WO2022175674A1

WO2022175674A1 - Modified human cells and uses thereof in the treatment of immune-mediated diseases

Info

Publication number: WO2022175674A1
Application number: PCT/GB2022/050445
Authority: WO
Inventors: James O. J. DAVIES; Calliope A. DENDROU
Original assignee: Oxford University Innovation Limited
Priority date: 2021-02-19
Filing date: 2022-02-18
Publication date: 2022-08-25

Abstract

The present invention relates to modified human cells and uses thereof in the treatment of immune-mediated diseases (IMDs), e.g. scleroderma, multiple sclerosis (MS) and Crohn's disease. In particular, the invention provides modified human cells wherein at least one allele of the TYK2 gene in the genomes of the cells has a mutation which reduces pro-inflammatory cytokine signalling in the cell. The invention also provides methods of autologous and allogeneic transplantation using such cells.

Description

MODIFIED HUMAN CELLS AND USES THEREOF IN THE TREATMENT OF

IMMUNE-MEDIATED DISEASES

The present invention relates to modified human cells, and populations of such cells, and uses thereof in the treatment of immune-mediated diseases (IMDs), e.g. scleroderma, multiple sclerosis (MS) and Crohn’s disease. In particular, the invention provides modified human cells wherein at least one allele of the TYK2 gene in the genomes of the cells has a mutation which reduces pro-inflammatory cytokine signalling in the cell. The invention also provides methods of autologous and allogeneic transplantation using such cells.

Immune-mediated diseases (IMDs) are conditions which result from abnormal activity of the body’s immune system, for example, wherein the immune system may over-react or start attacking the body. IMDs include scleroderma, multiple sclerosis (MS) and Crohn’s disease.

IMDs represent one of the most important classes of diseases, affecting around 10% of the population, and posing a substantial socio-economic burden.

Current treatment of IMDs generally consists of immunomodulation to suppress the active disease, rather than attempting to cure the disease indefinitely.

Treatment of scleroderma depends on the organs involved. Medications used include corticosteroids, methotrexate, and non-steroidal anti-inflammatory drugs (NSAIDs).

A number of medications have been approved for the treatment of MS including interferon beta-1 a, interferon beta-1 b, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, teriflunomide, dimethyl fumarate, alemtuzumab, ocrelizumab, siponimod, cladribine and ozanimod. Medications used to treat Crohn's disease include 5-aminosalicylic acid (5-ASA) formulations, prednisone, immunomodulators such as azathioprine, methotrexate, infliximab, adalimumab, certolizumab, vedolizumab, ustekinumab and natalizumab

Many IMD patients have successive relapses, they suffer a general decline in function over many years, and they can also experience adverse effects associated with long term drug regimens. It would be desirable, therefore, to find further methods to treat - and preferably to cure - such patients.

The advent of CRISPR-based gene-editing techniques has meant that correcting the genetic defects underlying the above-mentioned IMDs has become possible.

CRISPR/Cas9 recognizes specific DNA sequences with a 3’ “NGG” (the PAM site) in the genome; it introduces double-stranded breaks (DSBs) in a precise and efficient manner. These double-stranded breaks initiate a DNA damage response in the cell and they are repaired by one of two competitive pathways: non-homologous end joining (NHEJ) or homology-dependent repair (HDR, also known as homology-directed repair). The NHEJ pathway involves random insertion or deletions (indels) at the site of DNA damage, while the HDR pathway enables more precise modification, but it requires a homologous donor template for the repair.

Base editing is a form of genetic editing in which one base pair is permanently converted to another base pair at a target locus. Base editors are guided to their target by an associated guide RNA. Unlike other methods of genetic editing, base editing does not introduce any double-strand DNA breaks into the target DNA; it does not require non-homologous end joining or homology-directed repair methods; and also it does not require any donor DNA templates. For these reasons, base editing can introduce specific point mutations more efficiently while introducing less off-target insertions, deletions, translocations and other modifications than other methods of gene editing such as CRISPR-Cas9. Base editing has been demonstrated in bacteria, yeast, plants, mammals and human embryos. Base editing can achieve transitions in genomic DNA from (C to T, A to G; which can be used to convert G to A and T to C on the opposite strand). Interconversion of purine to pyrimidine is not possible at present (i.e. C to G or A to T).

The most common programmable base editors (BEs) are BE3s which comprise a catalytically impaired CRISPR-Cas9 mutant which is incapable of making double-strand breaks; a single-strand-specific cytosine deaminase that converts C to U within a window of around five nucleotides in the single-strand DNA bubble created by the Cas9; a uracil glycosylase inhibitor that prevents uracil excision and downstream processes that reduce base editing efficiency and product purity; and nickase activity to nick the non-edited DNA strand which directs cellular DNA repair processes to replace the G- containing DNA strand and complete the C-G to T-A conversion.

Adenine base editors (ABEs) that convert A-T to G-C have only recently been developed (Gaudelli et al., 2017). A seventh-generation evolved ABE (i.e. ABE7.10) was shown to have a conversion efficiency of around 50% in human cells with a product purity of at least 99.9%, and an indel rate of 0.1% or lower.

Prime editing uses a catalytically-impaired Cas9 endonuclease fused to a reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing a template DNA to facilitate the replacement of the target DNA. Prime editing mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. Prime editing is still an early-stage, experimental genome editing method; to date, it has been used in few therapeutic applications.

One or more of the above techniques may therefore be used to correct the genetic defects underlying the above-mentioned IMDs.

The inventors have had the insight to investigate whether it might not be necessary to correct the genetic defects underlying IMDs directly, i.e. whether it might be possible to overcome the effects of these diseases by other - indirect - means. It has previously been found that the single nucleotide polymorphism (SNP) rs34536443 is associated with a protective effect against about 20 common IMDs (Dendrou et al., 2016, Sci. Transl. Med. 8(363); Cortes et al., 2017, Nat. Genet. 49(1311); Cortes et al., 2020, Nat. Genet. 52(126)). Rs34536443 leads to the substitution of a highly conserved proline residue with an alanine at position 1104 (P1104A) in the kinase domain of the TYK2 polypeptide. The Pro1104Ala variant was found to reduce TYK2 activity in its homozygous state. The protection is mediated by reducing Type I interferon, IL-12 and IL-23 signalling, and the reduction in IMD development was reproducible in mice: animals homozygous for the orthologous Pro1124Ala substitution were completely protected from developing the multiple sclerosis-like disease experimental autoimmune encephalomyelitis.

A structural analysis of the TYK2 polypeptide by the inventors has resulted in the identification of a number of other TYK2 variants which reduce TYK2 activity yet further.

The inventors have now found that genome-editing techniques can be applied to the treatment of autoimmune disease by replacing a patient’s immune cell repertoire with TYK2 gene-edited immune cells which have impaired TYK2-dependent signalling. Such cells are highly protective against developing autoimmune disease.

One way to achieve this is through autologous cell transplantation, which involves the editing of haematopoietic stem cells, which then differentiate and repopulate the immune system with a different genotype. This might also be achieved by repopulating the immune system with autologous transplantation of edited lymphocytes instead of stem cells or by in vivo editing.

The rationale behind the current invention is therefore significantly different from current treatments which aim to suppress immune function in autoreactive cells.

It can be seen therefore that the current invention is based on a novel combination of a number of different technologies: a structural analysis of the TYK2 polypeptide which has identified TYK2 variants; the precision editing of the TYK2 gene; and the transplantation of immune cells with edited TYK2 genes into IMD patients.

