B2M gene binding molecules (or polynucleotides encoding these molecules) and/or pharmaceutical compositions comprising these cells or molecules are introduced into a subject via methods known in the art, e.g., by intravenous infusion, injection into a particular blood vessel such as the hepatic artery, or by direct tissue injection (e.g., muscle). In some embodiments, the subject is an adult having a disease or condition that can be treated or alleviated by the composition. In other embodiments, the subject is a pediatric subject to whom the composition is administered to prevent, treat, or ameliorate diseases and conditions (e.g., cancer, graft-versus-host disease, etc.).

In some aspects, compositions (including B2M-regulated cells of ACTR) also include exogenous antibodies-see also, U.S. patent application No. 15/357,772-in some aspects, antibodies can be used to assemble T cells comprising ACTR to prevent or treat disorders in some embodiments, the antibodies recognize antigens associated with tumor cells or with Cancer-associated processes, such as EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding antibodies, CD19, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, VEGF, VEGFR, α V β 3 and α 5 β 1 integrins, CD20, CD30, CD33, CD52, CTLA4, and cytoadhesin (encoscin) (remott et al) Nat 12:278) in other embodiments, antibodies that recognize infectious diseases, such as HIV.

In another aspect, provided herein are B2M DNA-binding domains (e.g., ZFPs, TALEs, and sgrnas) that bind to a target site in a B2M gene. In certain embodiments, the DNA binding domain comprises: ZFPs having identified helical regions in the order shown in any one of the rows in table 1; TAL-effector domain DNA-binding proteins having an RVD as shown in any one row in table 2B; and/or a sgRNA as shown in any one of the rows in table 2A. The DNA-binding protein may be associated with a transcriptional regulatory domain to form an engineered transcription factor that regulates B2M expression. Alternatively, these DNA-binding proteins can be associated with one or more nuclease domains to form engineered Zinc Finger Nucleases (ZFNs), TALENs, and/or CRISPR/Cas systems that bind to and cleave the B2M gene. In certain embodiments, the single guide rna (sgrna) of the ZFN, TALEN, or CRISPR/Cas system binds to a target site in the human B2M gene. The DNA-binding domain of a transcription factor or nuclease (e.g., ZFP, TALE, sgRNA) can bind to a target site in the B2M gene, which B2M gene includes 9, 10, 11, 12 or more (e.g., 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of any one of SEQ ID NOS: 6-48 and/or 137-205. The zinc finger protein may include 1, 2,3, 4,5, 6, or more zinc fingers, each zinc finger having a recognition helix that specifically contacts a target site in a target gene. In certain embodiments, the zinc finger protein comprises 4 or 5 or 6 fingers (referred to as F1, F2, F3, F4, F5, and F6, and from N-terminus to C-terminus arranged F1 to F4 or F5 or F6), such as shown in table 1. In other embodiments, a single guide RNA or TAL-effector DNA-binding domain may bind to the target site as set forth in any one of SEQ ID NOs 6-48 or 137-205 (or 12 or more base pairs within any one of SEQ ID NOs 6-48). Exemplary sgRNA target sites are shown in SEQ ID NOS: 16-48, and exemplary TALEN binding sites are shown in Table 2B (SEQ ID NO: 137-. Other TALENs can be designed to target the sites described herein using classical or atypical RVDs, as described in U.S. patent nos. 8,586,526 and 9,458,205. The nucleases described herein (including ZFPs, TALEs or sgRNA DNA-binding domains) are capable of generating genetic modifications in the B2M gene comprising any of SEQ ID NOs 6-48 and/or 137-205, modifications (insertions and/or deletions) comprised in any of these sequences (SEQ ID NOs 6-48 and/or 137-205) and/or modifications to the B2M gene sequence flanking the target site sequence shown in SEQ ID NOs 6-48 or 137-205, for example, modifications within exon 1 or exon 2 of the B2M gene located in one or more of the following sequences: GGCCTTA, TCAAATT, TCAAAT, TTACTGA and/or AATTGAA.

Also provided are fusion molecules comprising a DNA-binding domain that binds exon 1 or exon 2 of the B2M gene and a transcription regulatory domain or nuclease domain, wherein the DNA-binding domain comprises a Zinc Finger Protein (ZFP) as shown in any one of the rows in table 1 and a TALE-effector protein as shown in any one of the rows in table 2B or a single guide rna (sgrna) as shown in any one of the rows in table 2A.

Other proteins described herein can further include a cleavage domain and/or cleavage half-domain (e.g., wild-type or engineered fokl cleavage half-domain). Thus, in any of the nucleases (e.g., ZFNs, TALENs, CRISPR/Cas systems) described herein, the nuclease domain can comprise a wild-type domain or a nuclease half-domain (e.g., fokl cleavage half-domain). In other embodiments, the nuclease (e.g., ZFN, TALEN, CRISPR/Cas nuclease) comprises an engineered nuclease domain or half-domain, e.g., an engineered fokl cleavage half-domain that forms an obligate heterodimer, see, e.g., U.S. patent publication No. 20080131962.

In another aspect, the disclosure provides polynucleotides encoding any of the proteins, fusion molecules, and/or divisions thereof described herein (e.g., sgrnas or other DNA-binding domains) the polynucleotides may be part of a viral vector, a non-viral vector (e.g., a plasmid), or in mRNA form, while in another aspect, any of the polynucleotides described herein may further include sequences for targeted insertion into B2M, TCR α, and/or TCR β genes (donor, homology arm or patch (patch) sequences), while in another aspect, gene delivery vectors including any of the polynucleotides described herein are provided, in certain embodiments, the vectors are adenoviral vectors (e.g., Ad5/F35 vectors), or Lentiviral Vectors (LV) including integrative active or integration lentiviral vectors, or adeno-associated viral vectors (AAV) thus, viral vectors are also provided herein, including mRNA modified sequences encoding nucleases (e.g., ZFN or TALENs) and/or nuclease systems (e.g., mRNA/or mRNA systems) and/or modified sequences for targeted modification of mRNA sequences and/or mRNA sequences for targeted mRNA may be found in other embodiments, including mRNA capping mRNA sequences that may be introduced in mRNA capping mode for example, mRNA coding for mRNA, mRNA coding for mRNA, mRNA for example, mRNA coding for DNA capping, mRNA for coding for example, mRNA for coding for DNA, mRNA for coding for.

In yet another aspect, the present disclosure provides an isolated cell comprising any of the proteins, polynucleotides, and/or vectors described herein. In certain embodiments, the cell is selected from the group consisting of: stem/progenitor or T cells (e.g., CD 4)⁺T cells). In another aspect, the present disclosure provides a method of making a computer program productA cell or cell line of a cell or line of any of the proteins, polynucleotides and/or vectors described herein, i.e., a cell or cell line derived from (e.g., cultured from) a cell in which B2M has been inactivated by one or more ZFNs and/or in which a donor polynucleotide (e.g., ACTR, engineered TCR and/or CAR) has been stably integrated into the genome of the cell. Thus, progeny of the cells described herein may not themselves include the proteins, polynucleotides, and/or vectors described herein, but in these cells at least the B2M gene is inactivated and/or the donor polynucleotide is integrated into the genome and/or expressed.

In another aspect, described herein are methods of inactivating a B2M gene in a cell by introducing one or more proteins, polynucleotides, and/or vectors described herein into a cell as described herein. In any of the methods described herein, the nuclease may induce targeted mutations, deletions of cellular DNA sequences, and/or promote targeted recombination at a predetermined chromosomal locus. Thus, in certain embodiments, the nuclease deletes or inserts one or more nucleotides from or into the target gene. In some embodiments, the B2M gene is inactivated by nuclease cleavage followed by non-homologous end joining. In other embodiments, the genomic sequence in the target gene is replaced, for example, using a nuclease as described herein (or a vector encoding the nuclease) and a "donor" sequence that is inserted into the gene following targeted cleavage with the nuclease. The donor sequence can be present in a nuclease vector, in a separate vector (e.g., an AAV, Ad, or LV vector), or alternatively, it can be introduced into the cell using a different nucleic acid delivery mechanism.

Furthermore, any of the methods described herein can be performed in vitro, in vivo, and/or ex vivo. In certain embodiments, the method is performed ex vivo, e.g., to modify T cells so that they can be used as a therapeutic agent in an allogeneic setting to treat a subject (e.g., a subject having cancer). Non-limiting examples of cancers that may be treated and/or prevented include: lung cancer, pancreatic cancer, liver cancer, bone cancer, breast cancer, colorectal cancer, leukemia, ovarian cancer, lymphoma, brain cancer, etc.

Brief description of the drawings

FIG. 1 is a schematic representation of exon 1 and exon 2(SEQ ID NOS: 1 and 2) of the B2M gene targeted by nucleases. Boxes indicate 5 different cleavage regions flanking the ZFN binding (targeting) site (a (ggcctta), c (tcaaatt), d (tcaaat), e (ttactga), and g (aattgaa)).

FIGS. 2A and 2B show nuclease activity. Fig. 2A is a bar graph depicting the percentage of gene modification at each site in T cells treated with ZFNs at 2 or 6 μ G doses, which are specific for B2M sites A, C, D, E and G as shown in fig. 1. Fig. 2B depicts TALEN activity against the B2M gene in K562 cells.

Figure 3 depicts the percentage of HLA-negative T cells analyzed by FACS analysis after treatment with B2M-specific ZFN pairs.

FIGS. 4A-4E depict FACS results from cells treated with B2M and TCRA-specific ZFNs. Fig. 4A depicts the results without ZFN treatment, fig. 4B shows the results after only TCRA-specific ZFNs (96% knock out of CD3 signal), and fig. 4C shows the results after only B2M-specific ZFNs (92% knock out of HLA signal). Figure 4D is a schematic showing the location of cells with double knockouts (resulting in loss of HLA marker and CD3 marker). FIG. 4E shows the results after treatment of cells with TCRA-and CD 3-specific ZFNs, demonstrating double knockdown in 82% of these cells.

Figure 5 shows results from trac (tcra) and B2M double knockouts and targeted integration of donors into trac (tcra) or B2M loci.

Detailed Description

Disclosed herein are compositions and methods for producing cells in which expression of the B2M gene is modulated such that the cell no longer includes HAL type I on its cell surface. Cells modified in this manner can be used for therapy, e.g., transplantation, because the lack of B2M expression prevents or reduces HLA-based immune responses. In addition, other genes of interest may be inserted into cells in which the B2M gene has been manipulated and/or other genes of interest may be knocked out.

SUMMARY

The practice of the present methods, and the preparation and use of the compositions described herein, employ, unless otherwise indicated, conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related arts, which are within the skill of the art. These techniques are well described in the literature. See, e.g., Sambrook et al, Molecularcloning: A Laboratory Manual 2 nd edition, Cold Spring Harbor LABORATORY Press (Cold Spring Harbor LABORATORY Press), 1989, and 3 rd edition, 2001; ausubel et al, CURRENT protocol IN MOLECULAR BIOLOGY (New compiled MOLECULAR BIOLOGY laboratory Manual), New John Weley father, Inc. (John Wiley & Sons, New York)1987 and periodic updates; (ii) the book of the society of Methods IN Enzymolloy (METHODS IN ENZYMOLOGY), San Diego, Academic Press; wolffe, CHROMATIN STRUCTURE ANDFUNCTION (CHROMATIN Structure and function), 3 rd edition, academic Press, san Diego, 1998; (METHODS in enzymology), volume 304, "Chromatin" (p.m. wassarman and a.p. wolffe, eds.), academic press, san diego, 1999; and METHODS IN MOLECULAR BIOLOGY (METHODS IN MOLECULAR BIOLOGY), volume 119, "Chromatin protocol (METHODS IN Chromatin)" (p.b. becker, eds.), tomawa's himalayan press (humana press, totawa), 1999.

Definition of

The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in either a linear or cyclic configuration, in either single-or double-stranded form. For the purposes of this disclosure, these terms are not intended to limit the length of the polymer. The term can encompass known analogs of natural nucleotides, as well as nucleotides modified at the base, sugar, and/or phosphate moieties (e.g., phosphorothioate backbones). In general, analogs of a particular nucleotide have the same base-pairing specificity; that is, the analog of A will base pair with T.

The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acid.

"binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). As long as the interaction as a whole is sequence specific, it is not required that all components of the binding interaction be sequence specific (e.g., in contact with phosphate residues in the DNA backbone). Such interactions are generally characterized by dissociation constants (K)_d) Is 10^-6M^-1Or lower. "affinity" refers to the strength of binding: increased binding affinity with lower K_dAnd (6) associating. "non-specific binding" refers to non-covalent interactions that occur between a macromolecule (e.g., DNA) and any molecule of interest (e.g., an engineered nuclease) that are independent of the target sequence.

A "DNA binding molecule" is a molecule that can bind DNA. Such DNA binding molecules may be polypeptides, domains of proteins, domains within larger proteins, or polynucleotides. In some embodiments, the polynucleotide is DNA, while in other embodiments, the polynucleotide is RNA. In some embodiments, the DNA-binding molecule is a protein domain of a nuclease (e.g., a fokl domain), while in other embodiments, the DNA-binding molecule is a guide RNA component of an RNA-guided nuclease (e.g., Cas9 or Cfp 1).

A "binding protein" is a protein that is capable of non-covalent binding to another molecule. The binding protein is capable of binding to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), and/or a protein molecule (protein binding protein). In the case of a protein binding protein, it may bind to itself (forming homodimers, homotrimers, etc.) and/or it may bind to one or more molecules of one or more different proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding, and protein binding activities.

A "zinc finger DNA binding protein" (or binding domain) is a protein or domain within a larger protein that is capable of binding DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain that stabilize its structure through coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. Repeat domains, each comprising a Repeat Variable Diresidue (RVD), are involved in the binding of a TALE to its associated target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. TALE proteins can be designed to bind target sites using canonical or atypical RVDs within the repeat unit. See, for example, U.S. patent nos. 8,586,526 and 9,458,205, incorporated herein by reference in their entirety.

Zinc fingers and TALE DNA binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring zinc finger protein or by engineering amino acids involved in DNA binding (repeat variable diresidues or RVD regions). Thus, the engineered zinc finger protein or TALE protein is a non-naturally occurring protein. Non-limiting examples of methods of designing and selecting engineered zinc finger proteins and TALEs. A designed protein is a non-naturally occurring protein whose design/composition is largely on a reasonable basis. Reasonable criteria for design include applying substitution rules and computer algorithms to process existing ZFP or TALE designs (canonical and atypical RVDs) and information in database storage information that incorporates the data. See, for example, U.S. patent nos. 9,458,205; 8,586,526, respectively; 6,140,081, respectively; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

The "selected" zinc finger proteins, TALE proteins or CRISPR/Cas systems are not found in nature and their generation comes mainly from the use of empirical methods such as phage display, interaction traps or hybrid selection. See, e.g., U.S.5,789,538; U.S.5,925,523; U.S.6,007,988; U.S.6,013,453; U.S.6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

"TtAgo" is a prokaryotic algaeus (Argonaute) protein that is thought to be involved in gene silencing. TtAgo is derived from thermophilic bacteria (Thermus thermophilus). See, e.g., Swarts et al, supra, g.sheng et al, (2013) proc.natl.acad.sci.u.s.a.111, 652). The "TtAgo system" is all components required, including, for example, guide DNA for cleavage by TtAgo enzyme.

"recombination" refers to the process of exchanging genetic information between two polynucleotides. For the purposes of this disclosure, "Homologous Recombination (HR)" refers to the particular form in which this exchange occurs, e.g., through a homology-directed repair mechanism during repair of a double-strand break in a cell. This process, which requires nucleotide sequence homology, repairs the "target" molecule (i.e., the molecule that undergoes a double strand break) using a "donor" molecular template, and is also referred to as "non-cross-over gene conversion" or "short-path gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the fragmented target and donor, and/or "synthesis-dependent strand annealing" of genetic information that will become part of the target using donor resynthesis, and/or related processes. These specific HR typically result in a sequence change of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

In the methods of the present disclosure, one or more of the targeted nucleases described herein generate a Double Strand Break (DSB) at a predetermined site (e.g., a gene or locus of interest) in a target sequence (e.g., cellular chromatin), and a "donor" polynucleotide having homology to the nucleotide sequence in the break region can be introduced into the cell. The presence of DSBs has been shown to contribute to the integration of donor sequences. Optionally, the construct has homology to the nucleotide sequence within the break region. The donor sequence may be integrated by physical integration, or the donor polynucleotide may be used as a template for repair of a break by homologous recombination, resulting in introduction of all or part of the nucleotide sequence in the donor into cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in certain embodiments, converted to a sequence present in a donor polynucleotide. Thus, use of the terms "substitution" or "replacement" is understood to mean the replacement of one nucleotide sequence by another (i.e., the replacement of a sequence in the informational sense), without necessarily requiring the physical or chemical replacement of one polynucleotide by another.

Other zinc finger protein pairs may be used for other double-stranded cleavage of other target sites within the cell in any of the methods described herein.

In certain embodiments of the methods for targeted recombination and/or replacement and/or alteration of a sequence of a region of interest within cellular chromatin, chromosomal sequences are altered by homologous recombination using exogenous "donor" nucleotide sequences. If sequences homologous to the region of the break are present, the homologous recombination is stimulated by the presence of a double strand break in cellular chromatin.

In any of the methods described herein, the first nucleotide sequence ("donor sequence") can comprise a sequence that is homologous but not identical to a genomic sequence in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the region of interest sequence exhibits about 80-99% (or any integer therebetween) sequence identity to the genomic sequence to be replaced. In other embodiments, the homology between the donor and the genomic sequence is greater than 99%, e.g., a genomic sequence of greater than 100 consecutive base pairs differs from the donor by only 1 nucleotide. In some cases, non-homologous portions of the donor sequence can include sequences that are not present in the region of interest, thereby introducing new sequences into the region of interest. In these cases, the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000, which are homologous or identical to the sequence in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

Any of the methods described herein can be used to partially or completely inactivate one or more target sequences within a cell by targeted integration of a donor sequence that disrupts expression of a gene of interest. Cell lines having partially or fully inactivated genes are also provided.

In addition, the targeted integration methods described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, and one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence can produce one or more RNA molecules (e.g., small hairpin RNA (shrna), inhibitory RNA (rnai), microrna (mirna), etc.).

"cleavage" refers to the breaking of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of the phosphodiester bond. Both single-stranded and double-stranded cleavage can be employed, and double-stranded cleavage can result from different single-stranded cleavage events. DNA cleavage can result in blunt or staggered ends. In certain embodiments, the fusion polypeptide is used to target double-stranded DNA cleavage.

A "cleavage half-domain" is a polypeptide sequence that is capable of forming a complex with a second polypeptide (which may be the same or different) having cleavage activity, preferably double-stranded cleavage activity. The term "first and second cleavage half-domains"; "+ and-cleavage half-domains" and "right and left cleavage half-domains" are used interchangeably to refer to pairs of half-domains that cleave dimerization.

An "engineered cleavage half-domain" is a cleavage half-domain that is modified to form an obligate heterodimer with another cleavage half-domain (e.g., another engineered cleavage half-domain). See also, U.S. patent nos. 7,888,121; 7,914,796, respectively; 8,034,598, respectively; 8,623,618 and U.S. patent publication No. 2011/0201055, which are incorporated herein by reference in their entirety.

The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA; may be linear, cyclic or branched and may be single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. The donor sequence may be of any length, for example, from 2 to 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably 1,000 nucleotides in length (or any integer therebetween), more preferably 200 nucleotides in length and 500 nucleotides in length.

"chromatin" is a nucleoprotein structure comprising the genome of a cell. Cellular chromatin comprises nucleic acids, primarily DNA, and proteins, including histone and non-histone chromosomal proteins. Eukaryotic chromatin exists predominantly in the form of nucleosomes in which the nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two copies each of histones H2A, H2B, H3 and H4; and linker DNA extending between nucleosome cores (the length varies depending on the organism). Histone H1 molecules are typically associated with linker DNA. For the purposes of this disclosure, the term "chromatin" is intended to encompass all types of nuclear proteins, including prokaryotic and eukaryotic. Cellular chromatin includes chromosomal and episomal chromatin.

A "chromosome" is a chromatin complex comprising all or part of the genome of a cell. The genome of a cell is usually characterized by its karyotype, which is the collection of all chromosomes that comprise the genome of the cell. The genome of a cell may comprise one or more chromosomes.

An "episome" is a replicated nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

A "target site" or "target sequence" is a nucleic acid sequence that defines the portion of a nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding are present. For example, the sequence 5'GAATTC 3' is the target site for the Eco RI restriction endonuclease.

An "exogenous" molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical, or other means. "normally present in a cell" is determined with respect to a particular developmental mental state (mental state) and environmental conditions of the cell. Thus, for example, molecules that are only present during embryonic development of muscle are exogenous molecules to adult muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule relative to a cell that is not heat shocked. For example, an exogenous molecule can include a functional form of a malfunctioning endogenous molecule or a malfunctioning form of a normally functioning endogenous molecule.

The exogenous molecule may be a small molecule, such as produced by combinatorial chemistry, or a large molecule, such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the foregoing, or any complex comprising one or more of the foregoing, and the like. Nucleic acids include DNA and RNA, which may be single-stranded or double-stranded; may be linear, branched or cyclic; and may be of any length. See, for example, U.S. patent nos. 8,703,489 and 9,255,259. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. nos. 5,176,996 and 5,422,251. Proteins may include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

The exogenous molecule can be the same type of molecule as the endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, the exogenous nucleic acid can include an infectious viral genome, a plasmid or episome introduced into the cell, or contain a chromosome that is not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electrical transduction, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. The exogenous molecule may also be a molecule of the same type as the endogenous molecule but derived from a different species than the cell from which it was derived. For example, a human nucleic acid sequence can be introduced into a cell line originally derived from a mouse or hamster.

In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular stage of development under particular environmental conditions. For example, an endogenous nucleic acid can include a chromosome, mitochondrial genome, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules may include proteins, e.g., transcription factors and enzymes.

A "fusion" molecule is one in which two or more subunit molecules are linked (preferably covalently linked). The subunit molecules may be of the same chemical type or of different chemical types. Examples of the first type of fusion molecule can include, but are not limited to, fusion proteins (e.g., fusions between ZFPs or TALE DNA binding domains and one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the fusion proteins described above). Examples of the second class of fusion molecules include, but are not limited to: fusions between triplex-forming nucleic acids and polypeptides, and fusions between minor groove binders and nucleic acids. The term also includes systems in which a polynucleotide component is associated with a polynucleotide component to form a functional molecule (e.g., CRISPR/Cas system in which a single guide RNA is associated with a functional domain to regulate gene expression).

Expression of the fusion protein in the cell can result from delivery of the fusion protein into the cell or by delivery of a polynucleotide encoding the fusion protein to the cell, wherein the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. Expression of proteins in cells may also involve trans-splicing, polypeptide cleavage, and polypeptide ligation. Methods for delivering polynucleotides and polypeptides to cells are presented elsewhere in the disclosure.

For purposes of this disclosure, "gene" includes regions of DNA encoding a gene product (see above), as well as regions of DNA that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

A "safe harbor" locus is a locus within a genome in which a gene may not have any deleterious effect on the host cell. Most beneficial are safe harbor loci in which the expression of the inserted gene sequence is not disrupted by any read-through expression from adjacent genes. Non-limiting examples of safe harbor loci targeted by nucleases include: CCR5, CCR5, HPRT, AAVS1, Rosa and albumin. See, for example, U.S. patent nos. 8,771,985; 8,110,379, respectively; 7,951,925, respectively; U.S. patent publication numbers 20100218264; 20110265198, respectively; 20130137104, respectively; 20130122591, respectively; 20130177983, respectively; 20130177960, respectively; 20150056705 and 20150159172).

"Gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include modified RNA, proteins modified by processing such as capping, polyadenylation, methylation, and editing, and by processing such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

"Regulation" or "modification" of gene expression refers to an alteration in the activity of a gene. Modulation of expression may include, but is not limited to: gene activation and gene suppression, including by genetic modification via binding of exogenous molecules (e.g., engineering transcription factors). Modulation can be achieved by modification of the gene sequence via genome editing (e.g., cleavage, alteration, inactivation, random mutation). Gene inactivation refers to any reduction in gene expression compared to a cell that has not been modified as described herein. Thus, gene inactivation may be partial or complete.

