WO2016083811A1

WO2016083811A1 - Genome editing methods

Info

Publication number: WO2016083811A1
Application number: PCT/GB2015/053603
Authority: WO
Inventors: Philip Ashton-Rickardt
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2014-11-27
Filing date: 2015-11-26
Publication date: 2016-06-02
Anticipated expiration: 2017-05-27
Also published as: GB201421096D0

Abstract

The present invention relates to genome editing methods for introducing a mutation into DNA within a cell that encodes a protein referred to herein as Lymphocyte Expansion Molecule (LEM) (human C1ORF177, mouse-BC055111). The genomic DNA sequence encoding human LEM is provided as SEQ ID NO: 1 and the amino sequence of human LEM is provided as SEQ ID NO: 2. LEM has been shown to drive T cell proliferation and differentiation. In certain aspects, the invention relates to the use of a CRISPR/Cas9 system, TALEN or Zinc Finger Nuclease to modify the chromosomal sequence encoding LEM.

Description

Genome editing methods

The present invention relates to genome editing methods for introducing a mutation into a specific DNA sequence within a cell.

Targeted genome modification is a powerful tool for genetic manipulation of eukaryotic cells, embryos, and animals. Genome modification can be carried out using a variety of known technologies.

TALEN technology and Zinc Finger Technology are based on the use of engineered nuclease enzymes (zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs)). These chimeric nucleases contain programmable, sequence- specific DNA-binding modules linked to a nonspecific DNA cleavage domain. Each new genomic target requires the design of a new ZFN or TALEN comprising a novel sequence- specific DNA-binding module.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is currently the most commonly used RNA-Guided Endonuclease technology for genome engineering. There are two distinct components to this system: (1) a guide RNA (gRNA) and (2) an endonuclease, in this case the CRISPR associated (Cas) nuclease, Cas9. When the gRNA and the Cas9 are expressed in the cell, the genomic target sequence can be modified or permanently disrupted. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement to the target sequence in the genomic DNA. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of DNA causing a Double Strand Break (DSB). A DSB can be repaired through one of two general repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template (see https : /^'/www. ad dq e ne . o rq/cri s r/q u i de/ for further details).

In a first aspect, the invention provides a method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

(a) introducing into the cell (i) guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the gRNA hybridises to a target sequence within the chromosomal sequence or a target sequence in a sequence that is complementary to the chromosomal sequence;

(ii) one or more RNA-guided endonucleases or nucleic acid encoding the one or more RNA-guided endonucleases, and optionally

(iii) a nucleic acid repair template

(b) culturing the cell such that the gRNA directs the one or more RNA-guided

endonucleases to the target sequence where the one or more RNA-guided

endonucleases introduce a single-stranded break or a double-stranded break at a target site within the target sequence, and the single-stranded break or double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

The present invention relates to methods for editing a gene encoding a protein with previously unknown function. The protein is referred to herein as Lymphocyte Expansion Molecule (LEM) (human C10RF177, mouse-BC0551 11). The LEM gene was discovered using an unbiased forward genetic approach to identify mouse mutants with CTLs that are resistant to immunosuppression (see Example 1 for further details). The genomic DNA sequence encoding human LEM is provided as SEQ ID NO: 1 and the amino sequence of human LEM is provided as SEQ ID NO: 2. The present inventor has discovered that LEM can drive T cell proliferation and differentiation, in particular Cytotoxic T lymphocyte (CTL) proliferation and differentiation.

The present inventor has also discovered that an A1313G mutation in the human LEM nucleic acid sequence resulting in an E53G amino acid mutation in human LEM amino acid sequence (equivalent to a A1304G mutation in the mouse LEM nucleic acid sequence resulting in an E50G mutation in the mouse LEM amino acid sequence) is a gain-of-function mutation which gives rise to increased T cell proliferation and/or differentiation (a hyper-proliferation phenotype) relative to that elicited by the wild type gene. The present invention is based in part on methods for introducing this mutation and other mutations into the wild type LEM gene in mammalian cells, particularly human cells.

The term chromosomal sequence means an endogenous DNA sequence of a gene contained within the nucleus of a cell. In the present invention, the gene is that of LEM. Modifying the chromosomal sequence means making a change to the DNA sequence. The change may be in a coding region or non-coding region of the sequence. In certain embodiments, the method makes use of the well-known CRISPR-Cas genome editing system described in, for example WO 2013/176772, WO 2014/093718, WO 2014/093709, WO 2014/093622, WO 2014/093655, WO 2014/093701 , WO 2014/093712, WO 2014/093635, WO 2014/093595, WO 2014/093694, WO2014/093661 , WO

2014/018423, WO 2014/089290, WO 2014/099750, WO 2014/131833 and WO

2014/165825. For example the gRNA and endonuclease may be components of a CRISPR-Cas system.

The method facilitates the introduction of mutations into the genomic LEM sequence (SEQ ID NO: 1) in the cell. In some embodiments, the RNA- guided endonuclease comprises at least one nuclear localization signal. The nuclear localization signal facilitates entry of the endonuclease into the nuclei of eukaryotic cells.