It is therefore an object of the invention to provide modified human cells which reduce pro-inflammatory cytokine signalling in the cell. The invention also provides processes for producing such cells and methods of treatment of IMDs using such cells.

In one embodiment, the invention provides a modified human cell, wherein at least one allele of the TYK2 gene in the genome of the cell has a modification which reduces pro- inflammatory cytokine signalling in the cell.

Preferably, (i) the modification is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide in the cell; or (ii) the modification is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide in the cell.

In another embodiment, the invention provides a process for producing a modified human cell comprising a modified TYK2 gene, the process comprising the step:

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a DNA-targeting polypeptide, or

(ii) a nucleic acid molecule encoding a DNA-targeting polypeptide; under conditions such that, when present, the nucleic acid molecule encoding the DNA-targeting polypeptide is expressed in the cell to produce a DNA-targeting polypeptide, the DNA-targeting polypeptide is targeted to a target nucleotide sequence in the TYK2 gene, and wherein the DNA-targeting polypeptide modifies one or more nucleotides in the TYK2 gene, thereby producing a modified human cell comprising a modified TYK2 gene, wherein either:

(i) the target nucleotide sequence is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide; or (ii) the target nucleotide sequence is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide. In a preferred embodiment, the invention provides a process for producing a modified human cell comprising a modified TYK2 gene, the process comprising the step:

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a CRISPR polypeptide or a nucleic acid molecule coding therefor, and

(ii) a gRNA, wherein the gRNA is one which is capable of targeting the CRISPR polypeptide to a target nucleotide sequence in the TYK2 gene, or a nucleic acid coding therefor; under conditions such that, when present, the nucleic acid molecule coding for the CRISPR polypeptide is expressed in the cell to produce the CRISPR polypeptide, when present, the nucleic acid molecule coding for the gRNA is expressed in the cell to produce the gRNA, the CRISPR polypeptide is targeted by the gRNA to the target nucleotide sequence in the TYK2 gene, and wherein the CRISPR polypeptide modifies one or more nucleotides in the TYK2 gene, thereby producing a modified human cell comprising a modified TYK2 gene, wherein either:

(i) the target nucleotide sequence is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide; or

(ii) the target nucleotide sequence is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide.

In another embodiment, the invention provides a method of treating an immune- mediated disease (IMD) in a human subject, the method comprising administering an effective amount of modified human cells of the invention to a subject in need thereof.

In another embodiment, the invention provides a modified human cell of the invention or a composition of the invention, for use in treating an IMD. In another embodiment, the invention provides the use of a modified human cell of the invention in the manufacture of a medicament for treating an IMD.

In one embodiment, the invention relates to a modified human cell or a population of cells comprising one or more such modified human cells. The processes of the invention apply equally to producing modified cells and to producing populations of such modified cells. Preferably, the cells are ones which have been obtained from a human subject who is suffering from or at risk from an immune mediated disease (IMD), preferably an IMD as defined herein. In other embodiments, the cells are not ones which have been obtained from a human who is suffering from or at risk from an immune mediated disease (IMD). In some preferred embodiments, the cells are ones which have been obtained from the subject to be treated or a close relative thereof. The human may, for example, be 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80- 90, 90-100 or above 100 years old. The cells may be foetal cells, juvenile cells or adult cells. The cells may be isolated ones or purified ones. In particular, the cells of the invention may be in a form wherein the cells are isolated from red blood cells and/or platelets.

In some embodiments of the invention, the cells are haematopoietic cells. In some embodiments, the cells are myeloid cells or lymphoid cells, preferably lymphoid cells.

In some embodiments, the cells are immune cells, e.g. leukocytes.

Examples of suitable haematopoietic cells include haematopoietic stem cells, myeloid progenitor cells, lymphoid progenitor cells, lymphoblasts, T lymphocytes, B lymphocytes and monocytes.

Preferably, the cells are haematopoietic stem cells or leukocytes (e.g. lymphocytes or monocytes), most preferably haematopoietic stem cells.

In some embodiments, the cells are ones which have been selected for expression of a specific biomarker. For example, the cells may be CD34⁺ cells and/or CD133⁺ cells.

In other preferred embodiments, the haematopoietic cells are obtained from one of the following sources: a) Cord blood, e.g. collected at birth from the umbilical cord and/or placenta of a human subject; b) Bone marrow harvested directly from a human subject; c) Peripheral blood stem cells (e.g. collected by apheresis following administration of plerixafor or GCSF or chemotherapy); or d) An identical twin, twin transplant or sibling to the subject to be treated.

In other embodiments of the invention, the cells are stem cells, preferably stem cells which can give rise to immune cells (e.g. leukocytes). In some embodiments, the cells are induced pluripotent stem cells (iPSCs). In some embodiments, the stem cells are not embryonic stem cells or are cells which are not obtained from a human embryo.

In other embodiments of the invention, the cells are cells which are involved in an IMD in a subject (preferably the subject to be treated), e.g. fibroblasts or pancreatic islets cells.

The TYK2 gene encodes a non-receptor tyrosine kinase that is constitutively expressed across immune cell types. TYK2 has been reported to transduce signals downstream of Type I interferon (IFN), gp130, interleukin (IL)-10R2, IL-13Ra and IL-12Rp1 cytokine receptor families, and thus has a pleiotropic role in host responses to infection and in tumour surveillance (Schwartz DM, et al. "Type I/ll cytokines, JAKs, and new strategies for treating autoimmune diseases". Nat. Rev. Rheumatol. 2016; 12:25-36). TYK2 is constitutively expressed in all immune cell types and is thus amenable to editing, particularly in haematopoietic cells.

The human wild-type TYK2 polypeptide sequence is given herein as SEQ ID NO: 1.

The human wild-type TYK2 polypeptide-encoding sequence is given herein as SEQ ID NO: 2 or SEQ ID NO: 3. (SEQ ID NOs: 2 and 3 are two different versions of TYK2 mRNA, wherein the untranslated regions differ; both of these sequences encode the amino acid sequence given in SEQ ID NO: 1).

As used herein, the term “wild-type” TYK2 gene refers to the TYK2 gene which is present in the majority of the members of that species (e.g. humans) and which encodes a non-mutant form of the TYK2 polypeptide. As used herein, the term “TYK2 gene” refers to the region of DNA which codes for the TYK2 polypeptide and also to the associated regulatory regions (e.g. promoters, enhancers, repressors, etc.).

In most embodiments of the invention, the cells will be diploid, i.e. the cells will have two copies, i.e. alleles, of the TYK2 gene. At least one allele of the TYK2 gene has a modification in accordance with the invention. Preferably, both alleles of the TYK2 gene have modifications in accordance with the invention. The two alleles may have the same or different modifications in accordance with the invention. Each allele may independently have one or more than one (e.g. 2, 3, 4, or 5) modifications of the invention. In some embodiments of the invention, the cells are haploid.

The modifications are ones which reduce pro-inflammatory cytokine signalling in the cell.

The TYK2 gene encodes a non-receptor tyrosine kinase that is constitutively expressed across immune cell types. TYK2 is involved in pro-inflammatory cytokine signalling, including signally induced by type I interferon (IFN), interleukin-12 (IL-12) and interleukin-23 (IL-23). Preferably, the pro-inflammatory cytokine is type I IFN, IL-12 or IL-23.