A "region of interest" is any region of cellular chromatin that requires binding of an exogenous molecule, e.g., a gene or a non-coding sequence within or adjacent to a gene. Binding may be for the purpose of targeted DNA cleavage and/or targeted recombination. The region of interest can be present, for example, in a chromosome, episome, organelle genome (e.g., mitochondria, chloroplasts), or infectious viral genome. The region of interest may be in a coding region of the gene, a transcribed non-coding region such as a leader sequence, trailer sequence or intron, or upstream or downstream of the coding region in a non-transcribed region. The region of interest may be as small as a single nucleotide pair, or as long as 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

"eukaryotic" cells can include, but are not limited to, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells).

The terms "operatively connected" and "operatively connected" (or "operatively connected") are used interchangeably to refer to the juxtaposition of two or more components (e.g., sequence elements) arranged such that the components all function properly and allow at least one of the components to mediate an action exerted on at least one other component. For example, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Transcriptional regulatory sequences are typically operably linked in cis to a coding sequence, but need not be immediately adjacent to the sequence. For example, enhancers are transcriptional regulatory sequences operably linked to a coding sequence, although they are discontinuous.

For fusion polypeptides, the term "operably linked" may mean that each component functions the same in its connection to other components as it functions when not connected. For example, for a fusion polypeptide in which a DNA binding domain (e.g., ZFP, TALE) is fused to an activation domain, the DNA binding domain and activation domain are operably linked, in which fusion polypeptide the DNA binding domain portion is capable of binding to its target site and/or its binding site, and the activation domain is capable of up-regulating gene expression. When the DNA binding domain is fused to the cleavage domain in a fusion polypeptide, the DNA binding domain and the cleavage domain are operably linked if the DNA binding domain portion is capable of binding to its target site and/or its binding site and the cleavage domain is capable of cleaving DNA in the vicinity of the target site in the fusion polypeptide. Similarly, for a fusion polypeptide in which a DNA binding domain is fused to an activation or suppression domain, the DNA binding domain and the activation or suppression domain are operably linked if the portion of the DNA binding domain in the re-fusion polypeptide is capable of binding to its target site and/or its binding site and the activation domain is capable of up-regulating gene expression or the suppression domain is capable of down-regulating gene expression.

A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid that is not identical in sequence to a full-length protein, polypeptide, or nucleic acid, but retains the same function of the full-length protein, polypeptide, or nucleic acid. Functional fragments may have more, fewer, or the same number of residues as the corresponding native molecule, and/or may contain 1 or more amino acid or nucleotide substitutions. Methods for determining a function of a nucleic acid (e.g., encoding a function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of proteins are well known. For example, the DNA binding function of the polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be analyzed by gel electrophoresis. See Ausubel et al, supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assay, or complementation assay, either genetically or biochemically. See, e.g., Fields et al, (1989) Nature 340: 245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

A "vector" is capable of transferring a gene sequence to a target cell. "vector construct", "expression vector", "expression construct", "expression cassette" and "gene transfer vector" generally refer to a nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring a gene sequence to a target cell. Thus, the term includes cloning and expression vectors, as well as integration vectors.

"reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is readily detectable, which is not necessary but preferred in routine experimentation. Suitable reporter genes can include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. An "expression tag" includes a sequence encoding a reporter operably linked to a desired gene sequence to monitor expression of a gene of interest.

The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as laboratory animals such as rabbits, dogs, cats, rats, mice, and other animals. Thus, the term "subject" or "patient" refers to any mammalian patient or subject to which an expression cassette of the invention may be administered. Subjects of the invention include those suffering from a disorder or those at risk of developing a disorder.

The terms "treat" and "treatment" as used herein refer to reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the root cause, preventing the occurrence of symptoms and/or their root cause, and improving or eliminating injury. Cancer and graft-versus-host disease are non-limiting examples of conditions that can be treated using the compositions and methods described herein. Thus, "treating" and "treatment" include:

(i) preventing the disease or condition from occurring in a mammal, particularly when the mammal is predisposed to, but has not yet been diagnosed with, the condition;

(ii) inhibiting the disease or disorder, i.e., arresting its development;

(iii) alleviating, i.e., causing regression of, the disease or condition; or

(iv) Alleviating the symptoms caused by the disease or condition, i.e., alleviating pain without addressing the underlying disease or condition.

As used herein, the terms "disease" and "condition" are used interchangeably, or may be different, wherein a particular disease or condition may not have a known causative agent (and thus cause is not yet known), and thus has not yet been considered a disease but merely an undesirable-acting condition or syndrome, wherein a particular set of symptoms is more or less identified by a clinician.

"pharmaceutical composition" refers to a compound formulation of the present invention and to art-accepted vehicles for delivering biologically active compounds to a mammal (e.g., a human). Such media include pharmaceutically acceptable carriers, diluents or excipients, and the like.

An "effective amount" or "therapeutically effective amount" means: the amount of a compound of the present invention, when administered to a mammal, preferably a human, is sufficient to provide effective treatment of the mammal, preferably a human. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art, given their knowledge and the teachings of the present invention.

DNA binding domains

Described herein are compositions comprising a DNA binding domain that specifically binds to a target site in any gene, including an HLA gene or HLA regulator (including B2M gene). Any DNA binding domain can be used in the methods and compounds disclosed herein, including but not limited to zinc finger DNA binding domains, TALE DNA binding domains, DNA binding portions of CRISPR/Cas nucleases (sgrnas), or DNA binding domains from meganucleases (meganucleases). The DNA binding domain may bind to any target sequence within the gene, including, but not limited to, a target sequence of 12 or more nucleotides as set forth in any one of SEQ ID NOS 6-48.

In certain embodiments, the DNA binding domain comprises a zinc finger protein. Preferably, the zinc finger protein is non-naturally occurring, wherein it is engineered to bind to a selected target site. See, e.g., Beerli et al (2002) NatureBiotechnol.20: 135-141; pabo et al (2001) Ann. Rev. biochem.70: 313-340; isalan et al (2001) Nature Biotechnol.19: 656-660; segal et al (2001) curr. Opin. Biotechnol.12: 632-637; choo et al (2000) curr. Opin. struct. biol.10: 411-416; U.S. Pat. nos. 6,453,242; 6,534,261; 6,599,692, respectively; 6,503,717, respectively; 6,689,558, respectively; 7,030,215, respectively; 6,794,136, respectively; 7,067,317, respectively; 7,262,054, respectively; 7,070,934, respectively; 7,361,635, respectively; 7,253,273, respectively; and U.S. patent publication No. 2005/0064474; 2007/0218528, respectively; 2005/0267061, all of which are incorporated herein by reference in their entirety. In certain embodiments, the DNA binding domain comprises a zinc finger protein disclosed in U.S. patent publication No. 2012/0060230 (e.g., table 1), which is incorporated herein by reference in its entirety.

Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. The engineering methods may include, but are not limited to, rational design and different types of selection. For example, rational design includes the use of databases comprising trisomy (or tetrasome) nucleotide sequences and individual zinc finger amino acid sequences, wherein each trisomy or tetrasome nucleotide sequence is associated with one or more zinc finger amino acid sequences that bind that particular trisomy or tetrasome sequence. See, for example, U.S. Pat. nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety.

Exemplary selection methods include phage display and two-hybrid systems disclosed in U.S. Pat. nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568; and WO98/37186, WO 98/53057, WO 00/27878, WO 01/88197 and GB 2,338,237. Furthermore, enhancement of the binding specificity of zinc finger binding domains has been described, for example, in U.S. patent No. 6,794,136.

Furthermore, as disclosed in these and other references, the zinc finger domains and/or zinc finger proteins of the multi-fingers may be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are also see, e.g., U.S. patent nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. Furthermore, enhancement of the binding specificity of zinc finger binding domains has been described, for example, in U.S. patent No. 6,794,136.

Selecting a target site; ZFPs and methods for designing and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are described in detail in U.S. patent nos. 6,140,081; 5,789,538, respectively; 6,453,242; 6,534,261; 5,925,523, respectively; 6,007,988, respectively; 6,013,453, respectively; 6,200,759, respectively; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

Furthermore, as disclosed in these and other references, the zinc finger domains and/or zinc finger proteins of the multi-fingers may be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are also see, e.g., U.S. patent nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

In certain embodiments, the DNA binding domain is an engineered zinc finger protein that binds (in a sequence-specific manner) to a target site in the B2M gene or B2M regulatory gene and regulates B2M expression. In some embodiments, the zinc finger protein binds to a target site in B2M, while in other embodiments, the zinc finger binds to a target site in B2M.

ZFPs typically include at least three fingers. Some ZFPs include four, five, or six fingers. ZFPs that include three fingers typically recognize a target site that includes 9 or 10 nucleotides; ZFPs that include four fingers typically recognize target sites that include 12-14 nucleotides; whereas ZFPs with six fingers recognize target sites comprising 18-21 nucleotides. The ZFPs can also be fusion proteins comprising one or more regulatory domains, which can be transcriptional activation or repression domains.

In some embodiments, the DNA binding domain may be derived from a nuclease. For example, recognition sequences for homing endonucleases and meganucleases are known such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. See also U.S. patent No. 5,420,032; U.S. patent No. 6,833,252; belfort et al (1997) Nucleic Acids Res.25: 3379-3388; dujon et al (1989) Gene 82: 115-118; perler et al (1994) Nucleic Acids Res.22, 1125-1127; jasin (1996) Trends Genet.12: 224-228; gimble et al (1996) J.mol.biol.263: 163-; argast et al (1998) J.mol.biol.280: 345-353 and NEB corporation (New England Biolabs) catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target locations. See, e.g., Chevalier et al (2002) Molec. cell10: 895-905; epinat et al (2003) Nucleic acids sRs.31: 2952-2962; ashworth et al, (2006) Nature 441: 656-; paques et al (2007) CurrentGene Therapy 7: 49-66; U.S. patent publication No. 20070117128.

In other embodiments, the DNA binding domain comprises an engineered domain from a TAL effector similar to those derived from the plant pathogen Xanthomonas (Xanthomonas) (see Boch et al, (2009) Science326: 1509-; U.S. patent application nos. 20110301073 and 20110145940. Phytopathogens of the genus Xanthomonas (Xanthomonas) are known to cause a number of diseases in important crops. Pathogenicity of xanthomonas depends on a conserved type III secretion (T3S) system that injects more than 25 different effector proteins into plant cells. Among these injected proteins are transcriptional activator-like effectors (TALEs) that mimic plant transcriptional activators and manipulate plant transcriptomes (see Kay et al (2007) Science 318: 648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TALEs is AvrBs3 from Xanthomonas campestris pepper spot disease causing variety (Xanthomonas campestris pv. vesicatoria) (see Bonas et al (1989) Mol Gen Genet 218:127-136 and WO 2010079430). TALEs contain a centralized domain of tandem repeats, each repeat containing about 34 amino acids, which are critical to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcription activation domain (for a review see Schornack S et al (2006) J Plant Physiol163(3): 256-272). Furthermore, among the phytopathogenic bacteria, Ralstonia solanacearum, two genes called brg11 and hpx17 in the bacterial biovariant (biovar)1 strain GMI1000 and the bacterial strain RS1000 of biovariant 4 have been found to be homologous to the AvrBs3 family of Xanthomonas (see Heuer et al (2007) apple and Envir Micro 73(13): 4379-. These genes are 98.9% identical to each other in nucleotide sequence, but differ by a1,575 bp deletion in the hpx17 repeat domain. However, both gene products have less than 40% sequence identity to the AvrBs3 family protein of Xanthomonas (Xanthomonas).

The specificity of these TAL effectors depends on the sequence present in the tandem repeat. The repeat sequences comprise about 102 base pairs, and the repeats are typically 91-100% homologous to each other (Bonas et al, supra). The polymorphisms of the repeats are usually located at positions 12 and 13, and it appears that there is a one-to-one correspondence between the identity of the hypervariable diresidues (repeat variable diresidues or RVD regions) at positions 12 and 13 and the identity of consecutive nucleotides in the TAL effector target sequence (see Moscou and bogdanave, (2009) Science326:1501 and Boch et al (2009) Science326: 1509-. Experimentally, the natural codes for these TAL effector DNA recognition have been determined, thus the HD sequences (repeat variable diresidues or RVDs) at positions 12 and 13 result in binding for cytosine (C), NG binding T, NI binding A, C, G or T, NN binding a or G, and IG binding T. These DNA binding repeats have been assembled into proteins with novel combinations and numbers of repeats, resulting in artificial transcription factors that interact with novel sequences and activate expression of non-endogenous reporter genes in plant cells (Boch et al, supra). Engineered TAL proteins have been linked to fokl cleavage half-domains to generate TAL effector domain nuclease fusions (TALENs), including TALENs with atypical RVDs. See, for example, U.S. patent No. 8,586,526.