RNA-guided endonucleases generally comprise at least one nuclease domain and at least one domain that interacts with a guide RNA. An RNA-guided endonuclease is directed to a specific nucleic acid sequence (or target site) by a guide RNA. The guide RNA interacts with the RNA-guided endonuclease as well as the target site such that, once directed to the target site, the RNA-guided endonuclease is able to introduce a double-stranded break into the target site nucleic acid sequence. Since the guide RNA provides the specificity for the targeted cleavage, the endonuclease of the RNA-guided endonuclease is universal and can be used with different guide RNAs to cleave different target nucleic acid sequences. The target site has no sequence limitation except that the sequence is immediately followed (downstream) by a consensus sequence. This consensus sequence is also known as a Protospacer adjacent motif (PAM). Examples of PAM include, but are not limited to, NGG, NGGNG, and NNAGAAW (wherein N is defined as any nucleotide and W is defined as either A or T). A first region (at the 5' end) of the guide RNA is complementary to the PAM of the target sequence. Typically, the first region of the guide RNA is about 16 to 21 nucleotides in length.

The RNA-guided endonuclease can be derived from a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The CRISPR/Cas system can be a type I, a type II, or a type III system. Non- limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Casl Od, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1 , Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966.

In one embodiment, the RNA-guided endonuclease is derived from a type II CRISPR/Cas system. In specific embodiments, the RNA-guided endonuclease is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces

pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum,

Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus,

Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus

ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or

Acaryochloris marina.

For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motiff (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of DNA causing a Double Strand Break (DSB). As the skilled person will be aware, it is important that the repair template does not include the target sequence followed by the PAM, or the endonuclease may cut the repair template in addition to the target sequence.

Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, called 'nickases^: have been engineered. The Cas9 nickase can be targeted to a specific DNA sequence by gRNA but only cuts one strand of the target DNA, creating a single-strand break or 'nick'. The RuvC domain can be inactivated by a D10A mutation and the HNH domain can be inactivated by an H840A mutation. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a Double Strand Break (DSB), in what is often referred to as a 'double nick' or 'dual nickase' CRISPR system. A double-nick induced DSB can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. In some embodiments, the double stranded break made at the target site is generated by a double nick using one or more nickases. Such embodiments ma provide fewer off target effects and good efficiency.

The gRNA may be combination of the endogenous bacterial crRNA and tracr (trans- activating cr) RNA into a single chimeric guide RNA (gRNA) transcript. The gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA into a single transcript. The gRNA can be designed to target the mutations to a specific target sequence within the LEM sequence. gRNAs can be designed using free software available at the following URLs: http://crispr. m it. edu/

h††p7AA e-crisp orQ/E-CRiSP uesignerispr html

WtPj/Zwrny,

http://toois.flvcrispr.moibjo.wisc.edu/targetFinder/

CRISPR/Cas plasmids and protocols are available from Addgene, One Kendall Sq B7102, Cambridge MA 02139.

Modification of the chromosomal sequence is facilitated in part by the cell's own DNA repair processes. These include non-homologous end joining (NHEJ) and homology directed repair (HDR). During NHEJ repair, inDeis (insertions/deletions) may occur as a small number of nucleotides are either inserted or deleted at random at the doubles strand break (DSB) site. InDeis alter the Open Reading Frame (ORF) of the target gene, which may significantly change the amino acid sequence downstream of the DSB. Additionally, inDeis may introduce a premature stop codon either by creating one at the DSB or by shifting the reading frame to create one downstream of the DSB,

HDR can be used to make specific modifications to the target sequence. In order to do this, a repair template comprising the desired sequence may be provided. The HDR pathway can copy the sequence of the repair template to the cut target sequence. Thus, in certain embodiments, the method comprises introducing a repair template into the cell in addition to the endonuclease and the gRNA. In some embodiments, the repair template has a high degree of homology to the sequence immediately upstream and downstream of the DSB. The length and binding position of each homology arm is dependent on the size of the change being introduced. Thus, by utilising the HDR pathway and designing the gRNA and repair template appropriately, a specific mutation such as the A1313G gain of function mutation can be introduced into the LEM gene. The repair template may comprise a nucleic acid sequence that is substantially identical to a region flanking a target sequence in chromosomal DNA.

In some embodiments, the method comprises introducing at least one donor

polynucleotide into the cell. A donor polynucleotide comprises at least one donor sequence. In some aspects, a donor sequence of the donor polynucleotide corresponds to an endogenous or native chromosomal sequence. For example, the donor sequence can be essentially identical to a portion of the chromosomal sequence at or near the targeted site, but which comprises at least one nucleotide change. Thus, the donor sequence can comprise a modified version of the wild type sequence at the targeted site such that, upon integration or exchange with the native sequence, the sequence at the targeted chromosomal location comprises at least one nucleotide change. For example, the change can be an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides, or combinations thereof. As a consequence of the integration of the modified sequence, the cell can produce a modified gene product from the targeted chromosomal sequence.