As used herein, the term "reduce pro-inflammatory cytokine signalling" means that, in the presence of one or more of type I IFN, IL-12 and IL-23, there is a reduction in the level of phosphorylation of STAT1-5, STAT4 and STAT3, respectively, in cells of the invention compared to control cells.

In some embodiments, the term "reduce pro-inflammatory cytokine signalling" means that, in the presence of type I IFN, there is a reduction in the level of phosphorylation of STAT1-5 in cells of the invention compared to control cells.

Pro-inflammatory cytokine signalling in the cells may be assayed by assessing cytokine- induced TYK2 phosphorylation and STAT phosphorylation by Western blotting, STAT phosphorylation by flow cytometry, and cytokine-induced changes in gene expression. For variants that alter TYK2 expression, this expression may be assayed by measuring TYK2 mRNA and protein levels, using quantitative PCR or RNA sequencing and Western blotting, respectively.

In some embodiments, the control cells are ones which are identical to those of the invention except that all copies of the TYK2 genes in the control cells have wild-type TYK2 sequences and preferably wild-type epigenetic modifications. In other embodiments, the control cells are wild-type human cells, preferably of the same type as the modified cell.

Preferably, the reduction in pro-inflammatory cytokine signalling is at least 50%, 60%, 70%, 80%, or 90%, more preferably at least 80%.

The pro-inflammatory cytokine signalling is preferably reduced by: (i) altering the function of the TYK2 polypeptide, or (ii) reducing the expression level of the TYK2 polypeptide.

In some embodiments of the invention, the modification is a non-synonymous mutation in the TYK2-coding sequence, i.e. a nucleotide mutation that alters the wild-type amino acid sequence of the TYK2 polypeptide. Preferably, the mutation is a substitution at a position corresponding to a position selected from the group consisting of Val362Phe, Ne684Ser, Ne684Thr, Asn920Ser, Gly922Ala,, Ala931Asp, Ala931Val, Cys936Arg, His940Gln, Arg941 Leu, Gly943Val, Trp944Leu, Lys945Arg, Glu947Lys, Ne948Thr, Asp949Ala, Ne950Ser, Leu951Gln, Thr953Met, His956Arg, His956Gln, Glu957Lys, Tyr962Ser, Lys963Arg, Lys963Met, Gly964Ala, Cys966Gly, Cys966Tyr, Gln969Glu, Glu979Lys, Tyr980Asn, Asp988Val, Tyr989Cys, Arg992Trp, His993Tyr, His993Gln, Ser994Asn, lle995Val, Gly996Arg, Gln999Glu, Leu1001 Met, Leu1002Phe, Ala1004Gly, Gln1005Arg, Gln1005Leu, lle1007Met, His1015Gln, Ala1016Ser, His1018Gln, Ala1026Thr, Asn1028Ser, Asn1033Asp, Arg1035Ser, Arg1035Ser, Lys1038Asn, Pro1049Leu, Glu1050Lys, Glu1050Ala, Glu1050Asp, Pro1064Arg, Tyr1076Cys, Lys1077Gln, Tyr1080His, Tyr1080Cys, Phe1087Cys, Gly1088Arg, Tyr1092Phe, His1097Tyr, Ser1100Cys, Ser1103Gly, Ser1103Asn, Seri 103Thr and Pro1104Val. The mutation may also be Lys930Arg, Tyr1054His, Tyr1055Cys,Tyr1055His or Tyr1055Cys. The mutation may also be R987Q.

More preferably, the mutation is at a position in the TYK2 polypeptide sequence selected from those given in the following table or one of the mutant options given:

Table 1

The above positions relate to the TYK2 amino acid sequence as given in SEQ ID NO: 1.

Preferably, the mutation is Pro1104Ala, Ala928Val or Asp988Asn, most preferably Ala928Val. Another preferred mutation is R987Q. The combination of R987Q and D988N is also preferred. In some embodiments, the mutation is not P1104A, A928V or I684S.

In other embodiments of the invention, the modification is in a regulatory region of the TYK2 gene. Such modifications may indirectly reduce the pro-inflammatory cytokine signalling in the cell by reducing the expression level of TYK2 in the cell. Preferably, therefore, the modification is one which reduces the expression level of TYK2 in the cell. Preferably, the expression level is reduced compared to the expression level in a control cell in which the modification is not made (e.g. a wild-type cell). With regard to modifications in the regulatory regions of the TYK2 gene, the modification may be selected from insertions, deletions, substitutions of one or more nucleotides. In other embodiments, the modification is an epigenetic change, e.g. DNA methylation. In particular, the epigenetic change may be cytosine methylation or hydroxyl-methylation of DNA, or histone deacetylation.

The regulatory region may, for example, be a promoter, enhancer or a repressor.

Active regions in the TYK2 regulatory regions may be identified using chromatin conformation capture (3C) methods such as Next Generation Capture C (e.g.

WO201 7/068379 and WO2020/161485).

TYK2 expression levels may be assessed at the transcript (mRNA) level by quantitative PCR assays or RNA sequencing; protein-level expression may be assayed by Western blotting.

In some embodiments, the modification in the regulatory region of the TYK2 gene is in a region selected from the group consisting of chr19: 10,484,800-10,487,800; chr19: 10,489,994-10,492,729; chr19: 10,499,400-10,500, 150; chr19: 10,500,420-

10,501 ,170; chr19: 10,514,530-10,515,530; chr19: 10,515,830-10,516,830; chr19: 10,518,990-10,519,990; chr19: 10,522,630-10,523,630; chr19: 10,534,500-

10,536,500; chr19: 10,538,530-10,539,530; chr19: 10,542,319-10,543,320; chr19: 10,546,780-10,547,780; chr19: 10,571 ,500-10,572,500; chr19: 10,582,550-

10,583,050; and chr19: 10,589,320-10,590,320. (The aforementioned regions are from the human genome build GRCh37/hg19.)

Also provided is a population of cells comprising one or more modified human cells of the invention. The population may comprise a mixture of modified human cells of the invention and unmodified human cells (e.g. human cells which comprise wild-type TYK2 genes). The population may comprise a mixture of modified human cells of the invention of different cell types, e.g. a first cell type (e.g. modified haematopoietic stem cells) and a second cell type (e.g. modified lymphocytes). Preferably, at least 1%, 5%, 10% or 20% cells in the population are modified human cells of the invention. In some embodiments, the population does not comprise red blood cells or platelets.

In another embodiment, the invention provides a pharmaceutical composition comprising a population of cells comprising one or more modified human cells of the invention, optionally together with one or more adjuvants, carriers or diluents.

In another embodiment, the invention provides a TYK2 gene, wherein the nucleotide sequence of the TYK2 gene comprises:

(a) (i) a nucleotide sequence encoding an amino acid sequence having 95-99.5% amino acid sequence identity to SEQ ID NO: 1 , or

(ii) a nucleotide sequence having 90-99.9% nucleotide sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3; and wherein

(b) the nucleotide sequence of the TYK2 gene encodes an amino acid sequence comprising a non-wild-type amino acid at one or more positions corresponding to the positions identified in Table 1 , wherein, if the nucleotide sequence of the TYK2 gene encodes an amino acid sequence comprising a substitution at a position corresponding to position 928 or 1104 in SEQ ID NO: 1 , then the amino acid sequence additionally comprises a non-wild-type amino acid at one or more other positions (i.e. other than position 928 or 1104) corresponding to the positions identified in Table 1.