In some embodiments, the TALEN comprises an endonuclease (e.g., FokI) cleavage domain or cleavage half-domain. In other embodiments, the TALE-nuclease is a megatal. These large atmosphere TAL nucleases are fusion proteins that include a TALE DNA binding domain and a meganuclease cleavage domain. Meganuclease cleavage domains are active as monomers and do not require dimerization to obtain activity. (see Boissel et al, (2013) Nucl Acid Res:1-13, doi:10.1093/nar/gkt 1224).

In yet another embodiment, the nuclease comprises a compact TALEN. There are single-stranded fusion proteins that link the TALE DNA binding domain to the TevI nuclease domain. The fusion protein can act as a nickase that is localized by the TALE region, or can generate a double-strand break, depending on the position of the TALE DNA-binding domain relative to the TevI nuclease domain (see Berldeey et al (2013) Nat Comm:1-8DOI:10.1038/ncomms 2782). In addition, nuclease domains can also exhibit DNA-binding functionality. Any TALEN can be used in conjunction with other TALENs, e.g., one or more TALENs with one or more wide range TALEs (ctalens or FokI-TALENs).

Furthermore, as disclosed in these and other references, the zinc finger domains and/or the zinc finger proteins or TALEs of the multi-fingers can be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are also see, e.g., U.S. patent nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. Furthermore, enhancement of the binding specificity of zinc finger binding domains has been described, for example, in U.S. patent No. 6,794,136.

In certain embodiments, the DNA-binding domain is part of a CRISPR/Cas nuclease system comprising a single guide rna (sgrna) that binds to DNA. See, for example, U.S. patent No. 8,697,359 and U.S. patent publication nos. 20150056705 and 20150159172. The CRISPR (regularly clustered short palindromic repeats) locus encoding the RNA component of this system and the Cas (CRISPR-associated) locus encoding the protein (Jansen et al, 2002.mol. Microbiol.43: 1565;. Makarova et al, 2002.Nucleic Acids Res.30: 482. 496; Makarova et al, 2006.biol. direct 1: 7; Haft et al, 2005.PLoS Comp.biol.1: e60) constitute the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts comprise a combination of a CRISPR-associated (Cas) gene and a non-coding RNA element capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

Type II CRISPR is one of the well characterized systems and carries out targeted DNA double strand breaks in 4 consecutive steps. First, two non-coding RNAs, a pre-crRNA array and a tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNA comprising a separate spacer sequence. Third, the mature crRNA tracrRNA complex directs a functional domain (e.g., nuclease, such as Cas) to the target DNA via watson-crick base pairing, which is located between a spacer on the crRNA and a protospacer on the target DNA next to a Protospacer Adjacent Motif (PAM), which is an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to create a double strand break within the protospacer. The activity of the CRISPR/Cas system comprises 3 steps: (i) insertion of foreign DNA sequences into CRISPR arrays prevents future attacks by a process called "adaptation" (ii) expression of the protein of interest, and expression and processing of the array, followed by (iii) RNA-mediated interference with foreign nucleic acids. Thus, in bacterial cells, a number of so-called "Cas" proteins are involved in CRISPR/Cas system natural functions and play a role in functions such as insertion of foreign DNA and the like.

In certain embodiments, the Cas protein may be a "functional derivative" of a naturally occurring Cas protein. "functional derivatives" of a native sequence polypeptide are compounds that have qualitative biological properties in common with the native sequence polypeptide. "functional derivatives" include, but are not limited to, fragments of the native sequence or derivatives of the native sequence polypeptide and fragments thereof, provided that they have the common biological activity of the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" includes amino acid sequence variants of polypeptides, covalent modifications, and fusions thereof, such as derivatized Cas proteins. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, covalent modifications of Cas proteins, or fragments thereof. Cas proteins including Cas proteins or fragments thereof and derivatives of Cas proteins or fragments thereof may be obtained from cells or by chemical synthesis or by a combination of both methods. The cell can be a cell that naturally produces a Cas protein, or a cell that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein at a higher expression level or to produce a Cas protein from a heterologously introduced nucleic acid that encodes a Cas that is the same as or different from the endogenous Cas. In some cases, the cell does not naturally produce a Cas protein, but is genetically engineered to produce a Cas protein. In some embodiments, the Cas protein is a small Cas9 homolog for delivery via AAV vectors (Ran et al (2015) Nature510, p.186).

In some embodiments, the DNA binding domain is part of a TtAgo system (see, Swarts et al, supra; Sheng et al, supra). In eukaryotic cells, gene silencing is mediated by proteins of the algoracle (Ago) family. In this example, Ago bound to small (19-31nt) RNA. The protein-RNA silencing complex recognizes the target RNA via Watson-Crick base pairing, which is located between the small RNA and the target, and the endonuclease enzymatically cleaves the target RNA (Vogel (2014) Science 344: 972-973). In contrast, prokaryotic Ago proteins bind to small single-stranded DNA fragments and may serve to detect and remove foreign (usually viral) DNA (Yuan et al, (2005) mol.cell 19,405; Olovnikov et al (2013) mol.cell 51,594; Swarts et al, supra). Exemplary prokaryotic Ago proteins include: those from the species Pyrenophilus (Aquifexaeolius), Rhodobacter sphaeroides (Rhodobacter sphaeroides) and Thermus thermophilus (Thermusthermophilus).

One of the most well characterized prokaryotic Ago proteins is one from thermus thermophilus (Ttago; Swarts et al, supra). TtAgo is associated with 15nt or 13-25nt single stranded DNA fragments having a 5' phosphate group. This "guide DNA" bound by TtAgo can be used to guide the protein-DNA complex to bind to the watson-crick complementary DNA sequence in a third party DNA (third-party) molecule. Once the sequence information in these guide DNAs can allow identification of the target DNA, the TtAgo-guide DNA complex cleaves the target DNA. This mechanism is also supported by the results of the TtAgo-guided DNA complex, despite binding to its target DNA (g.sheng et al, supra). Ago (RsAgo) from rhodobacter sphaeroides has similar properties (Olivnikov et al, supra).

Exogenous guide DNA of any DNA sequence can be loaded onto the TtAgo protein (Swarts et al, supra). Because the specificity of TtAgo cleavage is guided by the guide DNA, a TtAgo-DNA complex formed with exogenous and recognizer-specific (endogenous-specific) guide DNA will therefore guide TtAgo target DNA cleavage to target DNA specific for the complementary recognizer. In this way, one can generate a targeted double-stranded break in the DNA. The use of a TtAgo-guided DNA system (or a homologous Ago-guided DNA system from other organisms) would allow targeted cleavage of genomic DNA within a cell. Such cleavage may be single-stranded or double-stranded. For cleaving mammalian genomic DNA, it is preferable to use a form of TtAgo codon optimized for expression in mammalian cells. Furthermore, cells may be treated preferably with TtAgo-DNA complexes formed in vitro, wherein the TtAgo is fused to a cell-penetrating peptide. Furthermore, the TtAgo protein can be preferably used in a form that has been altered by mutagenesis to have an improved activity at 37 ℃. Ago-RNA-mediated DNA cleavage can be used to affect all outcomes including gene knock-out, targeted gene addition, gene correction, target gene deletion using technical standards in the art for the development of DNA breaks.

Thus, any DNA binding domain may be used.

Fusion molecules

Also provided are fusion molecules that include a DNA-binding domain (e.g., ZFP or TALE, CRISPR/Cas component, such as a single guide RNA) associated with a heterologous regulatory (functional) domain (or functional fragment thereof) as described herein. Common domains include, for example: transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzymes and their related factors and modifiers; DNA rearranging enzyme and its related factor and modifier; chromatin-associated proteins and their modifiers (e.g., kinases, acetylases, and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their related factors and modifiers. Such fusion molecules include transcription factors, which include a DNA binding domain and a transcription regulatory domain as described herein, and nucleases, which include a DNA binding domain and one or more nuclease domains.

Suitable domains (transcriptional activation domains) for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al, J.Virol.71,5952-5962(1997)), the nuclear hormone receptor (see, e.g., Torchia et al, curr. Opin. cell. biol.10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko and Barik, J.Virol.72: 5610-; liu et al, Cancer Gene ther.5:3-28(1998)), or artificial chimeric functional domains, such as VP64(Beerli et al (1998) Proc. Natl. Acad. Sci. USA95: 14623-33), and the degradation determinant (degron) (Molinari et al, (1999) EMBO J.18, 6439-6447). Additional exemplary activation domains include Oct 1, Oct-2A, Sp1, AP-2 and CTF1(Seipel et al, EMBO J.11, 4961 4968(1992) and p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. see, e.g., Robyr et al (2000) mol. Endocrinol.14: 329-; Collingwood et al (1999) J.mol. Endocrinol.23: 255-275; Leo et al (2000) Gene 245: 1-11; Manteuffel-Cymborskowa (1999) Acta. Acchi.46: 77-89; McKena et al (1999) J.Sterorem.Biochem.69: 3-12; Devk et al (1999) Ochium. 46: 77-89; McKenna et al (1999) J.Steror.Biochem.11; Biochel.283: 12; Devk et al (1999) Acta. Scirk et al (2000) and C25. J.11; McKernar. J.11. J.7. J.J.11. Biochem.11; McKernar.23-Oc.35, III, 7, III, 7, III (1991) Genes Dev.5: 298-; cho et al (1999) Plant mol.biol.40: 419-429; ullmason et al (1999) Proc.Natl.Acad.Sci.USA 96: 5844-; Sprenger-Haussels et al, (2000) plantatJ.22: 1-8; gong et al (1999) Plant mol. biol.41: 33-44; and Hobo et al (1999) Proc.Natl.Acad.Sci.USA 96:15, 348-.

It will be appreciated by those skilled in the art that in the formation of a fusion protein (or nucleic acid encoding the same) between a DNA binding domain and a functional domain, the activation domain or molecules that interact with the activation domain are suitable as functional domains. Basically, any molecule capable of recruiting an activation complex and/or activation activity (e.g., histone acetylation) to a target gene can be used as the activation domain of the fusion protein. Insulator domains, localization domains and chromatin remodeling proteins such as ISWI-containing domains and/or methyl binding domain proteins suitable for use as functional domains in fusion molecules are described, for example, in U.S. patent No. 7,053,264.

Exemplary repressing domains may include, but are not limited to, KRAB A/B, KOX, TGF- β -inducible early genes (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B), Rb and MeCP2, see, e.g., Bird et al (1999) Cell 99: 451-454; Tyler et al (1999) Cell 99: 443-446; Knoepfler et al (1999) Cell 99: 447-450; and Robertson et al Gene (2000) Nature t.25:338-342 other exemplary repressing domains include, but are not limited to, ROM2 and AtHD2A. see, e.g., Chem et al (1996) Plant Cell 8: 305-321; and Wu et al (2000) Plant J.22: 19-27.

Fusion molecules are constructed by cloning and biochemical coupling methods well known to those skilled in the art. The fusion molecules include a DNA binding domain (e.g., ZFP, TALE, sgRNA) and a functional domain (e.g., a transcription activation or repression domain). The fusion molecule also optionally comprises a nuclear localization signal (e.g., T-antigen from SV40 medium) and an epitope tag (e.g., FLAG and hemagglutinin). The fusion protein (and the nucleic acid encoding it) is designed such that the translational reading frame between the fusion components is preserved.

The fusion between the functional domain (or functional fragment thereof) polypeptide component on the one hand and the non-protein DNA binding domain (e.g. antibiotics, intercalators, minor groove binders, nucleic acids) on the other hand is constructed by biochemical coupling methods known to the person skilled in the art. See, for example, the Pierce Chemical company (Pierce Chemical company) (rockford, il). Methods and compositions for generating fusions between minor groove binders and polypeptides have been described. Mapp et al (2000) Proc. Natl. Acad. Sci. USA 97: 3930-. In addition, the single guide RNA of the CRISPR/Cas system is associated with functional domains to form active transcription regulators and nucleases.