In other aspects, the donor sequence of the donor polynucleotide corresponds to an exogenous sequence. As used herein, an "exogenous" sequence refers to a sequence that is not native to the cell or embryo, or a sequence whose native location in the genome of the cell or embryo is in a different location. For example, the exogenous sequence can comprise protein coding sequence, which can be operably linked to an exogenous promoter control sequence such that, upon integration into the genome, the cell or embryo/animal is able to express the protein coded by the integrated sequence. Alternatively, the exogenous sequence can be integrated into the chromosomal sequence such that its expression is regulated by an endogenous promoter control sequence. In other iterations, the exogenous sequence can be a transcriptional control sequence, another expression control sequence, an RNA coding sequence, and so forth. Integration of an exogenous sequence into a chromosomal sequence is termed a "knock in." As can be appreciated by those skilled in the art, the length of the donor sequence can and will vary. For example, the donor sequence can vary in length from several nucleotides to hundreds of nucleotides to hundreds of thousands of nucleotides. In some embodiments, the donor sequence in the donor polynucleotide is flanked by an upstream sequence and a downstream sequence, which have substantial sequence identity to sequences located upstream and downstream, respectively, of the targeted site in the chromosomal sequence. Because of these sequence similarities, the upstream and downstream sequences of the donor polynucleotide permit homologous recombination between the donor polynucleotide and the targeted chromosomal sequence such that the donor sequence can be integrated into (or exchanged with) the chromosomal sequence.

The method can comprise introducing two RNA-guided endonucleases (or encoding nucleic acid) and two guide RNAs (or encoding DNA) into a cell or embryo, wherein the RNA-guided endonucleases introduce two double-stranded breaks in the chromosomal sequence. The two breaks can be within several base pairs, within tens of base pairs, or can be separated by many thousands of base pairs.

In some embodiments, nucleic acid encoding the gRNA and/or the endonuclease may be introduced into the cell. The nucleic acid may be mRNA or DNA. The DNA may be provided as part of a vector. The vector may comprise DNA encoding the gRNA and/or the endonuclease. The components may be introduced into the cell by any suitable means, for example by electroporation, nucleofection, lipofectamine-mediated transfection or lentiviral vectors.

In some embodiments, the cell is a T cell. The method may be carried out ex vivo or in vitro. Thus, the method of the present invention may form part of an adoptive T cell therapy treatment in which T cells are extracted from a patient, subjected to the method of the invention and re-infused into the patient. Methods of adoptive T cell therapy are known to those skilled in the art and discussed in, for example, WO 2006/000830.

The method of the invention can be used to facilitate the introduction of mutations into the LEM gene which enhance the pro-proliferative/pro-differentiation activity of LEM. Such mutations may, for example reduce degradation of LEM mRNA or increase the expression of LEM. Thus, the method of the invention may be used alone or in combination with other treatments to treat or preventing a disease or disorder that would benefit from stimulation of T cell proliferation and/or differentiation such as cancer and chronic viral infection. Examples of cancers which the present invention can be used to prevent or treat include solid tumours and leukaemias, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumour, in situ, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukaemia (e.g., B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV-ll-associated, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid), histiocytosis malignant, Hodgkin disease,

immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumours, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumour, adeno-carcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumour, gynandroblastoma, hepatoma, hidradenoma, islet cell tumour, Leydig cell tumour, papilloma, Sertoli cell tumour, theca cell tumour, leiomyoma, leiomyosarcoma, myoblastoma, mymoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma,

ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma,

paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma,

lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma, phyllodes, fibrosarcoma, hemangiosarcoma, leimyosarcoma,

leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell), neoplasms (e.g., bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia, and other conditions in which cells have become immortalised or transformed. In particular, the present invention is useful in the treatment of malignant melanoma, renal carcinoma, prostate cancer, lung cancer, breast cancer and hepatocellular carcinoma. The invention could be used in combination with other treatments, such as chemotherapy, cryotherapy, hyperthermia, radiation therapy, and the like. The method may be useful in the treatment or prevention of viral infection such as those cause by human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus and the hepatitis B and C viruses (HBV, HCV).

In other embodiments, the method may be used to introduce mutations which reduce the activity or expression of LEM. Such methods may be useful to treat or prevent a disease or disorder that would benefit from a reduction in T cell proliferation and/or differentiation. Examples of disorders involving unwanted or excessive proliferation and/or differentiation of T cells such as CTLs include autoimmune diseases and inflammatory diseases such Alopecia Areata, Anklosing Spondylitis, Antiphospholipid Syndrome, Autoimmune

Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Inner Ear Disease, Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Thrombocytopenic Purpura (ATP), Behcet's Disease, Bullous Pemphigoid,

Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue Syndrome Immune, Deficiency Syndrome (CFIDS), Chronic Inflammatory Demyelinating Polyneuropathy, Cicatricial Pemphigoid, Cold Agglutinin Disease, CREST Syndrome, Crohn's Disease, Dego's Disease, Dermatomyositis, Dermatomyositis - Juvenile, Discoid Lupus, Essential Mixed Cryoglobulinemia, Fibromyalgia - Fibromyositis, Grave's Disease, Guillain-Barre, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin Dependent Diabetes (Type I), Juvenile Arthritis, Lupus, Meniere's Disease, Mixed connective Tissue Disease, Multiple Sclerosis,

Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglancular Syndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo and Wegener's Granulomatosis.