Preferably, the non-wild-type amino acid is one of the preferred mutant options.

The invention also provides a vector or plasmid comprising a TYK2 gene of the invention or comprising a nucleic acid molecule encoding a TYK2 polypeptide of the invention.

In another embodiment, the invention provides a TYK2 polypeptide, wherein the amino acid sequence of the TYK2 polypeptide comprises: (a) an amino acid sequence having 95-99.5% amino acid sequence identity to SEQ ID NO: 1 ; and

(b) the amino acid sequence of the TYK2 polypeptide comprises a non-wild-type amino acid at one or more of the positions corresponding to the positions identified in Table 1 , wherein, if the amino acid sequence of the TYK2 polypeptide comprises a substitution at a position corresponding to position 928 or 1104 in SEQ ID NO: 1 , then the amino acid sequence additionally comprises a non-wild-type amino acid at one or more other positions (i.e. other than position 928 or 1104) corresponding to the positions identified in Table 1.

Preferably, the non-wild-type amino acid is one of the preferred mutants.

Recombinant methods for the production of the nucleic acid molecules, polypeptides and cells are well known in the art (e.g. “Molecular Cloning: A Laboratory Manual” (Fourth Edition), Green, MR and Sambrook, J., (updated 2014)).

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a DNA-targeting polypeptide, or

(ii) a nucleic acid molecule encoding a DNA-targeting polypeptide; under conditions such that, when present, the nucleic acid molecule encoding the DNA- targeting polypeptide is expressed in the cell to produce a DNA-targeting polypeptide, the DNA-targeting polypeptide is targeted to a target nucleotide sequence in the TYK2 gene, and wherein the DNA-targeting polypeptide modifies one or more nucleotides in the TYK2 gene, thereby producing a modified human cell comprising a modified TYK2 gene, wherein either:

(i) the target nucleotide sequence is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide; or (ii) the target nucleotide sequence is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide.

In a preferred embodiment, the invention provides a process for producing a modified human cell comprising a modified TYK2 gene, the process comprising the step:

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a CRISPR polypeptide or a nucleic acid molecule coding therefor, and

Step (a) comprises introducing components (i) and (ii) into a human cell comprising a TYK2 gene. Human cells have been defined above. In diploid cells, the TYK2 gene will be present in two copies in the cell’s chromosomes.

The DNA-targeting polypeptide is a polypeptide which is capable of targeting a target nucleotide sequence in the TYK2 gene and of modifying one or more nucleotides in the TYK2 gene. Examples of DNA-targeting polypeptides include CRISPR polypeptides, TALENs and zinc-finger proteins. As used herein, the term "CRISPR polypeptide" refers to one or more polypeptides which are capable of forming a complex with a CRISPR RNA, e.g. a guide RNA (gRNA), single guide RNA (sgRNA) or prime editor guide RNA (pegRNA). As used herein, the term “prime editor” includes double-prime editor.

In particular, in some embodiments of the invention, the term “CRISPR polypeptide” refers to the use of two or more (e.g. two) CRISPR polypeptides.

When complexed with a CRISPR RNA, the CRISPR polypeptide becomes one which is capable of being targeting to a target nucleotide sequence which has a nucleotide sequence which is complementary to that of the spacer element in the CRISPR RNA.

The DNA-targeting polypeptide may be replaced by a nucleic acid molecule encoding the DNA-targeting polypeptide. Similarly, the CRISPR polypeptide may be replaced by a nucleic acid molecule encoding the CRISPR polypeptide; and the gRNA may be replaced by a nucleic acid molecule encoding the gRNA.

The aforementioned nucleic acid molecules may be provided, for example, in the form of vectors, plasmids or recombinant virus genomes (e.g. adeno-associated viruses).

The aforementioned nucleic acid molecules are expressed in the cells to produce the DNA-targeting polypeptide, the CRISPR polypeptide and the gRNA, as appropriate.

Most preferably, the CRISPR polypeptide is a base editor, a prime editor, a double prime editor, or a Cas9 (e.g. a Cas9 capable of creating double-stranded breaks in target DNA or a dCas9).

As used herein, the term “base editor” refers to an enzyme which is capable of binding to a specific DNA sequence and which can chemically convert nucleotides of one specific type in a DNA molecule to a different specific type (e.g. C to T or A to G, resulting in G to A or T to C on the opposite strand). These usually comprise Cas9 linked to a base editor protein such as the APOBEC or Adenine Base Editor protein, but other programmable nucleic acid binding proteins could be used. In some embodiments, the base editor is a programmable nucleic acid binding protein (e.g. an impaired CRISPR-Cas9 mutant) which is capable of being targeted to a target (DNA) sequence. In some embodiments, the base editor is an enzyme which comprises a catalytically impaired CRISPR-Cas9 mutant which is incapable of making double-strand breaks. Base editors are enzymes that combine programmable nucleic acid binding with an ability to change the nucleic acid bases at the target sequence. To date, base editors have been described that deaminate cytosine resulting in conversion to thymine or deaminate adenine resulting in conversion to guanine. Examples of base editors include cytosine deaminating editors (C:G to T:A), e.g. AID-CRISPR-Cas9 (Nishida et al., 2016); BE3 (Komor et al., 2016); BE4 and BE-Gam (Komor et al., 2017); BE4max (Koblan et al. , 2018); and AncBE4max (Koblan et al. , 2018). Other examples of base editors include adenine deaminating editors (A:T to G:C). Preferably, the base editor is an adenine base editor (ABE). These convert A:T to G:C. Examples of preferred ABEs include Cas9-ABE7.10 ((Gaudelli et al., 2017); US 2018/0073012); xCas9-ABE7.10 (Hu et al., 2018); and ABEmax (Koblan et al., 2018). Preferably, the mutations A928V and D988N are carried out by base-editing.

Prime editing involves three main components:

1 . A prime editing guide RNA (pegRNA), capable of (i) identifying the target nucleotide sequence to be edited, and (ii) encoding new genetic information that replaces the targeted sequence. The pegRNA consists of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.

2. A fusion protein comprising a Cas9 nickase fused to a reverse transcriptase. Preferably, the Cas9 nickase is Cas9 H840A nickase. Preferably, the reverse transcriptase is Moloney murine leukaemia virus (MMLV) reverse transcriptase.

Cas9 H840A nickase: the Cas9 enzyme contains two nuclease domains that can cleave DNA sequences: a RuvC domain that cleaves the non-target strand; and a HNH domain that cleaves the target strand. The introduction of a H840A substitution in Cas9 inactivates the HNH domain. With only the RuvC functioning domain, the catalytically- impaired Cas9 introduces a single strand nick.

M-MLV reverse transcriptase: This synthesizes DNA from the single-stranded RNA template.

3. A single guide RNA (sgRNA) that directs the Cas9 nickase portion of the fusion protein to nick the non-edited DNA strand.

Preferably, the mutation Pro1104Ala is carried out by prime-editing or double-prime editing (www.biorxiv.org/content/10.1101/2021.11.01 466790v1.full.pdf).

In some embodiments, the CRISPR polypeptide has nuclease, preferably endonuclease, activity. In such embodiments, the CRISPR polypeptide may, for example, be a wild-type Cas9 or Cas12a (Cpf1), or a variant or derivative thereof which has endonuclease activity.