In certain embodiments, the target site is present in a accessible region of cellular chromatin. The reach regions may be determined, for example, as described in U.S. patent nos. 7,217,509 and 7,923,542. If the target site is not present in a accessible region of cellular chromatin, one or more accessible regions may be generated as described in U.S. patent nos. 7,785,792 and 8,071,370. In other embodiments, the DNA-binding domain of the fusion molecule is capable of binding to cellular chromatin, regardless of whether its target site is located in a accessible region. For example, the DNA binding domain is capable of binding to a linker DNA and/or a nucleosome DNA. Examples of "pioneer" DNA binding domains of this type can be found in certain steroid receptors and hepatocyte nuclear factor 3(HNF3) (Cordingley et al (1987) Cell 48: 261-.

The fusion molecule can be formulated with a pharmaceutically acceptable carrier, as known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 17 th edition, 1985; and U.S. Pat. nos. 6,453,242 and 6,534,261.

The functional components/domains of the fusion molecule may be selected from any of a variety of different components that are capable of affecting gene transcription once the fusion molecule is bound to a target sequence via its DNA binding domain. Thus, functional components may include, but are not limited to, various transcription factor domains, such as activators, repressors, co-activators, co-repressors, and silencers.

Additional exemplary functional domains are disclosed, for example, in U.S. Pat. nos. 6,534,261 and 6,933,113.

Functional domains that are modulated by exogenous small molecules or ligands can also be selected. For example, can adoptTechniques in which the functional domain is only in the extra RheoChem^TMThe ligand assumes its active conformation in the presence of ligand (see e.g. US 20090136465). Thus, ZFPs can be operably linked to a functional domain that can be modulated, wherein the activity of the resulting ZFP-TF is controlled by an external ligand.

Nuclease enzymes

In certain embodiments, the fusion molecule comprises a DNA-binding domain associated with a cleavage (nuclease) domain. Thus, genetic modification can be achieved using nucleases, e.g., engineered nucleases. Engineered nuclease technology is based on the engineering of naturally occurring DNA binding proteins. For example, engineering of homing endonucleases with modulated DNA binding specificity has been described. Chames et al, (2005) Nucleic Acids Res 33(20) e 178; arnould et al, (2006) J.mol.biol.355: 443-458. In addition, engineering of ZFPs has been described. See, for example, U.S. patent nos. 6,534,261; 6,607,882, respectively; 6,824,978, respectively; 6,979,539, respectively; 6,933,113, respectively; 7,163,824, respectively; and 7,013,219.

In addition, ZFPs and/or TALEs can be fused to nuclease domains to produce ZFNs and TALENs, which are functional entities that are able to recognize their intended nucleic acid target through their engineered (ZFP or TALE) DNA binding domain and cause the DNA to be cleaved via nuclease activity in the vicinity of the DNA binding site.

Thus, the methods and compositions described herein have broad application and may involve any nuclease of interest. Non-limiting examples of nucleases include meganucleases, TALENs, and zinc finger nucleases. Nucleases can comprise heterologous DNA binding and cleavage domains (e.g., zinc finger nucleases; meganuclease DNA binding domains with heterologous cleavage domains) or alternatively, the DNA binding domain of a naturally occurring nuclease can be altered to bind to a selected target site (e.g., meganucleases engineered to bind differently than the associated binding site).

In any of the nucleases described herein, the nuclease can include an engineered TALE DNA binding domain and a nuclease domain (e.g., an endonuclease and/or meganuclease domain), also referred to as a TALEN. Methods and compositions for engineering these TALEN proteins to interact robustly and site-specifically with user-selected target sequences have been disclosed (see, U.S. patent No. 8,586,526). In some embodiments, the TALEN comprises an endonuclease (e.g., FokI) cleavage domain or cleavage half-domain. In other embodiments, the TALE-nuclease is a megatal. These large atmosphere TAL nucleases are fusion proteins that include a TALE DNA binding domain and a meganuclease cleavage domain. Meganuclease cleavage domains are active as monomers and do not require dimerization to obtain activity. (see Boissel et al, (2013) Nucl Acid Res:1-13, doi:10.1093/nar/gkt 1224). In addition, nuclease domains can also exhibit DNA-binding functionality.

In yet another embodiment, the nuclease comprises a compact talen (ctalen). There are single-stranded fusion proteins that link the TALE DNA binding domain to the TevI nuclease domain. The fusion protein can act as a nickase that is localized by the TALE region, or can generate a double-strand break depending on where the TALE DNA binding domain is located relative to the TevI nuclease domain (see, Berldeey et al (2013) Nat Comm:1-8DOI:10.1038/ncomms 2782). Any TALEN can be used in combination with other TALENs (e.g., one or more TALENs with one or more large range TALs (ctalens or FokI-TALENs)) or other DNA cleaving enzymes.

In other embodiments, the nuclease comprises a meganuclease (homing endonuclease) or portion thereof that exhibits cleavage activity. Naturally occurring meganucleases recognize cleavage sites of 15-40 base pairs and are generally divided into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. The recognition sequence is known. See also, U.S. patent No. 5,420,032; U.S. patent No. 6,833,252; belfort et al (1997) Nucleic Acids Res.25: 3379-3388; dujon et al (1989) Gene 82: 115-118; perler et al (1994) Nucleic Acids Res.22, 1125-1127; jasin (1996) Trends Genet.12: 224-228; gimble et al (1996) J.mol.biol.263: 163-; argast et al (1998) J.mol.biol.280: 345-353 and NEB corporation (New England Biolabs) catalog.

DNA binding domains from naturally occurring meganucleases, mainly from the LAGLIDADG family, have been used to facilitate site-specific genomic modifications in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to modification of homologous Genes that retain meganuclease recognition sequences (Monet et al (1999), biochem. Biophysics. Res. Common.255:88-93) or to pre-engineered genomes into which recognition sequences have been introduced (Route et al (1994), mol.cell.biol.14: 8096-106; Chilton et al (2003), Plant physiology.133: 956-65; Puchta et al (1996), Proc.Natl.Acad.Sci.USA 93: 5055-60; Rong et al (2002), Genes Dev.16: 1568-81; Gouble et al (1996), J.Gene.8 (5): Med.616: Med.622: 5055-meds 106; Rong et al (2002). Thus, attempts have been made to engineer meganucleases to exhibit novel binding specificities at medically or biotechnologically relevant sites (Porteus et al (2005), Nat. Biotechnol.23: 967-73; Sussman et al (2004), J. mol. biol.342: 31-41; Epinat et al (2003), Nucleic Acids Res.31: 2952-62; Chevalier et al (2002) mol. Cell10: 895-905; Epinat et al (2003) Nucleic Acids Res.31: 2952-2962; Ashworth et al (2006) Nature 441: 656-659; Paques et al (2007) Current Gene Therapy 7: 49-66; U.S. patent publication Nos. 20070117128, 20060206949, 20060153826, 20060078552 and 20040002092). In addition, a naturally occurring or engineered DNA-binding domain from a meganuclease, which can be operably linked to a heterologous DNA-binding domain (e.g., ZFP or TALE), can be operably linked to a cleavage domain from a heterologous nuclease (e.g., FokI) and/or a cleavage domain from a meganuclease.

In other embodiments, the nuclease is a Zinc Finger Nuclease (ZFN) or TALE DNA binding domain-nuclease fusion (TALEN). ZFNs and TALENs include DNA binding domains (zinc finger proteins or TALE DNA binding domains) engineered to bind to target sites and cleavage domains or cleavage half-domains in selected genes (e.g., from restriction and/or meganucleases as described herein).

As detailed above, the zinc finger binding domain and TALE DNA binding domain may be engineered to bind to a selected sequence. See, e.g., Beerli et al (2002) Nature Biotechnol.20: 135-) -141; pabo et al (2001) Ann. Rev. biochem.70: 313-340; isalan et al (2001) Nature Biotechnol.19: 656-660; segal et al (2001) curr. Opin. Biotechnol.12: 632-637; choo et al (2000) curr. Opin. struct. biol.10: 411-416. Engineered zinc finger binding domains or TALE proteins may have novel binding specificities compared to naturally occurring zinc finger proteins. The engineering methods may include, but are not limited to, rational design and different types of selection. For example, rational design includes the use of databases comprising trisomy (or tetrasome) nucleotide sequences and individual zinc fingers or TALE amino acid sequences, wherein each trisomy or tetrasome nucleotide sequence is associated with one or more amino acid sequences of a zinc finger or TALE repeat unit that binds that particular trisomy or tetrasome sequence. See, for example, U.S. Pat. nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety.

Selecting a target site; and methods for designing and constructing fusion proteins (and polynucleotides encoding the same) are known to those skilled in the art and are described in detail in U.S. Pat. nos. 7,888,121 and 8,409,861, both of which are incorporated herein by reference in their entirety.

Furthermore, as disclosed in these and other references, the zinc finger domains, TALEs, and/or zinc finger proteins of the multi-fingers can be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are found, for example, in U.S. patent nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. See also U.S. patent No. 8,772,453.

Thus, nucleases such as ZFNs, TALENs and/or meganucleases can include any DNA binding domain as well as any nuclease (cleavage) domain (cleavage domain, cleavage half-domain). As described above, the cleavage domain may be heterologous to the DNA-binding domain, e.g., a zinc finger or TAL effector DNA-binding domain and a cleavage domain from a certain nuclease or a meganuclease DNA-binding domain and a cleavage domain from another nuclease. The heterologous cleavage domain may be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, catalog 2002-; and Belfort et al (1997) Nucleic Acids Res.25: 3379-338. Other enzymes known to cleave DNA (e.g., S1 nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease; see also Linn et al, Nucleases (nuclease), Cold spring harbor Laboratory Press (Cold spring harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains and cleavage half-domains.

Similarly, cleavage activity requires that the dimerized cleavage half-domain can be derived from any nuclease or portion thereof, as described above. In general, if a fusion protein comprises a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein comprising two cleavage half-domains may be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). Furthermore, it is preferred that the target sites of the two fusion proteins are arranged relative to each other such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that enables the cleavage half-domains to form a functional cleavage domain (e.g., by dimerization). Thus, in certain embodiments, the adjacent edges of these target sites are separated by 5-8 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or nucleotide pairs (e.g., 2-50 nucleotide pairs or more) may be interposed between the two target sites. In general, the site of cleavage is between the target sites, but may be located 1 or more kilobases from the cleavage site, including between 1-50 base pairs (or any value therebetween, including 1-5, 1-10, and 1-20 base pairs), 1-100 base pairs (or any value therebetween), 100-500 base pairs (or any value therebetween), 500-1000 base pairs (or any value therebetween), or even more than 1kb from the cleavage site.

Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site) and cleaving DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fok I catalyzes double-stranded cleavage of DNA, one 9 nucleotides from its recognition site and the other 13 nucleotides from its recognition site. See, for example, U.S. Pat. nos. 5,356,802, 5,436,150, and 5,487,994; and Li et al, (1992) Proc. Natl. Acad. Sci. US A89: 4275-4279; li et al, (1993) Proc. Natl. Acad. Sci. USA 90: 2764-; kim et al (1994a) Proc. Natl.Acad.Sci.USA 91: 883-887; kim et al (1994b) J.biol.chem.269:31,978-31, 982. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, with or without engineering.

Fok I is an exemplary type IIS restriction enzyme whose cleavage domain is separable from the binding domain. This particular enzyme is active as a dimer. Bitinaite et al, (1998) Proc. Natl. Acad. Sci. USA95:10,570-10, 575. Thus, for the purposes of this disclosure, the portion of the Fok I enzyme used by the fusion protein is considered to be the cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted cellular sequence replacement using zinc finger-Fok I fusions, two fusion proteins each containing one FokI cleavage half-domain can be used to reconstitute the catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains may also be used. Parameters for targeted cleavage and targeted sequence changes using zinc finger-Fok I fusions are provided elsewhere in the application.

The cleavage domain or cleavage half-domain can be any portion of the protein that retains cleavage activity or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary type IIS restriction enzymes are described in International publication WO 07/014275, which is incorporated herein by reference in its entirety. Other restriction enzymes also comprise separable binding and cleavage domains, and these are contemplated by the present invention. See, e.g., Roberts et al (2003) Nucleic Acids Res.31: 418-420.

In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) whose homodimerization is minimized or prevented, e.g., as described in U.S. patent nos. 7,914,796; 8,034,598 and 8,623,618; and U.S. patent publication No. 20110201055, all of which are incorporated herein by reference in their entirety. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are targets for affecting dimerization of the Fok I cleavage half-domains.

Exemplary engineered Fok I cleavage half-domains capable of forming obligate heterodimers include the pairs: the first cleavage half-domain includes mutations at amino acid residues 490 and 538 of Fok I, and the second cleavage half-domain includes mutations at amino acid residues 486 and 499.