Alternative technologies for cleaving DNA at a desired site are available and can be used to generate mutations at a specific site in the LEM DNA sequence. TALEN technology is one such example. TALEN (Transcription activator-like effector nuclease) systems are a fusion of TALEs derived from the Xanthomonas spp. to a restriction endonuclease Fokl. By modifying the amino acid repeats in the TALEs, users can customize TALEN systems to specifically bind target DNA and induce cleavage by the nuclease between the two distinct TAL array binding sites. A variety of plasmid kits are available from Addgene, One Kendall Sq B7102 , Cambridge MA 02139 and allow for the creation of custom repeat arrays for easy TALEN preparation. The different TALEN tool kits use various cloning techniques and protocols to enable custom TALEN design and preparation. Further information on TALEN-based genome editing can be found in WO 2014/134412.

In a second aspect, the invention provides a method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

(a) introducing into the cell a TALEN protein or nucleic acid encoding the TALEN protein, and optionally a nucleic acid repair template, wherein the TALEN protein comprises a plurality of TAL effector repeat sequences and an endonuclease domain, the effector repeat sequences being configured to direct the endonuclease domain to a target sequence in the chromosomal sequence,

(b) culturing the cell such that the TALEN protein binds to the target sequence and introduces a double strand break at a target site within the target sequence, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

A further technology that can be used to modify a gene sequence at a specific site is Zinc Finger Nuclease (ZFN) technology. Zinc finger nuclease (ZFN) technology utilizes a Fokl nuclease as the DNA-cleavage domain and binds DNA by engineered Cys2His2 zinc fingers. Specific zinc fingers recognize different nucleotide triplets and dimerize the Fokl nuclease. The activated nuclease introduces a double stranded break between the two distinct zinc finger binding sites, which prompts recombination and modification of the genome. Plasmids and protocols for ZFN genome editing are available at Addgene , One Kendall Sq B7102, Cambridge MA 02139. Further information on ZFN technology is available in EP2806025.

Thus, in a third aspect, the invention provides a method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

(a) introducing into the cell a Zinc Finger Nuclease (ZFN) or nucleic acid encoding the ZFN, and optionally a nucleic acid repair template, the ZFN comprising

(i) a DNA binding domain comprising a zinc finger domain which binds a target sequence within the chromosomal sequence; (ii) a Fok I cleavage domain; and optionally

(iii) a nuclear localization signal (NLS); and

(b) culturing the cell such that the ZFN binds to the target sequence and introduces a double strand break at a target site within the target sequence, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

Both TALEN and ZFN processes introduce a double stranded break in the target sequence which can be repaired by cellular DNA repair processes in the way described in relation to CRISPR methods.

Nucleic acid encoding the ZFN or TALEN proteins may be provided in a vector. The vector may be, for example an adenovirus vector, a parvovirus vector, a herpes virus vector or a retroviral vector.

In certain embodiments of the invention (any aspect of the invention), the chromosomal sequence comprises or consists of SEQ ID NO: 1. The target sequence may include residue 1313 of SEQ ID NO: 1. The gRNA may be configured to bind a region of SEQ ID NO: 1 or a sequence complimentary to SEQ ID NO: 1 and target the mutation/modification to residue 1313 of SEQ ID NO: 1. The modification made using a method of the invention may be an A1313G mutation in SEQ ID NO: 1. The modification may result in an E53G mutation in the human LEM amino acid sequence (SEQ ID NO: 2).

In a further aspect, the invention provides a T cell comprising a chromosomal sequence encodes LEM, wherein the chromosomal sequence has one or more modifications introduced by the method of any preceding claim. The chromosomal sequence may comprise or consist of SEQ ID NO: 1.

In a further aspect, the invention provides a T cell having a mutation in a chromosomal sequence, wherein the chromosomal sequence comprises or consists of SEQ ID NO: 1 and the mutation is an A1313G mutation.

The invention also provides a population of T cells as defined herein.

All references cited herein are incorporated by reference to the fullest extent permitted by law. Description of the Figures

In the Figures, the term "Retro" is used to denote a mutant version of LEM.

Figure 1 : human LEM genomic sequence (SEQ ID NO: 1)

Figure 2: human LEM amino acid sequence (SEQ ID NO: 2)

Figure 3: Phenotype of Retro homozygous mutant mice.

Figure 4A: CD8⁺ T cell intrinsic effect of Retro mutation.

Figure 4B: Retro-mutant CTLs are hyper-proliferative in vivo.

Figure 5: CTLL-2 cells with Retro genes.

Figure 6: Retro mRNA levels for CTL in vitro.

Figure 7: Retro mRNA levels for CTL in vivo.

Figure 8A: CD8 T cells from Retro mutant mice were hyper-proliferative as evidence by BrdlT incorporation.

Figure 8B: Retro mutant CTL transduced by Retro shRNA show reduced proliferation relative to CTL transduced with scrambled shRNA.

Figure 8C: Retro mRNA levels reduced using Retro shRNA

Figure 9: Retro mutant mice have increased CTL immunity to melanoma

Figure 10: Affinity of Retro binding to RNA containing an ARE motif.

Figure 11 : Over expression of hRetro increases expansion of Jurkat T cells.