Examples of CRISPR polypeptides which may be used in this regard include SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl , LbCpfl , VQR SpCas9, EQR SpCas9, VRER SpCas9, RHA FnCas9 and KKH SaCas9 (see Komor et ai, CRISPR- Based Technologies for the Manipulation of Eukaryotic Genomes, Cell (2017), http://dx.doi.Org/10.1016/j.cell.2016.10.044).

Cas9 is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to reduce or abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a gRNA-programmed manner. Such dCas9 polypeptides, when fused to another polypeptide or polypeptide domain, can target that polypeptide or domain to virtually any DNA sequence simply by co-expression with an appropriate CRISPR RNA.

In other embodiments, therefore, the CRISPR polypeptide is nuclease-deficient, e.g. dCas9. Lack of nuclease activity may be assessed using a Surveyor assay to detect DNA repair events (Pinera et at. Nature Methods (2013) 10(10):973-976). The CRISPR polypeptide is unable to cleave dsDNA but it retains the ability to target and bind the DNA.

In some embodiments, the CRISPR polypeptide is preferably a Cas9, Cas12 or Cas13 polypeptide.

The function of the gRNA is to target the prime editor, base editor or other CRISPR polypeptide to the desired nucleotide sequence of the TYK2 gene. The gRNA is therefore one which is capable of binding to a cognate base editor, prime editor or other CRISPR polypeptide, as appropriate. In one embodiment, a gRNA is a chimeric RNA which is formed from a crRNA and a tracrRNA such as those which have been used in CRISPR/Cas systems (Jinek et al., 2012). The term gRNA is well accepted in the art.

In some embodiments, wherein the base editor or prime editor or other CRISPR polypeptide comprises Cas9 or an analogue or a variant thereof, the gRNA is a RNA which is capable of binding to Cas9, or to analogues or variants thereof.

The gRNA is generally made up of the ribonucleotides A, G, T and U. Modified ribonucleotides, deoxyribonucleotides, other synthetic bases and synthetic backbone linkages (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), etc.) may also be used.

The gRNA comprises a targeting RNA sequence. The targeting sequence has a degree of sequence identity with the region of DNA in the TYK2 gene which includes the target nucleic acid sequence.

The gRNA may be a prime editing guide RNA (pegRNA), i.e. one which is capable of identifying the target site and providing a template DNA to facilitate the replacement of the target DNA. The modified nucleotides may be located within the target nucleotide sequence or outside the target nucleotide sequence (e.g. in the vicinity of the target nucleotide sequence). In some embodiments (e.g. processes involving the use of HDR), more than one gRNA may be used. For example, 1 , 2, 3, or 4 (preferably 2) gRNAs, or nucleic acids coding therefor, may be introduced into the human cell.

Preferably, the degree of sequence identity between the targeting RNA sequence and the target nucleotide sequence is at least 80%, more preferably at least 90%, 95%, 99% or 100%.

Preferably, the targeting RNA sequence is 14-30 nucleotides, more preferably 20-30 nucleotides in length.

In some embodiments of the invention, the nucleotide sequence of the PAM site in the target DNA is one which has been modified compared to the wild-type PAM nucleotide sequence in order to increase efficiency of the base-editing process. Such a modification is one which does not affect the function of the TYK2 polypeptide.

Any non-synonymous mutation is preferably produced by base editing or prime editing.

With regard to the editing of the regulatory regions of the TYK2 gene, specific nucleotide changes are less important (than for the non-synonymous mutations). In this case, the mutations may be selected from insertions, deletions, substitutions of one or more nucleotides. Consequently, any suitable method may be used including conventional Cas9 (or other programmable nucleases such as TALEN, Zn finger etc.), base editors or prime editors.

Genome-editing methods are well known in the art (e.g. Cong et al., 2013 Science Vol. 339, Issue 6121 , pp. 819-823; DOI: 10.1126/science.1231143; Komor et al., 2016 Nature volume 533, pages 420-424; Gaudelli et al., 2017, Nature volume 551 , pages 464-471 ; Gilbert et al., 2013 Volume 154, Issue 2, Pages 442-451).

In some embodiments, the term “under conditions such that” means a Step (b) of “culturing the human cell or cells under conditions such that”. Homology-directed repair (HDR) methods may also be used to introduce desired changes (modifications) in the TYK2 gene. Such methods might generally not be considered to be sufficiently efficient to edit all of the TYK2 genes in a population of cells. However, the double-stranded cuts which are produced but not repaired by Cas9 in an HDR method will be repaired by non-homologous end joining (NHEJ) methods. This leads to the production of a mixture of edited (HDR) and non-functional (NHEJ) TYK2 alleles in the population of cells, as well as some non-edited TYK2 genes.

Overall, the result is a population of cells with a phenotype which is similar to that obtained in a population of cells with (fully) edited TYK2 genes, and hence a phenotype which is usable to treat IMDs.

In some embodiments, therefore, the CRISPR polypeptide is one which has nuclease, preferably endonuclease, activity. In such embodiments, the CRISPR polypeptide may, for example, be a wild-type Cas9 or Cas12a (Cpf1), or a variant or derivative thereof which has endonuclease activity.

In such embodiments, two gRNAs are introduced into the human cell, targeting CRISPR polypeptides to nucleotide sequences which are upstream and downstream, respectively, of the target nucleotide sequence in the TYK2 gene; and thus promoting the production by the CRISPR polypeptide of double-stranded breaks upstream and downstream of the target nucleotide sequence in the TYK2 gene.

Thus Step (a)(ii) relates to introducing two or more gRNAs, wherein the gRNAs are ones which are capable of targeting CRISPR polypeptides to two or more nucleotide sequences in the TYK2 gene which flank the target nucleotide sequence, or one or more nucleic acids coding therefor.

In such embodiments, Step (a) additionally comprises the step of introducing into the human cell comprising a TYK2 gene or population of such cells: (iii) a double-stranded donor DNA. The donor DNA comprises a modified fragment of a TYK2 gene, wherein the modification is in the target nucleotide sequence, i.e. the nucleotide sequence of the donor DNA differs from that the corresponding sequence of the cellular TYK2 gene at the target nucleotide sequence, at least.

The donor DNA comprises a nucleotide sequence of the TYK2 gene which spans the desired target nucleotide sequence in the TYK2 gene. The nucleotide sequence of the donor DNA differs from the nucleotide sequence of the TYK2 gene at positions corresponding to the one or more nucleotides in the cellular TYK2 gene which are to be modified.

The nucleotide sequence of the donor DNA will be such that it promotes homologous recombination (double cross-over) between the donor DNA and the TYK2 gene in the human cell genome under HDR conditions. This results in the replacement of the target nucleotide sequence in the TYK2 gene with donor DNA by HDR, thereby producing a modified human cell comprising a modified TYK2 gene or a population of such cells.

In particular, the 5’ and 3’ arms of the donor DNA will be such that they promote homologous recombination (double cross-over) by HDR between those arms and corresponding regions of the TYK2 gene in the human cell genome which flank the target nucleotide sequence.

The appropriate lengths of the donor DNAs and the level of sequence identity between the arms of the donor DNA and the cellularTYK^ gene can readily be established by methods which are well known in the art.