Thus, in one embodiment, the mutation at position 490 replaces glu (e) with lys (k); mutation at position 538 to Lys (K) for Iso (I); mutation at position 486 replaces gln (q) with glu (e); whereas a mutation at position 499 replaces iso (i) with lys (k). Specifically, engineered cleavage half-domains described herein were prepared by mutating positions 490(E → K) and 538(I → K) in one cleavage half-domain to produce the engineered cleavage half-domain named "E490K: I538K" and by mutating positions 486(Q → E) and 499(I → L) in the other cleavage half-domain to produce the engineered cleavage half-domain named "Q486E: I499L". Engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. See, for example, U.S. patent nos. 7,914,796 and 8,034,598, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In certain embodiments, the engineered cleavage half-domain comprises mutations at positions 486, 499, and 496 (numbered according to wild-type fokl), e.g., mutations that replace the wild-type gln (q) residue at position 486 with a glu (e) residue, the wild-type iso (i) residue at position 499 with a leu (l) residue, and the wild-type asn (n) residue at position 496 with an asp (d) or glu (e) residue (also referred to as "ELD" and "ELE" domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490, 538 and 537 (numbered relative to wild-type fokl), for example mutations that replace the wild-type glu (e) residue at position 490 with a lys (k) residue, the iso (i) residue wild-type at position 538 with a lys (k) residue, and the wild-type his (h) residue at position 537 with a lys (k) or arg (r) residue (also referred to as "KKK" and "KKR" domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type fokl), e.g., a mutation that replaces the wild-type glu (e) residue at position 490 with a lys (k) residue, and replaces the wild-type his (h) residue at position 537 with a lys (k) residue or a arg (r) residue (also referred to as "KIK" and "KIR" domains, respectively). See, e.g., U.S. patent nos. 7,914,796; 8,034,598, and 8,623,618, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In other embodiments, the engineered seven domains include "Sharkey" and/or "Sharkey" mutations (see Guo et al, (2010) j.mol.biol.400(1): 96-107).

Alternatively, nucleases can be assembled in vivo at a nucleic acid target site using a technique known as "split-enzyme" (see, e.g., U.S. patent publication No. 20090068164). The components of such a cleavage enzyme may be expressed on another expression construct or may be linked in an open reading frame with the individual components separated from each other, e.g., the components are separated by a self-cleaving 2A peptide or IRES sequence. The module may be a separate zinc finger binding domain or a domain of a meganuclease nucleic acid binding domain.

Nucleases (e.g., ZFNs and/or TALENs) can be screened for activity prior to use, for example, in yeast-based chromosome systems as described in U.S. patent No. 8,563,314.

In certain embodiments, the nuclease comprises a CRISPR/Cas system. The CRISPR (regularly clustered short palindromic repeats) locus encoding the RNA component of this system and the Cas (CRISPR-associated) locus encoding the protein (Jansen et al, 2002.mol. Microbiol.43: 1565-1575; Makarova et al, 2002.Nucleic Acids Res.30: 482-496; Makarova et al, 2006.biol. direct 1: 7; Haft et al, 2005.PLoS Compout. biol.1: e60) constitute the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts comprise a combination of a CRISPR-associated (Cas) gene and a non-coding RNA element capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

Type II CRISPR is one of the well characterized systems and carries out targeted DNA double strand breaks in 4 consecutive steps. First, two non-coding RNAs, a pre-crRNA array, and a tracrRNA are transcribed by the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the process of the pre-crRNA to mature crRNA containing a separate spacer sequence. Third, the mature crRNA tracrRNA complex directs Cas to the target DNA via watson-crick base pairing, which is located between the spacer on the crRNA and the protospacer on the target DNA next to the Protospacer Adjacent Motif (PAM), which is an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to create a double strand break within the protospacer. The activity of the CRISPR/Cas system comprises 3 steps: (i) insertion of foreign DNA sequences into CRISPR arrays prevents future attacks by a process called "adaptation" (ii) expression of the protein of interest, and expression and processing of the array, followed by (iii) RNA-mediated interference with foreign nucleic acids. Thus, in bacterial cells, a number of so-called "Cas" proteins are involved in CRISPR/Cas system natural functions and play a role in functions such as insertion of foreign DNA and the like.

In some embodiments, the CRISPR-Cpf1 system is used. The CRISPR-Cpf1 system identified in francisella species (Francisellaspp) is a type 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many respects, including in their guide RNA and substrate specificity (see Fagerlund et al, (2015) Genom Bio 16: 251). The main difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize tracrRNA, and therefore only crRNA is required. FnCpf1crRNA is 42-44 nucleotides in length (19 nucleotide repeats and 23-25 nucleotide spacers) and contains a single stem loop that is resistant to sequence changes that maintain secondary structure. Furthermore, Cpf1crRNA is significantly shorter than the about 100 nucleotides engineered sgRNA required for Cas9, and the PAM requirement for FnCpfl is to displace 5 '-TTN-3' and 5 '-CTA-3' on the strand. While both Cas9 and Cpf1 produce double-strand breaks in the target DNA, Cas9 uses its RuvC-and HNH-like domains to create blunt-ended nicks within the seed sequence of the guide RNA, Cpf1 uses RuvC-like domains to create malposition nicks out of the seed. Since Cpf1 generated the malposition nicks away from the critical seed region, NHEJ will not disrupt the target site, thus ensuring that Cpf1 can continue to cleave the same site until the desired HDR recombination event occurs. Thus, in the methods and compositions described herein, it is understood that the term "Cas" includes Cas9 and Cpf1 proteins. Thus, as used herein, "CRISPR/Cas system" refers to CRISPR/Cas and/or CRISPR/Cpf1 systems, including nuclease and/or transcription factor systems.

In certain embodiments, the Cas protein may be a "functional derivative" of a naturally occurring Cas protein. "functional derivatives" of a native sequence polypeptide are compounds that have qualitative biological properties in common with the native sequence polypeptide. "functional derivatives" include, but are not limited to, fragments of the native sequence or derivatives of the native sequence polypeptide and fragments thereof, provided that they have the common biological activity of the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses amino acid sequence variants of the polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, covalent modifications of Cas proteins, or fragments thereof. Cas proteins including Cas proteins or fragments thereof and derivatives of Cas proteins or fragments thereof may be obtained from cells or by chemical synthesis or by a combination of both methods. The cell can be a cell that naturally produces a Cas protein, or a cell that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein at a higher expression level or to produce a Cas protein from a heterologously introduced nucleic acid that encodes a Cas that is the same as or different from the endogenous Cas. In some cases, the cell does not naturally produce a Cas protein, but is genetically engineered to produce a Cas protein.

Exemplary CRISPR/Cas nuclease systems targeting TCR genes are disclosed, as well as other genes, for example, in U.S. patent publication No. 20150056705.

The nuclease may produce one or more double-stranded and/or single-stranded cuts in the target site. In certain embodiments, the nuclease comprises a catalytically inactivated cleavage domain (e.g., FokI and/or Cas protein). See, for example, U.S. patent nos. 9,200,266; 8,703,489, and Guillinger et al (2014) Nature Biotech.32(6): 577-582. The catalytically inactive cleavage domain may be combined with a catalytically active structure as a nickase to produce single-stranded cleavage. Thus, two nicking enzymes can be used in combination to produce double-stranded cleavage in a specific region. Other nickases are known in the art, e.g., AMcCaffery et al (2016) Nucleic Acids Res.44(2): e11.doi:10.1093/nar/gkv878. electronically published 10/19/2015.

Delivery of

Proteins (e.g., transcription factors, nucleases, TCR and CAR molecules), polynucleotides and/or compositions comprising the proteins and/or polynucleotides described herein can be delivered to a target cell by any suitable means, including, for example, by injection of the protein and/or mRNA components.

Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines produced by such cells include T cells, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, as well as insect cells such as Spodoptera frugiperda (Sf), or fungal cells such as yeast (Saccharomyces), Pichia pastoris (Pichia) and Schizosaccharomyces pombe (Schizosaccharomyces). In certain embodiments, the cell line is a CHO-K1, MDCK, or HEK293 cell line. Suitable cells also include stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells (iPS cells), hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.

Methods of delivering proteins comprising the DNA binding domains described herein can be described, for example, in U.S. Pat. nos. 6,453,242; 6,503,717, respectively; 6,534,261; 6,599,692, respectively; 6,607,882, respectively; 6,689,558, respectively; 6,824,978, respectively; 6,933,113, respectively; 6,979,539, respectively; 7,013,219, and 7,163,824, the disclosures of which are incorporated herein by reference in their entirety.

DNA binding domains and fusion proteins comprising these DNA binding domains described herein can also be delivered using vectors comprising sequences encoding one or more DNA binding proteins. In addition, other nucleic acids (e.g., donors) can also be delivered via these vectors. Any vector system can be used, including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, adeno-associated virus vectors, and the like. See also, for example, U.S. Pat. nos. 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219; and 7,163,824, which are incorporated herein by reference in their entirety. In addition, it will be appreciated that any of these vectors may include one or more DNA binding protein-encoding sequences and/or other suitable nucleic acids, as appropriate. Thus, when one or more of the DNA binding proteins described herein are introduced into a cell, and the other DNA, if appropriate, they may be carried by the same vector or a different vector. When multiple vectors are employed, each vector may include sequences encoding one or more DNA binding proteins, as well as other nucleic acids as desired.

Traditional viral and non-viral based transgenic approaches can be used to introduce nucleic acids encoding engineered DNA binding proteins into cells (e.g., mammalian cells) and target tissues, and to co-introduce other nucleotide sequences as desired. Such methods can also be used to administer nucleic acids (e.g., encoding DNA binding proteins and/or donors) to cells in vitro. In certain embodiments, the nucleic acid is administered for in vivo or ex vivo gene therapy applications. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with a delivery vehicle (e.g., liposomes, lipid nanoparticles, or poloxamers). Viral vector delivery systems include DNA and RNA viruses that have episomes or integrated genomes after delivery to cells. For a review of gene therapy, see Anderson, Science 256: 808-; nabel and Felgner, TIBTECH 11:211-217 (1993); mitani and Caskey, TIBTECH 11:162-166 (1993); dillon, TIBTECH 11: 167-; miller, Nature 357:455-460 (1992); van Brunt, Biotechnology 6(10):1149-1154 (1988); vigne, reactive Neurology and Neuroscience 8:35-36 (1995); kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); haddada et al, published in microbial and immunological enthusiasm (Current Topics in Microbiology and Immunology) Doerfler and B.H.m. (eds.) (1995); and Yu et al, Gene Therapy 1:13-26 (1994).

Methods for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced DNA uptake. The sonoporation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids. In a preferred embodiment, the one or more nucleic acids are delivered in the form of mRNA. It is also preferred to use capped mrnas to increase translation efficiency and/or mRNA stability. An ARCA (anti-inversion cap analogue) cap or a variation thereof is particularly preferred. See U.S. patent nos. 7,074,596 and 8,153,773, which are incorporated herein by reference.

Other exemplary nucleic acid delivery systems include those provided by Amaxa biosystems (cologne, germany), macitet (Maxcyte, Inc.), rocwell, ma, BTX molecular delivery systems (holliston, ma), and Copernicus Therapeutics Inc (see, e.g., US 6008336). Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787 and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam)^TM，Lipofectin^TMAnd Lipofectamine^TMRNAiMAX). Suitable highly efficient receptor-recognizing lipofections of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Can be delivered to cells (ex vivo administration) or target tissues (in vivo administration).

Preparation of nucleic acid complexes (including targeted liposomes, such as immunoliposome complexes) is well known to those skilled in the art (see, e.g., Crystal, Science 270:404- & 410 (1995); Blaese et al, Cancer GeneTher.2:291- & 297 (1995); Behr et al, Bioconjugate chem.5:382- & 389 (1994); Remy et al, Bioconjugate chem.5:647- & 654 (1994); Gao et al, Gene Therapy 2:710- & 722 (1995); Ahmad et al, Cancer Res.52:4817- & 4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344,4,235,871,4,261,975,4,485,054,4,501,728,4,774,774,085, 4,837,028 and 4,946,787).

Other methods of delivery include packaging the nucleic acid to be delivered into an EnGeneIC Delivery Vector (EDV). These EDVs are specifically delivered to a target tissue using a bispecific antibody, where one arm of the antibody is specific for the target tissue and the other arm is specific for the EDV. The antibody brings the EDV to the surface of the target cell, and then the EDV enters the cell by endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al (2009) Nature Biotechnology27(7): p 643).