Figure 12: Increased development of primary and memory CD8 T cells after vaccination of Retro mutant mice. Figure 13: In vivo validation of the causative Retro mutation. Examples

In the following examples, the term "Retro" is used to denote a mutant version of LEM.

Example 1 - Identification of a positive-regulator of tumour immunity

Many targets that show animal model efficacy not only control immunosuppression of anti- tumour CTLs but also immunosuppression of anti-viral CTL responses (e.g. PD-1 , IL-2, OX-40) . A model system of immunosuppression during chronic LCMV infection was used to identify new pathways for the control of T cell immunosuppression. An unbiased forward genetic approach to identify mouse mutants with CTLs that are resistant to immuno-suppression was used. Using ethyl-nitrosourea (ENU) mutagenesis random mutations in the sperm of C57BL/6J (Charles River sub-strain) male mice (GO) were generated. GO mice were bred to C57BL/6J wild-type females to obtain a G1 generation mice derived from an ENU-mutated sperm with a unique spectrum of point mutations (about 2,500 heterozygous mutations per G1). Two subsequent crosses brought mutations to homozygosity in G3 animals. Infection of mice with the clone 13 variant of LCMV (LCMV C13) results in chronic infection because of physical deletion of protective CTLs specific for the np396 epitope . 403 G3 mice were screened for mutants that were resistant to the deletion of np396 specific CTLs after LCMV C13 infection.

G3 mice were identified from one G1 x G2 breeding pair that had a >8-fold increase in the number of np396-specific CTLs, increased cytolytic activity (based on surface CD 107a expression) and the down-regulation of PD-1 expression . G3 with this phenotype were called Retro mice. The Retro phenotype was inherited as a semi- dominant trait and homozygous mutant Retro mice were generated. Analysis of Retro homozygous mutant mice revealed that they had about 20-fold greater number of np396 specific CTLs compared to wild-type (Figure 3) and a corresponding 10⁴-fold decrease in the titer of LCMV C13 in the spleen (data not shown). This increased immunity to LCMVC13 came at the cost of increased immunopathology because there was a substantial increase in mortality of Retro homozygous mice (data not shown) . Naive Retro mutant mice harbour increased levels of activated T cells and exhibit signs of autoimmunity late in life (data not shown). Example 2 - Increase in np396-specific CTL is cell-autonomous to CD8 T cells

1x10e7 CD8 cells were isolated by CD8a (Ly2) beads (Miltenyi Biotec) from donor Retro Thy1.2 and WT Thy1.2 mouse spleens and intravenously injected into 3-4 WT B6 recipients Thy 1.1 mice of 6-8 weeks old with sterile PBS used as a control. After 24 hour recovery from the injection, recipient mice were infected with 1x10e6 pfu LCMV C13. On day 8 post infection, spleens were taken from the mice and standard staining with CD8 antibody, LCMV NP396 and GP33 tetramers were performed for measurement of CD8NP396 and CD8GP33 antigen specific cells using FACS on a CyAn ADP (Dako).

This experiment showed that the increase in the number of np 396-specific CTLs after LCMV C13 infection was a trait that was cell-autonomous to CD8 T cells (Figure 4A).

Example 3 - Retro homozygous mutant CTL proliferated more than wild-type after LCMV C13 infection

4-5 Retro homozygous mice fed on BrdU containing H₂0 were intravenously infected with 1x10e6 pfu LCMV C13 using WT B6 as controls. On day 8 post infection, splenocytes were isolated and stained with anti-CD8 antibody and LCMV NP396 and GP33 tetramers following standard BrdU and AnnexinV stainings according to the manufacture's protocol (eBioscience). The frequencies of BrdU/Annexin V positive in antigen specific CD8 T cells were measured by standard FACS on a CyAn ADP (Dako) using proper gating.

This study revealed that Retro homozygous mutant CTLs proliferated more than wild-type after LCMV C13 infection (Figure 4B). No difference in the percentage of cells undergoing apoptosis as indicated by annexin V staining was observed.

Example 4 - The identification of the Retro mutation.

Genomic DNA from Retro homozygous mutant mice was subjected to whole exome next generation sequencing. Comparison with the wild-type C57BL/6 reference exome sequence (sub-strain: C57BL/6J) identified 8 homozygous single nucleotide polymorphisms (SNP) that would give synonymous amino acid sequencing revealed that 4/8 of the Retro-associated SNP were also present in the genome of wild-type C57BL/6 sub-strain (Charles River) used for our ENU mutagenesis. The 4 ENU-generated SNP were then analysed in the progeny of mice to see which one segregated with the Retro phenotype. Only one Retro mutant allele was found to be homozygous in every mouse exhibiting the Retro phenotype (> 4-fold increase in level of np396+ CD8+ cells on day 8 of C13 infection). This homozygous SNP was in the gene BC0551 11 on chromosome 4 resulting in a glutamate (E) to glycine (G) change at amino acid 50 (E50G).

Example 5 - Validation of BC055111 as the LEM gene.