In particular, the invention provides a process for producing a population of cells comprising human cells with modified (i.e. non-wild-type) TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

(i) CRISPR polypeptides with nuclease activity, or a nucleic acid molecule coding therefor, and (ii) two or more gRNAs, wherein the gRNAs are ones which are capable of targeting the CRISPR polypeptides to nucleotide sequences in the TYK2 gene which flank a target nucleotide sequence, or one or more nucleic acids coding therefor; and

(iii) a donor DNA comprising a modified fragment of a TYK2 gene; and (b) culturing the population of human cells under conditions such that, when present, the nucleic acid molecule coding for the CRISPR polypeptide is expressed in the cells to produce the CRISPR polypeptides, when present, the nucleic acid molecule(s) coding for the gRNA is expressed in the cells to produce the gRNAs,

CRISPR polypeptides are targeted by the gRNAs to make double-stranded breaks in the TYK2 gene either side of the target nucleotide sequence, and wherein the target nucleotide sequence in the TYK2 gene is replaced by HDR with the nucleotide sequence of the donor DNA, thereby producing a population of cells comprising human cells with modified TYK2 genes, wherein either:

In this embodiment, the resultant population of cells may comprise or consist of:

(i) cells comprising TYK2 genes having the nucleotide sequence of the donor DNA in the target nucleotide sequence (i.e. cells which have been modified by HDR);

(ii) cells comprising an indel (insertion or deletion) in the TYK2 gene (i.e. cells which have been modified by NHEJ); and

(iii) cells comprising an unmodified, i.e. cellular, wt, TYK2 gene (i.e. cells which were not modified by HDR or NHEJ).

The invention also provides a population of cells, as defined above. A yet further embodiment of the invention relates to combining the use of a base editor or a prime editor, as described above, together with a CRISPR polypeptide having nuclease activity (e.g. Cas9). In such embodiments, the base editor or prime editor will make a desired edit in the TYK2 gene. Unedited TYK2 genes may be cut by the CRISPR polypeptide having nuclease activity (e.g. Cas9), and repaired by a NHEJ mechanism, resulting in non-functional TYK2 genes. The overall phenotype of a population of such cells would be a reduction in TYK2 polypeptide expression.

The invention there also provides a process of the invention, as defined herein, wherein the CRISPR polypeptides which are introduced into the cells (or nucleic acid(s) coding therefor) are a base editor or a prime editor, together with a CRISPR polypeptide having nuclease activity (e.g. a Cas9).

In some preferred embodiments, the CRISPR polypeptides are a base editor and a CRISPR polypeptide having nuclease activity (e.g. a Cas9). In other embodiments, the CRISPR polypeptides are a prime editor and a CRISPR polypeptide having nuclease activity (e.g. a Cas9).

The CRISPR polypeptide having nuclease activity, and the base editor or the prime editor may target the same or different target nucleotide sequences. In particular, the target specificity (within the TYK2 gene) of the CRISPR polypeptide having nuclease activity will be less constrained if the intention of this polypeptide is merely to knockout the function of the TYK2 gene.

In particular, the invention provides a process for producing a population of cells comprising human cells with modified TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

(i) a CRISPR polypeptide having nuclease activity (e.g. Cas9), and a base editor polypeptide, or nucleic acid molecules encoding one or both polypeptides; and

(ii) one or more gRNAs, wherein the gRNAs are ones which are capable of targeting the CRISPR polypeptide and/or the base editor to target nucleotide sequences in the TYK2 gene, or one or more nucleic acids coding therefor;

(b) culturing the population of human cells under conditions such that, when present, the nucleic acid molecules coding for the CRISPR polypeptide and base editor are expressed in the cells to produce the CRISPR polypeptides and base editor polypeptides, when present, the nucleic acid molecule(s) coding for the gRNAs are expressed in the cells to produce the gRNAs, the CRISPR polypeptides are targeted by one or more of the gRNAs to make double- stranded breaks in the target nucleotide sequences in the TYK2 genes in a first portion of the population of cells, and the base editor polypeptides are targeted by one or more of the gRNAs to make base edits in the target nucleotide sequences in the TYK2 genes in a second portion of the population of cells, thereby producing a population of cells comprising human cells with modified TYK2 genes, wherein either:

(i) cells comprising TYK2 genes which have been base edited by the base editor polypeptide;

(iii) cells comprising an unmodified, i.e. cellular, wt, TYK2 gene (i.e. cells which were not modified by CRISPR polypeptide having nuclease activity or base editor polypeptide).

The invention also provides a population of cells, as defined above. In particular, the invention provides a process for producing a population of cells comprising human cells with modified TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

(i) a CRISPR polypeptide having nuclease activity (e.g. Cas9), and a prime editor polypeptide, or nucleic acid molecules coding one or both polypeptides; and

(ii) one or more gRNAs (e.g. a gRNA and a pegRNA), wherein the gRNAs are ones which are capable of targeting the CRISPR polypeptide and/or the prime editor to target nucleotide sequences in the TYK2 gene, or one or more nucleic acids coding therefor;

(b) culturing the population of human cells under conditions such that, when present, the nucleic acid molecules coding for the CRISPR polypeptide and prime editor are expressed in the cells to produce the CRISPR polypeptides and the prime editor polypeptides, when present, the nucleic acid molecule(s) coding for the gRNAs are expressed in the cells to produce the gRNAs, the CRISPR polypeptides are targeted by one or more of the gRNAs to make double- stranded breaks in the target nucleotide sequences in the TYK2 genes in a first portion of the population of cells, and the prime editor polypeptides are targeted by one or more of the gRNAs to make prime edits in the target nucleotide sequences in the TYK2 genes in a second portion of the population of cells, thereby producing a population of cells comprising human cells with modified TYK2 genes, wherein either:

(i) the target nucleotide sequence(s) is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide; or

(ii) the target nucleotide sequence(s) is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide.

In this embodiment, the resultant population of cells may comprise or consist of: (i) cells comprising TYK2 genes which have been prime edited by the prime editor polypeptide;

(iii) cells comprising an unmodified, i.e. cellular, TYK2 gene (i.e. cells which were not modified by CRISPR polypeptide having nuclease activity or prime editor polypeptide).

The invention also provides a population of cells, as defined above.

There are several forms in which the DNA-targeting polypeptide and nucleic acid molecules can be delivered into human cells, e.g. by: a) DNA (e.g. in plasmid form) b) mRNA (e.g. the polypeptide and/or guide RNA) c) Protein-gRNA complex (e.g. CRISPR polypeptide/gRNA complex) d) Viral vectors (e.g. AAVs).

The DNA-targeting polypeptide and nucleic acid molecules may be introduced into the human cells using, for example, one or more of the following methods: electroporation, lipofection, viral transduction or nanoparticles.

Suitable conditions for genome modifications are readily known in the art (e.g. (Gaudelli et al., 2017). In particular, procedures which are used for CRISPR/Cas9 (e.g. Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery, 2018, Ed. Krishnarao Appasani, Cambridge University Press; and references therein) may be adapted for use in the processes disclosed herein.

This process may be done in any suitable vessel, e.g. test tube, Eppendorf tube, tissue culture flask, etc.

In other embodiments of the invention, the process additionally comprises, prior to Step (a), the step of modifying the nucleotide sequences of one or more PAM sites in the vicinity of the target nucleotide sequence in order to increase the efficiency of the modifying process. In most embodiments of the invention, the process will not be carried out on a single nucleic acid molecule or single gene; in general, the process will be applied to a population of nucleic acid molecules or genes, which may be present within a population of cells. The population of cells may be homogeneous or heterogeneous.