The use of RNA or DNA virus based systems to deliver nucleic acids encoding engineered DNA binding proteins and/or the required donor (e.g., CAR or ACTR) takes advantage of highly evolved processes to target the virus to specific cells in the body and transport the viral load to the nucleus. The viral vector may be administered directly to the patient (in vivo) or it may be used to treat cells in vitro and the modified cells administered to the patient (ex vivo). Conventional virus-based systems for delivering nucleic acids include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene delivery. Integration into the host genome can be carried out using retroviral, lentiviral, and adeno-associated viral gene delivery methods, typically resulting in long-term expression of the inserted transgene. In addition, high transduction efficacy has been observed in many different cell types and target tissues.

The tropism of retroviruses can be altered by introducing foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and generally produce high viral titers. The choice of retroviral transgene system depends on the target tissue. Retroviral vectors contain cis-acting long terminal repeats with the ability to package foreign sequences up to 6-10kb in length. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J.Virol.66:2731-2739 (1992); Johann et al, J.Virol.66:1635-1640 (1992); Sommerfelt et al, Virol.176:58-59 (1990); Wilson et al, J.Virol.63:2374-2378 (1989); Miller et al, J.Virol.65:2220-2224 (1991); PCT/US 94/05700).

In applications where transient expression is preferred, an adenovirus-based system may be employed. Adenovirus-based vectors are capable of achieving extremely high transduction efficiencies in many cell types and do not require cell division. With this vector, high titers and high levels of expression have been obtained. The vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also useful for transduction of cells with target nucleic acids, e.g., for in vitro generation of nucleic acids and peptides, and for in vivo and ex vivo Gene Therapy (see, e.g., West et al, Virology160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyckan et al, J.Clin.invest.94:1351 (1994); construction of recombinant AAV vectors is described in various publications, including U.S. Pat. No. 5,173,414; Tratschin et al, mol.cell.biol.5: 3251-42 60 (1985); Tratschin et al, mol.cell.4: 2072-2081 (1984); Hertzumankan and Muzyckan, PNA 3281: 3266-AS 822; and Virol. J.647 J.389; 1989: 039).

At least six viral vector methods are currently available for gene delivery in clinical trials, using methods involving complementation of defective vectors by insertion of genes into helper cell lines to generate transducible agents.

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85: 3048-. PA317/pLASN is the first therapeutic vector for gene therapy trials. (Blaese et al, Science 270: 475-. Transduction efficacy of 50% or more has been observed in MFG-S packaged vectors. (Ellem et al, Immunol Immunother.44(1):10-20 (1997); Dranoff et al, hum. Gene ther.1:111-2 (1997)).

Recombinant adeno-associated viral vectors (rAAV) are a promising alternative gene delivery system based on defective and non-pathogenic parvovirus adeno-associated type 2 viruses. All vectors were derived from plasmids that only retained the inverted terminal repeats of AAV145bp flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery are key features of this vector system due to integration into the genome of the transduced cell. (Wagner et al, Lancet 351: 91171702-3 (1998), Kearns et al, Gene ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAVrh10, and pseudotyped AAV, such as AAV2/8, AAV2/5, and AAV2/6, can also be used in accordance with the invention.

Replication-defective recombinant adenovirus vectors (Ad) can be generated at high titers and readily infect a variety of different cell types. Most adenoviral vectors are engineered such that the transgene replaces the Ad E1a, E1b, and/or E3 genes; the replication deficient vector is then propagated in human 293 cells that provide the function of the deleted trans gene. Ad vectors can transduce various types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. One example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunization with intramuscular injection (Sterman et al, hum.7:1083-9 (1998)). Other examples of the use of adenoviral vectors for transgenics in clinical trials include Rosenecker et al, Infectin 24: 15-10 (1996); sterman et al, hum. Gene Ther.9: 71083-one 1089 (1998); welsh et al, hum.Gene ther.2:205-18 (1995); alvarez et al, hum. Gene ther.5: 597-; topf et al, Gene ther.5: 507-; sterman et al, hum. Gene ther.7:1083-1089 (1998).

Packaging cells are used to form viral particles capable of infecting host cells. The cells include 293 cells, which package adenovirus and AAV, and ψ 2 cells or PA317 cells, which package retrovirus. Viral vectors for gene therapy are typically produced by a producer cell line that packages nucleic acid vectors into viral particles. The vector will typically contain the minimal viral sequences required for packaging and subsequent integration into the host (if feasible), with the other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The lost viral function is provided in trans by the packaging cell line. For example, AAV vectors for gene therapy typically process only Inverted Terminal Repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome. Viral DNA is packaged into cell lines, which contain helper plasmids encoding the other AAV genes, rep and cap, but lacking ITR sequences. The cell lines were also infected with adenovirus (as an adjuvant). Helper viruses facilitate replication of AAV vectors and expression of AAV genes from helper plasmids. Helper plasmids are not packaged in significant quantities because of the lack of ITR sequences. Contamination of the adenovirus can be reduced, for example, by heat treatment (adenovirus is more susceptible to heat treatment than AAV). In addition, AVV can be produced using a baculovirus system (see, e.g., U.S. Pat. nos. 6,723,551 and 7,271,002).

Purification of AAV particles by the 293 or baculovirus system typically involves growing virus-producing cells and then collecting viral particles from the cell supernatant, or lysing the cells and collecting virus from the crude lysate. AAV is then purified by methods known to those skilled in the art, including ion exchange chromatography (see, e.g., U.S. patent nos. 7,419,817 and 6,989,264), ion exchange chromatography and CsCl density centrifugation (e.g., PCT publication WO2011094198a10), immunoaffinity chromatography (e.g., WO2016128408), or purification using AVB agarose (e.g., GE Healthcare life sciences).

In many gene therapy applications, it is desirable to deliver gene therapy vectors to specific tissue types with a high degree of specificity. Thus, viral vectors can be modified to have specificity for a given cell type by expressing the ligand as a protein located on the outer surface of the virus fused to a viral coat protein. Ligands are selected that have affinity for receptors known to be present on the cell type of interest. For example, Han et al, Proc.Natl.Acad.Sci.USA 92:9747-9751(1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that this recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for almost any selected cellular receptor. Although the above description applies primarily to viral vectors, the same principles apply to non-viral vectors. The vector may be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical administration, as described below. Alternatively, the vector may be delivered ex vivo to cells, such as explanted cells from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, which are then reimplanted into the patient, typically after selection of cells that have taken into the vector.

Ex vivo cell transfection for diagnosis, study, inhibition, or for gene therapy (e.g., by re-infusion of transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a subject organism, transfected with a DNA binding protein nucleic acid (gene or cDNA), and reinfused back into the subject organism (e.g., a patient). A variety of cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et alAnimal cell Manual of basic techniques for cultivationIn the Culture of Animal Cells, A Manual of Basic Technique (third edition 1994)) the references cited herein are for a discussion of how to isolate and Culture Cells from patients).

The use of stem cells in one embodiment, the advantage of using stem cells in ex vivo protocols for cell transfection and gene therapy is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (e.g., a donor of cells) where they will be transplanted into the bone marrow methods for the in vitro differentiation of CD34+ cells into clinically important immune cell types using cytokines such as GM-CSF, IFN- γ, and TNF- α are known (see Inaba et al, J.Exp.Med.176: 1693-.

Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells can also be isolated from bone marrow cells by screening the bone marrow cells with antibodies that bind to unwanted cells, such as CD4⁺And CD8+ (T cells), CD45⁺(panB cells), GR-1 (granulocytes), and Iad (differentiated antigen presenting cells) (see Inaba et al, J.Exp.Med.176:1693-1702 (1992)).

In some embodiments, stem cells that have been modified can be used. For example, neuronal stem cells made anti-apoptotic, wherein the stem cells further comprise a ZFP TF of the invention, can be used as therapeutic compositions. Resistance to apoptosis can result, for example, from knock-out of BAX and/or BAK in stem cells by using BAX-or BAK-specific ZFNs (see, U.S. patent application No. 12/456,043), or those that interfere with caspases, again using, for example, caspase 6-specific ZFNs. These cells can be transfected with ZFP TFs known to modulate TCR.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) comprising therapeutic DNA binding proteins (or nucleic acids encoding such proteins) can also be administered directly into the body for in vivo cell transduction. Alternatively, naked DNA may be administered. Administration is by any conventional route commonly used for introducing molecules and ultimately contacting blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods of administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be utilized to administer a particular composition, one route will generally provide a more direct and more effective response than another route.

Methods for introducing DNA into hematopoietic stem cells are disclosed, for example, in U.S. patent No. 5,928,638. Can be used to introduce transgenes into hematopoietic stem cells (e.g., CD 34)⁺Cell) includes adenovirus type 35.

Suitable vectors for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors. See, e.g., Ory et al (1996) Proc. Natl. Acad. Sci. USA 93: 11382. 11388; dull et al (1998) J.Virol.72: 8463-8471; zuffery et al (1998) J.Virol.72: 9873-; follenzi et al, (2000) Nature Genetics 25: 217-222.

The pharmaceutically acceptable carrier will depend, in part, on the particular composition being administered and the particular method used to administer the composition. Thus, a variety of suitable pharmaceutical composition formulations are available, as described below (see, e.g., Remington's pharmaceutical Sciences; 17 th edition, 1989).

As noted above, the disclosed methods and compositions can be used with any type of cell, including, but not limited to, prokaryotic cells, fungal cells, archaeal cells, plant cells, insect cells, animal cells, vertebrate cells, mammalian cells, and human cells, including any type of T cells and stem cells. Suitable cell lines for protein expression are known to those of skill in the art and may include, but are not limited to, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), PerC6, insect cells such as Spodoptera frugiperda (Sf), and fungal cells such as yeast (Saccharomyces), Pichia (Pichia) and Schizosaccharomyces (Schizosaccharomyces). Progeny, variants and derivatives of these cell lines may also be used.

Applications of

The disclosed compositions and methods may be used for any application where modulation of B2M expression and/or functionality is desired, including, but not limited to, therapeutic and research applications where modulation of B2M is desired. For example, the disclosed compositions can be used in vivo and/or ex vivo (cell therapy) to disrupt the expression of endogenous B2M in T cells modified for adoptive cell therapy to express one or more exogenous CARs, exogenous TCRs, exogenous ACTRs, or other cancer-specific receptor molecules, thereby treating and/or preventing cancer. Furthermore, in such a setting, modulation of intracellular B2M expression may eliminate or substantially reduce the risk of unwanted cross-reactivity with healthy, non-targeted tissues (i.e., graft versus host response).

Methods and compositions also include stem cell compositions in which the B2M gene is modulated (modified) within the stem cell, and the cell further includes an ACTR and/or a CAR and/or an isolated or engineered TCR. For example, B2M knockout or knock-down regulated allogeneic hematopoietic stem cells can be introduced into HLA-mismatched patients after bone marrow ablation. These altered HSCs will allow for re-colonization (re-colonization) of the patient, but will not cause potential GvHD. The introduced cells may also have other alterations to help treat the underlying disease during subsequent treatments (e.g., chemotherapy resistance). HLA-free cells also have application as "off the shelf" therapies in the case of emergency treatment of trauma patients.

The methods and compositions of the invention may also be used to design and implement in vitro and in vivo models, for example, animal models of B2M and/or HLA and related disorders, which allow for the study of these disorders.

All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art that various changes and modifications may be made without departing from the spirit or scope of the invention. The above description and examples are, therefore, not to be taken in a limiting sense.

Examples

Example 1: design of B2M-specific nucleases

B2M-specific ZFNs were constructed to achieve site-specific introduction of double-strand breaks at the B2M gene. Mainly comprises Urnov et al (2005) Nature 435(7042), 646-; 25(11) 1298-. The ZFN pairs target different sites in the constant region of the B2M gene (see fig. 1). Recognition helices and target sequences for exemplary ZFN pairs are shown below in tables 1 and 2. The target sites for the B2M zinc finger design are shown in column one. Nucleotides in target sites targeted by ZFP recognition helices are indicated in uppercase; nucleotides that are not targeted are indicated in lower case letters. Linkers for joining the FokI nuclease domain and ZFP DNA binding domain are also shown (see, U.S. patent publication No. 20150132269). For example, the amino acid sequence of the domain linker L0 is the DNA binding domain-QLVKS-FokI nuclease domain (SEQ ID NO: 3). Similarly, the amino acid sequence of domain linker N7a was FokI nuclease domain-SGTPHEVGVYTL-DNA binding domain (SEQ ID NO:4), and N6a was FokI nuclease domain-SGAQGSTLDF-DNA binding domain (SEQ ID NO: 5).