The in vivo findings discussed above show that Retro mutant mice harbouring the BC05511 1 E50G mutation have increased levels of CTLs due to increased proliferation. To validate the E50G mutation in BC05511 1 as the causative mutation for the Retro- phenotype, wild-type or E50G mutant BC0551 11 were overexpressed as open reading frames (ORF) in the mouse CTLL-2 T cell line by MIGR1 retrovirus transduction. Over- expression of wild-type BC0551 11 resulted in a 14-fold increase in the expansion of CTLL-2 cells compared to empty vector controls (Figure 5). CTLL-2 cells transduced with E50G mutant BC05511 1 expanded about 3-times more than cells with wild-type BC05511 1. Real-time PCR revealed that the level of BC05511 1 mRNA was the same in CTLL-2 cells over-expressing E50G mutant versus wild-type ORF (data not shown).

Therefore, wild-type LEM promotes the proliferation of CTLs and the E50G mutation is a gain-of function phenotype. It was concluded that E50G mutation in BC055111 is the causative mutation of the Retro phenotype.

Example 6- Increased Retro gene expression in Retro mutant CTLs.

The expression of Retro mRNA in CD8 T cells was examined after activation by anti-CD3 and antiCD28 antibodies. 24 well plates were coated with monoclonal anti-CD3 antibody ^g/ml) and (2 μg/ml) monoclonal anti-CD28 antibody at 4°C overnight. Primary CD8 T cells (magnetic bead sorted) from both wild-type and Retro mutant mice (1.5 x10⁶) were cultured in RPMI-10% FCS on antibody coated plates and IL2 (5ng/ml). The relative Retro mRNA level determined by real-time PCR is normalized by the level in naive CD8 T cells and the GAPDH internal control.

A modest (1.2-fold) up-regulation of BC05511 1 mRNA on day 4 of culture was observed, which then diminished to the naive level by day 6. However, when CD8 T cells from Retro mutant mice were examined, a far greater up-regulation (3-fold) of BC0551 11 on both day 4 and day 6 (2-fold) over the naive level was observed (Figure 6). The in vivo relevance of this observation was confirmed when a 6-fold up-regulation in BC0551 11 mRNA in anti- LCMV CTLs in Retro mutant compared to a 2-fold in wild-type mice was also observed (Figure 7). Data shown is for magnetic bead sorted CD8 T cells on day 8 of infection with LCMV C13 (10⁶ pfu/i.v.). The relative Retro mRNA level determined by real-time PCR is normalized by the level in naive CD8 T cells and the GAPDH internal control. Mean values from 6 mice are shown.

As observed in vivo (Figure 4), after activation (by TCR/CD28 stimulation), CD8 T cells from Retro mutant mice were hyper-proliferative (as evidenced by BrdU incorporation) (Figure 8A). Briefly, CD8 T cells from wild-type and Retro mutant mice were cultured on anti-CD3 and anti-CD28 antibodies (as in Figure 8B) for 3 days, then pulsed with BrdU and on day 4 the % that stained positive with anti- BrdlT antibody (PE-secondary antibody) then determined. Mean values from 4 wells are shown.

To determine if the increased level of BC0551 11 mRNA contributed to the hyper- proliferation of Retro CD8 T cells, BC0551 11 mRNA was knocked down in Retro mutant CTLs. CD8 T cells from Retro mutant mice were cultured on anti-CD3 and anti-CD28 antibodies for 2 days then transduced with lentivirus (GIPZ vector, Open Biosystems) (MOI= 30: 1) encoding shRNA encoding either scrambled shRNA or shRNA specific for BC05511 1 (according to manufacturer's protocol). On day 3 cells were pulsed with BrdU and on d4 the % of GFP+ cells (5-20%) that stained positive with anti-BrdU+ antibody (PE-secondary antibody) was determined. Mean values from 4 wells are shown in Figure 8B.

CD8 T cells from Retro mutant mice were cultured on anti-CD3 and anti-CD28 antibodies for 2d then transduced with lentivirus then on day 4 GFP⁺ cells purified by FACS. The relative Retro mRNA level was determined by real-time PCR compared to GAPDH internal control and expressed as % of the level in GFP⁺ cells transduced with scrambled shRNA. Mean values from 4 wells are shown in Figure 8C.

The proliferation of Retro mutant CTLs transduced by Retro shRNA was reduced by about 3-fold (Figure 8B ) when the level of BC05511 1 mRNA in day 4 Retro mutant CTLs was knocked down by 70% (Figures 8C). It was concluded that the increased expression of BC05511 1 mRNA contributes to the Retro phenotype of CTL hyper-proliferation. Example 7 - Retro mutant mice have increased CTL immunity to melanoma

Some negative checkpoints that control CTL-immunity to chronic viral infection also control CTLimmunity to cancer, the best known being the PD-1/PD-L1 axis. Therefore CTL-immunity to malignant melanoma was examined in the B16 melanoma transplantation model. Mice were injected (i.v.) with B16- F10 melanoma cells (C57BL/6 origin) (3x10⁵) then after 5 weeks melanomas analyzed in the lungs. Tumours were excised and digested and purified on a ficol gradient and the % of CD3⁺ CD8⁺ (CTL) determined.