Within this population of nucleic acid molecules/cells, depending on the codon in question and the targeted nucleotide sequence, the DNA-targeting polypeptide (e.g. CRISPR polypeptide) may modify different numbers of nucleotides within a single codon. In particular, due to the processivity of base editors, occurrences of 2 or 3 relevant nucleotides within the codon are more likely all to be edited.

In particular, there is provided a process of the invention wherein the process is applied to a population of nucleic acid molecules, TYK2 genes, vectors or cells and wherein at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the target nucleic acid molecules in the population of nucleic acid molecules, vectors or cells have been modified.

The processes of the invention may be carried out ex vivo, in vitro or in vivo.

In one embodiment, the process for producing a modified human cell is carried out in vitro. In this embodiment, the DNA-targeting polypeptide (e.g. CRISPR polypeptide or base editor) may be introduced into the human cells in the form of mRNA or as a polypeptide. This introduction may be done by electroporation, for example; nanoparticles or recombinant viral vectors may also be used.

Prior to use, the modified cells may be cultured and/or clonally expanded in order to increase the number of cells.

The cells may also be tested, e.g. prior to use in therapy, for one or more of the following: (i) the presence of the desired modification; (ii) to ascertain the extent of any off-target (undesired) modifications; and (iii) to ascertain the presence of any undesired DNA templates or viral vector DNA.

The reduced level of TYK2 kinase activity and the reduced level of expressible TYK2 polypeptide are levels which are reduced compared to the corresponding levels obtainable from a wild-type TYK2 polypeptide (e.g. SEQ ID NO: 1) in a control cell, under controlled conditions.

In one embodiment, the modified human cells of the invention are for use and/or are suitable for use in transplantation into a human subject in order to treat an IMD.

The processes of the invention may also be carried out in vivo.

With regard to such uses, the chosen genome editing strategy for IMDs should ideally deliver the following: it should reduce the risk for de novo disease relapse and secondary IMD development; it should not lead to increased risk for other unmanageable diseases; and for maximum efficiency and minimum manipulation, there should be a single target in the genome.

In yet a further embodiment, therefore, the invention provides a method of treating an immune-mediated disease (IMD) in a human subject, the method comprising administering an effective amount of a composition comprising modified human cells of the invention to a subject in need thereof.

The invention also provides a modified cell of the invention or a composition of the invention for use in treating an IMD. The invention also provides the use of a modified cell of the invention in the manufacture of a medicament for treating an IMD.

Autologous cell transplantation (particularly of haematopoietic cells) enables therapeutic "rebooting" of the human subject’s immune system.

Preferably, the method of treatment comprises the steps: (a) obtaining cells from a human subject, wherein the human subject is one who is suffering from an IMD;

(b) modifying, ex vivo, at least one allele of the TYK2 gene in the genome of the cells to produce modified cells of the invention; and

(c) transplanting the modified cells into the human subject.

Preferably, modifying Step (b) is carried out using a process of the invention.

Preferably, the cells are haematopoietic cells, more preferably, haematopoietic stem cells.

The invention may also be carried out using allogeneic transplantation. In another embodiment, the method of treating a human subject suffering from an IMD, the method comprising the steps:

(a) obtaining cells from a first human subject, wherein the human subject is one who is not suffering from an IMD;

(c) transplanting the modified cells into a second human subject, wherein the second human subject is one who is suffering from an IMD.

Preferably, the first and second subjects are related subjects (e.g. wherein the first subject is a twin, sibling, parent, grandparent or first cousin of the second subject or vice versa).

In the transplanting steps, all or a portion of the modified cells may be transplanted.

Preferably, modifying Step (b) is carried out using a process of the invention. Preferably, the cells are haematopoietic cells, more preferably, the cells are haematopoietic stem cells.

Preferably, prior to transplantation Step (c), the subject who is to receive the transplant is treated with chemotherapy or a combination of chemotherapy and radiation therapy in order to ablate immune cells (including those pathogenic immune cells that contribute to disease). For example, the subject may be treated with cyclophosphamide (e.g. about 200mg/kg) and anti-thymocyte globulin.

The process of the invention may also be implemented in vivo, e.g. using viral vectors (e.g. adeno-associated virus (AAV) vectors) to introduce the necessary TYK2 gene modifying components into cells.

In yet another embodiment, therefore, the invention provides a method of treating a human subject suffering from an IMD, the method comprising the step:

(A) performing a process for producing a modified human cell of the invention, wherein in Step (a) of the process, a nucleic acid molecule encoding the DNA-targeting polypeptide or CRISPR polypeptide (and optionally a nucleic acid molecule encoding a gRNA) are encoded within a recombinant viral vector, and the recombinant viral vector is introduced into the subject. Preferably, the cells are human monocytes. Preferably, the recombinant viral vector is a recombinant AAV.

Preferably the immune-mediated disease (IMD) is selected from the group consisting of Addison’s disease, alopecia, ankylosing spondylitis, Crohn’s disease, Graves’ disease, hypothyroidism, juvenile idiopathic arthritis, multiple sclerosis, pernicious anaemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, and vitiligo. In some embodiments, the IMD is COVID-19. In some embodiments, the IMD is not rheumatoid arthritis.

The modified cells of the invention may also be used in other cases where immune cells are transferred into patients and it is desired to reduce the likelihood of IMD development in the patients without affecting the capacity to fight off infection and malignancy. In yet another embodiment, therefore, the invention provides a method of preventing or reducing the risk of graft versus host disease, the method comprising administering an effective amount of modified cells of the invention to a patient in need thereof.

Also provided is a method of allogeneic transplantation, the method comprising administering an effective amount of modified cells of the invention to a patient in need thereof.

As genetic variation that correlates with increased TYK2 expression has been described to be associated with the risk of life-threatening COVID-19 (Pairo-Castineira et al.,

2020, Nature PM ID: 33307546), the invention also provides a method for reducing risk for this disease.

The invention also provides a modified cell obtained or obtainable by a process of the invention.

Preferably, the process and method steps of the invention are carried out in the order specified.

The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the levels of kinase activity different TYK2 variants in HEK293 cells after stimulation with Type I interferon.

Figure 2 shows the codon modifications needed to produce the variants A928V and D988N. (Figure 2 includes SEQ ID NOs: 4-5.)

Figure 3 shows immune cell regulatory regions in the vicinity of the TYK2 gene that may modify gene expression.

Figure 4A shows A928V base editing in the human Jurkat cell line. (Figure 4A includes SEQ ID NOs: 6-10.) Figure 4B shows A928V base editing in primary human T-cells. (Figure 4B includes SEQ ID NOs: 11-14.)

Figure 4C shows A928V orthologue (mouse A925V) base editing in the mouse BAF-3 cell line. (Figure 4C includes SEQ ID NOs: 15-17.)

Figure 5A shows R987Q-D988N base editing (with the BE4-max-NG editor) in the human Jurkat T cell line. (Figure 5A includes SEQ ID NOs: 18-22.)

Figure 5B shows R987Q-D988N base editing in primary human T cells. (Figure 5B includes SEQ ID NOs: 23-27.)

Figure 6 shows P1104A HDR in the human Jurkat cell line. (Figure 6 includes SEQ ID NOs: 28-29.)