Table 1: B2M zinc finger design

All ZFNs were tested and found to bind their target site and to be active as nucleases.

Guide RNAs for the streptococcus pyogenes (s. pyogenes) CRISPR/Cas9 system were also constructed for targeting the B2M gene. The target sequences in the B2M gene are indicated in table 2A below, along with guide RNA sequences. All guide RNAs were tested in CRISPR/Cas9 system and found to be active. The lower case "G" at the 5' end of some guide sequences indicates an added G nucleotide that acts in the PAM sequence.

Table 2A: guide RNA for human B2M constant region

TALENs were used to target the B2M locus and are shown in table 2B below. All TALENs were tested in K562 cells and were considered active (see table 2C and fig. 2B).

Table 2B: TALENs specific for B2M

TALENs from table 2B were tested at different concentrations of each TALEN at each reaction of 25, 100 or 400 ng. All TALENs tested were considered to bind their target site and were considered to have nuclease activity; exemplary data are shown in table 2C and fig. 2B.

Table 2 activity of TALEN pairs in k562 cells

Thus, the nucleases described herein (e.g., nucleases comprising ZFP, TALE or sgRNA DNA binding domains) bind to their target site and cleave the B2M gene, thus making genetic modifications in the B2M gene comprising any of SEQ ID NOS: 6-48 or 137-; modifications within 1-50 (e.g., 1-10) base pairs of these gene sequences; modification between target sites of paired target sites (for dimers); and/or modifications within one or more of the following sequences: GGCCTTA, TCAAATT, TCAAAT, TTACTGA, and/or AATTGAA (see, fig. 1).

Furthermore, when associated with one or more transcriptional regulatory domains, all DNA binding domains (ZFPs, TALEs, and sgrnas) bind to their target sites and also form actively engineered transcription factors.

Example 2: B2M-specific ZFN Activity in T cells

The nuclease activity of B2M-specific ZFN pairs was tested in human T cells. mRNA encoding ZFNs was transfected into purified T cells. Briefly, T cells were obtained from leukapheresis (leukapheresis) products and purified using the CliniMACS system (CD4 and CD8 double selection) of american whirlpool (Miltenyi). The cells were then activated using Dynabeads (thermo fisher) according to the manufacturer's protocol. 3 days after activation, cells were transfected with two doses of mRNA (2 or 6 μ g total of the two ZFNs) using a BTX 96-well electrotransformation apparatus (BTX) according to standard protocols. The cells were then expanded for an additional 7 days, for a total of 10 days after activation. Cells were removed on day 7 and targeted B2M modifications were analyzed using deep sequencing (Miseq, llminda corporation (Illumina)) and FACs analysis was performed on day 10 using HLA-a, -B and-C staining.

B2M-specific ZFN pairs were active in T cells all and resulted in mean values of 89% and 83% for 6 μ g and 2 μ g mRNA doses, respectively (see, fig. 2). The pairs and positions used (shown in fig. 1) are listed in table 3 below.

Table 3: B2M specific ZFN pairs and target sites

Similarly, T cells treated with ZFNs lost HLA a, B and C expression, with FACS analysis showing an average of 81% and 67% HLA negative T cells at 6 μ g and 2 μ g mrna dose, respectively (see, fig. 3).

Example 3: in vitro Activity of guide RNAs against B2M

In these experiments, Cas9 was provided on the pVAX plasmid and sgrnas were provided on the plasmid under the control of the U6 promoter. Plasmids were mixed at 100ng each or 400ng each and mixed with 2e5 cells per round. Cells were transfected using the Amaxa system. Briefly, Amaxa transfection kit was used and transfected using standard Amaxa shuttle (shuttle) protocol. After transfection, cells were allowed to resuscitate at room temperature for 10 min and then resuspended in pre-warmed RPMI. Cells were then grown at 37 ℃ under standard conditions. Genomic DNA was isolated 7 days post transfection and subjected to MiSeq analysis.

The data shown below (table 4) represent the percent indels (insertions and deletions) detected at two doses of guide RNA and represent the cleavage induced by the different guide RNAs at the targeted site. The numbers represent the average of two experiments. All guides are active.

Table 4: activity of CRISPR/Cas System on B2M

Example 4: double knockout of B2M and TCR in Primary T cells

The B2M pair described herein was also tested in combination with ZFNs for TCRA (see table 5 below, and U.S. provisional patent applications 62/269,365 and 62/306,500). Cells were obtained and processed as described in example 2. mRNA encoding ZFN pairs (SBS #57017/SBS #57327 for B2M and SBS #55254/SBS #55248 for TCRA) were electroporated into cells using the Maxcyte apparatus according to the manufacturer's instructions. Briefly, T cells were activated at day 0 and treated with ZFN-encoding mRNA at day 3, where the cell density was 3e7 cells/mL. Electroporation was followed by overnight cold shock (cold shock) at 30 ℃. On day 4, cells were counted and assayed for viability, diluted to 0.5e6 cells/mL and transferred to 37 ℃. On day 7, cells were counted and re-analyzed, then re-diluted to 0.5e6 cells/mL. At days 10 and 14, fractions of the cells were harvested for FACS and MiSeq deep sequencing analysis.

Table 5: TCRA ZFN

The cells were divided into 4 groups: no ZFN control, only TCRA ZFN, only B2M ZFN, and TCRA ZFN + B2 MZFN. High proportion of cleavage was achieved by FACS analysis for ZFN pools for TCRA (180 μ g/μ L ZFN mRNA) alone or for B2M (180 μ g/μ L ZFN mRNA) alone (96% CD3 marker for TCRA ZFN, and 92% HLA marker knockout for B2M ZFN (fig. 4)). When cells were treated with both types of ZFN pairs (both 180 μ g/μ L), 82% of the cells lost CD3 and HLA markers.

Similar groups of cells were also treated with different amounts of the indicated TCRA-specific ZFNs (60-250ug/uL) plus 60ug/mL of B2MZFN, and on days 10 and 14, MiSeq deep sequencing (llmingda) and FACs analysis were performed with the results shown in table 6 below. The results indicate that a high rate of double knockouts detected by NHEJ mediated insertions and deletions were observed with these ZFNs.

Table 6: FACS and miSeq analysis of TCRA/B2M double knockouts

Thus, this data demonstrates that double knock-out of B2M and TCRA inactivates 2 genes located within or adjacent to 1-50 (e.g., 1-10) base pairs (including between the paired target sites) of the target and/or target sequence and/or the nuclease cleavage site described herein, including the B2M sequence TCAAAT (site D in fig. 1) and the TCRA sequence CCTTC, between the two target sequences for the SBS #55254/SBS #55248TCRA specific pair.

Example 5: double knock-out of B2M and TCRA with targeted integration

Nucleases as described above (see, example 4) were used to inactivate B2M and TCRA (see, example 5) and the donor (transgene) was introduced into the TCRA or B2M locus via targeted integration. In this experiment, the TCRA-specific ZFN pair was SBS #55266/SBS #53853, which included the sequence TTGAAA between the TCRA-specific ZFN target sites (table 5), and the B2M pair was SBS #57332/SBS #57327 (table 1), which included the sequence TCAAAT between the B2M-specific ZFN target sites.

Briefly, T cells (AC-TC-006) were thawed and activated in X-vivo15T cell culture medium (day 0) with CD3/28dynabeads (1:3 cell: bead ratio). After 2 days of culture (day 2), AAV donors (including GFP transgene and homology arms of TCRA or B2M gene) were added to the cell culture, except that the control group without donor was also maintained. The following day (day 3), TCRA and B2M ZFNs were added via mRNA delivery to the following 5 groups:

(a) group 1 (TCRA and B2M ZFN only, no donor): TCRA 120 ug/mL: B2M is only 60 ug/mL;

(b) group 2(TCRA and B2M ZFN and donors with TCRA homology arms): TCRA 120 ug/mL; B2M 60ug/mL and AAV (TCRA-hPGK-eGFP-clone E2)1E5 vg/cell;

(c) group 3(TCRA and B2M ZFN and donors with TCRA homology arms): TCRA 120 ug/mL; B2M 60 ug/mL: and AAV (TCRA-hPGK-eGFP-clone E2)3E4 vg/cell;

(d) group 4(TCRA and B2M ZFN and donor with B2M homology arm): TCRA 120 ug/mL; B2M 60ug/mL and AAV (pAAV B2M site D hPGK GFP)1E5 vg/cell;

(e) group 5(TCRA and B2M ZFN and donor with B2M homology arm): TCRA 120 ug/mL; B2M 60ug/mL and AAV (pAAV B2M site D-hPGK GFP)3E4 vg/cell.

All experiments were performed at a cell density of 3e7 cells/ml using the protocol described in us application No. 15/347,182 (extreme cold shock) and were incubated at 30 ℃ overnight to cold shock after electroporation.

The next day (day 4), cells were diluted to 0.5e6 cells/ml and transferred to culture at 37 ℃. After 3 days (day 7), the cells were again diluted to 0.5e6 cells/ml. After 3 and 7 more days of culture (day 10 and 14, respectively), cells were harvested for FACS and MiSeq analysis (diluted to 0.5e6 cells/ml).

As shown in fig. 5, GFP expression indicates that targeted integration was successful and a genetically modified cell was obtained that included B2M and TCRA modifications (insertions and/or deletions) within the nuclease site (or within 1-50 base pairs of the nuclease target site, including within TTGAAA and TCAAAT and/or between paired target sites).

Experiments were also performed in which CAR transgenes were integrated into the B2M and/or TCRA locus to create a dual B2M/TCRA knockout expressing CAR.

All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that various changes and modifications may be made without departing from the spirit or scope of the invention. The above description and examples are, therefore, not to be taken in a limiting sense.

Claims (13)

1. An isolated cell wherein expression of the β 2 microglobulin (B2M) gene is regulated by modifying exon 1 or exon 2 of the B2M gene.

2. The cell according to claim 1, wherein the modification is performed by an exogenous fusion molecule comprising a DNA binding domain and a functional domain, wherein the DNA binding domain binds to a target site as set forth in any one of SEQ ID NO 6-48 or 137-205.

3. The cell of claim 1, wherein the cell comprises an insertion and/or deletion within one or more of SEQ ID NO 6-48 or 137-; 6-48 or 137-205 (flanking genomic sequence) within 1-10 base pairs; or GGCCTTA, TCAAAT, TCAAATT, TTACTGA and/or AATTGAA.

4. The cell of any one of claims 1-3, further comprising an inactivated T cell receptor gene, PD1, and/or CTLA4 gene.

5. The cell of any one of claims 1-4, further comprising a transgene encoding a Chimeric Antigen Receptor (CAR), a transgene encoding an antibody-coupled T cell receptor (ACTR) and/or a transgene encoding an engineered TCR.

6. The cell of any one of claims 1-5, wherein the cell is a lymphocyte, stem cell, or progenitor cell.

7. The cell of claim 6, wherein the cell is a T cell, an Induced Pluripotent Stem Cell (iPSC), an embryonic stem cell, a Mesenchymal Stem Cell (MSC), or a Hematopoietic Stem Cell (HSC).

8. A pharmaceutical composition comprising the cell of any one of claims 1-7.

9. A fusion molecule comprising a DNA-binding domain that binds exon 1 or exon 2 of the B2M gene and a transcription regulatory domain or nuclease domain, wherein the DNA-binding domain comprises a Zinc Finger Protein (ZFP) as set forth in any one of rows in table 1, a TALE-effector protein as set forth in any one of rows in table 2B, or a single guide rna (sgrna) as set forth in any one of rows in table 2A.

10. A polynucleotide encoding the fusion molecule of claim 9.

11. The polynucleotide of claim 10, wherein the polynucleotide is a viral vector, a plasmid, or an mRNA.

12. A method of treating or preventing cancer, the method comprising administering the cell of any one of claims 1-7 or the pharmaceutical composition of claim 8 to a cancer subject.

13. Use of the cell of any one of claims 1-7, the pharmaceutical composition of claim 8 or the fusion molecule of claim 9 or the polynucleotide of claim 10 for treating a subject with cancer.