Figure 9 (left panel) shows that Retro homozygous mutant mice generated about 6-times more CTLs in TIL compared to wild-type. The middle panel of Figure 9 is a picture of lungs showing melanoma tumour foci in black. The right panel of Figure 9 shows the number of melanoma tumour foci in female or male mice in WT (n=6) or Retro (n=6) mice. There was about 5-fold fewer tumours in the lungs of Retro mice compared to wild-type. *** p<0.001. This experiment is representative of 3 others. Although it is difficult to compare with other independent studies the magnitude of increase in the level of CTLs in TIL from B16-BL6 melanoma compares favourably with the 3-fold increase induced by G VAX vaccination followed by PD-1 and CTLA-4 antibody combined blockage.

Example 8 - LEM is an RNA-binding protein

Wild-type or E50G BC0551 11 ORFs (constructed by GeneArt, Life Technologies, Invitrogen) were cloned into the pEX6 vector and GSTRetro produced in E. coli then purified on GST beads to >90% purity. GST-Retro protein (74 kD, 150ng) was incubated with biotin-labeled 5'-UUUAUUUAUUAUU-3' (over a range of concentrations) as in Barreau, 2005 #308 [23]. Then the binding of labeled oligoribonucleotide to Retro was measured after filtration through nitrocellulose followed by washing using a slot blotter. RNA bound to Retro on filters was visualized by probing with streptavidin-HRP and developed with ECL. The relative signal was determined by densitometry on slot signals. Relative signal was determined from the binding of biotin labeled oligoribonucleotide alone (filled circles in Figure 10) or in the presence of a 100-fold molar excess of un-labeled oligoribonucleaotide (open circle in Figure 10). These assays revealed that affinity (Kd) of binding between recombinant mouse LEM and an oligoribonucleotide containing an ARE motif was about 153 nM (Figure 10). It was also found that the E50G mutant version of LEM bound to the ARE oligoribonucleotide with a higher affinity (Kd = 34nM). It was concluded that the E50G mutation increases the affinity of LEM for this particular RNA species. The affinity of LEM binding to the ARE motif is within the affinity range of bone fide RNA-BPs with specificity for mRNA

Example 9 - Overexpression of wild type human LEM increases expansion of Jurkat T cells.

Flag-C1orf177 was commercially synthesized into the pcDNA3.1 (+) vector (GeneART technologies, Invitrogen). Flag-C1orf177 coding sequences were excised from pcDNA3.1 (+)-C1orf177 by digestion with Xhol and cloned into the MIGR1 vector. Constructs were verified by automated DNA sequencing for correct orientation. MIGR1- C1orf177 was transiently transfected into the Phoneix packaging line using calcium phosphate method (CAPHOS-1 KT, Sigma) in a 100mm TC plate. Twenty four hours following transfection, media was removed and the cells washed gently with PBS, and fresh media re-applied. Cells were transferred to a 32°C, 5% C0₂ incubator and left overnight. The following morning polybrene (5ug/ml) was added to the virus containing media and gently agitated. Virus containing media was removed and filtered through a 0.45uM filter to avoid cell carry over. Fresh media was applied to the Phoenix cells. Virus containing media was added to 5x105 /ml Jurkat cells and centrifuged at 2250rpm for 90 mins at 37°C and left at 32°C 5% in a TC incubator for eight hour incubation. Following incubation, the Jurkat media was removed, and replaced with fresh virus containing media, spun and kept at 32°C overnight. The cycle of viral infection was continued for three days dependent on the condition of the phoenix cells. After this time transduction was measured as % GFP⁺ in Jurkat cells (typically 5-20%).

The number of transduced cells was determined (GFP⁺) on day 0, then every 3 days until day 12. The fold expansion was determined by dividing through by the starting number on day 0. Figure 11 shows that over-expression of wild-type human LEM increases the expansion of Jurkat cells. Data shown is the mean ± sem (n=4 wells).

Example 10- Increased development of memory CD8 T cells in Retro mutant mice Infection of mice with the WE strain of LCMV gives an acute infection that results in a robust primary CD8 T cell response, long-term immunological memory and viral clearance. The development of memory T cells benefits vaccination. Thus infection of mice with the WE strain of LCMV can effectively vaccinate against subsequent challenge with a strain of LCMV that would give chronic infection in un-vaccinated mice (e.g. LCMV C13 strain). In this Example, LCMV WE infection was used as a measure of the effect of the Retro mutation on memory T cell development and therefore the potential to increase the efficacy of vaccination.

CB57 BL/6 wild type and Retro homozygous mutant mice were infected with LCMV WE (200pfu i.p.) Flow- cytometry after staining with MHC-tetramers containing either the gp33 or np396 peptide antigens of LCMV (Lymphocytic Choriomenigitis virus) and anti-CD8 antibody was used to measure the percentage of CD8+ T cells specific for gp33 or np396 of total T cells in peripheral blood leucocytes over time.

The results are shown in Figure 12A (n=4 mice). Clonal bursts of CD8 T cells specific for both the gp33 and np396 antigens can be seen from day 8 post infection. In mice having the Retro mutation, the percentage of gp33 and np396-specifc CD8 T cells out of total T cells was significantly increased relative to the percentage observed in the wild type mice.

Subsequently, the number of gp33 and np396-specific central memory CD8 T cells was measured in the spleen after 21 days. The results are shown in Figure 20B (n=4 mice). It can be seen that the Retro mutant mice had significantly more central memory CD8 T cells than wild type mice 21 days post-infection.

Example 11 - In vivo validation of the causative Retro mutation

In order to examine whether BC05511 1 (the mouse Retro gene) directly controls CTL immunity to LCMV, mice with a targeted BC0551 11 1 null allele were generated. Mice harbouring the BC05511 i ^1a allele were obtained from the KOMP (Knock-out mouse project) repository then bred with transgenic mice constitutively expressing Cre recombinase under the control or the Ella promotor to delete exons 2-4 after recombination between loxp sites. The position of the E50G mutation in exon 2 is indicated in Figure 13A by the asterisk. Inter-crossing generated mice harbouring different combinations of BC05511 1 alleles (illustrated in Figure 13B). The generation of the BC05511 i ^1b null allele was verified by PCR (using primers that spanned the exons deleted by Cre recombination) followed by Western blot (anti-BC05511 1 antibody from Santa Cruz Biotech, 1/200 dilution) which showed a 50% reduction for BC0551 11 protein in BC055111^E50Glm" compared to BC055111^E50G/E50G mice (Figure 13B).

Mice harbouring the different allele combinations set out in Figure 13B were infected with LCMV C13. On day 8 the level of np396⁺ CD8 T cells in the spleen was determined by tetramer staining and flow- cytometry. The results are shown in Figure 13C which indicates that the E50G/E50G allele combination produced the highest number of np396⁺ CD8 T cells and the wt/null combination produced the lowest number of np396⁺ CD8 T cells In addition, it can be seen that the E50G/null mutation produced fewer np396⁺ CD8 T cells than the E50G/wt allele combination.

The cytotoxic activity (E/T =10: 1 , np 396-pulsed EL4 targets) of the T cells of each mouse type was determined using standard CTL assays. A pattern of results similar to that shown in Figure 13C was observed, with the E50G/E50G combination giving rise to the highest cytotoxic activity, the wt/null combination having the lowest cytotoxic activity and the E50G/null combination producing lower cytotoxic activity than the E50G/wt combination (see Figure 13D).

Finally, the titre of LCMV was determined for each allele combination using a PCR assay (n= 6-10 mice. *** p<0.001). The results are shown in Figure 13E. Mice with the E50G/E50G allele combination achieved the lowest viral titre and mice with the wt/null combination had the highest viral titre. Mice with the E50G/null allele combination had a higher viral titre than mice with the E50G/wt combination.

In summary, it has been determined that in BC05511 i^E50G/nul1 mice, the null allele negates the Retro gain-of function phenotype in trans, indicating that the E50G mutation is the culpable mutation for the Retro phenotype. In addition, BC055111 ^wt/nu!l mice had decreased anti-LCMV CTL immunity compared to ΒΟ055111^ΜΑΜ mice indicating that BC055111 is a positive regulator of CTL expansion. This also indicates that ablation of Retro expression (in this case by gene knock-out) can reduce the expansion of T lymphocytes in vivo.

Claims

1. A method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

(a) introducing into the cell

(i) guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the gRNA hybridises to a target sequence within the chromosomal sequence or a target sequence in a sequence that is complementary to the chromosomal sequence;

(iii) a nucleic acid repair template

(b) culturing the cell such that the gRNA directs the one or more RNA-guided

endonucleases to the target sequence where the one or more RNA-guided

endonucleases introduce a single-stranded break or a double-stranded break at a target site within the target sequence, and the single stranded-break or double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

2. The method of claim 1 , wherein the RNA-guided endonuclease comprises at least one nuclear localization signal.

3. The method of claim 1 or claim 2, wherein the RNA-guided endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) endonuclease, optionally Cas9.

4. The method of any preceding claim, wherein the guide RNA comprises a guide sequence and trans-activating cr (tracr) sequence.

5. The method of any preceding claim, wherein the nucleic acid encoding the RNA- guided endonuclease is mRNA or DNA.

6. The method of claim 5, wherein the DNA is part of a vector that further comprises a sequence encoding the guide RNA.

7. The method of any preceding claim, wherein the cell is a T cell.

8. The method of any preceding claim, wherein the method is carried out ex vivo or in vitro.

9. A method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

10. A method of modifying a chromosomal sequence in a mammalian cell, wherein the chromosomal sequence encodes LEM, the method comprising:

(i) a DNA binding domain comprising a zinc finger domain which binds a target sequence within the chromosomal sequence;

(ii) a Fok I cleavage domain; and optionally

(iii) a nuclear localization signal (NLS); and

11. The method of any preceding claim, wherein the chromosomal sequence comprises or consists of SEQ ID NO: 1.

The method of claim 11 , wherein the target sequence includes residue 1313 of ID NO: 1.

13. The method of claim 11 or claim 12, wherein the modification is an A1313G mutation in SEQ ID NO: 1.

14. A T cell comprising a chromosomal sequence encodes LEM, wherein the chromosomal sequence has one or more modifications introduced by the method of any preceding claim.

15. The T cell of claim 14, wherein the chromosomal sequence comprises or consists of SEQ ID NO: 1.

16. A T cell having a mutation in a chromosomal sequence, wherein the chromosomal sequence comprises or consists of SEQ ID NO: 1 and the mutation is an A1313G mutation.