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 : Editing of TYK2 with base editors

Production of the missense variants, such as A928V is performed by base editing. The base editing requires a Cas nickase or Cas fused to a deaminase that makes the edit, a gRNA targeting Cas to a specific locus, and a target base for editing within the editing window specified by the Cas protein. The A928V edit, for example, is performed using C-G to T-A editors (CBEs), as this edit involves converting cytosine to thymine (which only requires deamination of the base), and this is done using a canonical spCas9 NGG PAM sequence. Targeted oligonucleotide capture and high-throughput sequencing are then used to interrogate the sites of the required modification in cells that have undergone editing, and editing efficiency is estimated by sequencing. Off-target effects of gRNAs are profiled by in silico prediction using Cas-OFFinder, and experimental approaches, such as the use of CIRCLE-seq or CHANGE-seq, are used to define off- target sites comprehensively. RNA-seq is used to monitor for RNA editing effects of the base editor proteins.

Example 2: Assaying levels of TYK2 kinase activity

TYK2 kinase activity was assayed upon stimulation of transfected HEK-293 cells with interferon beta. The transfected cells expressed different TYK2 protein variants (or the non-variant protein). These variants included P1104A, A928V, and other variants that based on our structural investigations were predicted to alter kinase activity without compromising protein stability. The impact of these protein-coding variants on TYK2 function was assayed by measuring TYK2 phosphorylation as a direct readout of TYK2 kinase activity.

The results are shown in Figure 1. This data demonstrates that there was TYK2 expression and function in the transfected cells, i.e. that the protein-coding variants are likely to function by having an altered kinase activity and that variants with a very low pTYK2/TYK2 ratio do not just have very low TYK2 protein level expression. The A928V and D988N variants displayed a level of relative TYK2 phosphorylation that is most similar to that of P1104A.

Example 3: Identification of active TYK2 promoter and enhancer sequences

Regulatory regions within the vicinity of the TYK2 gene, including its promoter, were identified by DNase hypersensitivity assays, ATAC-Seq, and ChIP-seq. The regions that regulate TYK2 expression, as opposed to the expression of other genes in the genomic region, were further defined by NG Capture C, as this enabled the identification of the regions that interact with the TYK2 promoter. In silico interrogation of these regions, along with experimental validation, then allowed for (i) the determination of the precise DNA sites that specific transcription factors and other regulatory proteins bind to, and (ii) the determination of which modifications of these DNA sites would perturb the binding of regulatory proteins, thereby altering TYK2 gene expression. Figure 3 shows a schematic diagram showing regulatory regions (shown as peaks at the bottom of the Figure) in the vicinity of the TYK2 gene. NG Capture C provided information relating to which of these regions designated by the peaks interacts with the TYK2 promoter, and thus regulates the expression of the gene

Example 4: Editing of promoter and other regulatory sequences

Editing of specific regulatory sequences is performed by base editing, or other approaches. The base editing requires a Cas nickase or Cas fused to a deaminase that makes the edit, a gRNA targeting Cas to a specific locus, and a target base for editing within the editing window specified by the Cas protein. C-G to T-A editors (CBEs) or A-T to G-C base editors (ABEs) are used depending on the exact sequence to be modified. Targeted oligonucleotide capture and high-throughput sequencing are then used to interrogate the sites of the required modification in cells that have undergone editing, and editing efficiency is estimated by sequencing. Off-target effects of gRNAs are profiled by in silico prediction using Cas-OFFinder, and experimental approaches, such as the use of CIRCLE-seq or CHANGE-seq, are used to define off-target sites comprehensively.

Example 5: Demonstration of editing capacity and efficiency

Base editing of TYK2 A928V and of the murine orthologue TYK2 A925V was performed. Figure 4A shows that A928V base editing in the human Jurkat T cell line was performed and that this had an -70% efficiency. Figure 4B shows that A928V editing in primary human T cells was performed with an -50% efficiency. Figure 4C shows that the murine orthologue A925V was edited, with an -30% efficiency in the mouse BAF-3 cell line.

Example 6: Base editing of TYK2 R987Q-D988N

The double mutant R987Q-D988N was produced in human cells. Figure 5A shows R987Q-D988N base editing (with the BE4-max-NG editor) in the human Jurkat T cell line has an -80% efficiency. Figure 5B shows R987Q-D988N base editing in primary human T cells can be performed with an -50% efficiency. Example 7: Editing the P1104A variant by HDR and NHEJ

The TYK2 variant P1104A variant is produced in human cells using an established homology directed repair (HDR) mechanism, using Cas9 together with a suitable donor DNA template. Whilst this HDR mechanism is inefficient, when the donor DNA is not inserted into the cellular genome by HDR, the Cas9 makes a deletion in the target sequence by non-homologous end joining (NHEJ) which leads to a non-functioning TYK2 allele. This results in two populations of alleles: those in which TYK2 gene has been deleted or rendered non-functional; and those which have the P1104A protective genotype.

To outline the idea numerically, if there is 30% homology directed repair, 65% of the remaining cells will have a deletion (the overall Cas9 cutting efficiency is often in the order of 95%) and 5% of alleles will be unedited. This would lead to 9% homozygous P1104A, 40% heterozygous deletion / P1104A, 42% homozygous deletion, 3% heterozygous WT/deletion and <1% WT (assuming both alleles are edited independently). This would essentially lead to a P1104A-like phenotype, since nearly all of the remaining cells will be inert.

In order to illustrate this concept, P1104A editing was performed by CRISPR/Cas9 homology directed repair (HDR) in human Jurkat T cells. This had -80% efficiency, as shown in Figure 6.

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Diogo et al., 2015, PLoS One 10(e0122271)

SEQUENCES

The Sequence Listing filed with this patent application is fully incorporated herein as part of the description.

SEQ ID NO: 1

TYK2 amino acid, Human

SEQ ID NO: 2

TYK2 nucleotide (cDNA), human

> GRCh38.p13_ ENST00000525621.6

SEQ ID NO: 3

TYK2 nucleotide (cDNA), human

> GRCh38.p13_ ENST00000264818.10

Sequence Listing Free Text

<210> 9 <223> A928V sequence (Fig 4A)

<210> 10 <223> Negative control (Fig4A)

<210> 14 <223> Fig 4B

<210> 21 <223> R987Q-D988N <210> 26 <223> Fig 5B

<210> 27 <223> Fig 5B

<210> 29 <223> Negative control - Fig 6

Claims

1 . A process for producing a modified human cell comprising a modified TYK2 gene, the process comprising the step:

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a DNA-targeting polypeptide, or

2. A process as claimed in claim 1 , the process comprising the step:

(a) introducing, into a human cell comprising a TYK2 gene,

(i) a CRISPR polypeptide or a nucleic acid molecule coding therefor, and

3. A process for producing a population of cells comprising human cells with modified (i.e. non-wild-type) TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

(i) CRISPR polypeptides with nuclease activity, or a nucleic acid molecule coding therefor, and

(ii) two or more gRNAs, wherein the gRNAs are ones which are capable of targeting the CRISPR polypeptides to nucleotide sequences in the TYK2 gene which flank a target nucleotide sequence, or one or more nucleic acids coding therefor; and

(iii) a donor DNA comprising a modified fragment of a TYK2 gene; and

(b) culturing the population of human cells under conditions such that, when present, the nucleic acid molecule coding for the CRISPR polypeptide is expressed in the cells to produce the CRISPR polypeptides, when present, the nucleic acid molecule(s) coding for the gRNA is expressed in the cells to produce the gRNAs,

4. A process as claimed in claim 2, wherein the CRISPR polypeptide is a base editor, a prime editor or a double prime editor.

5. A process for producing a population of cells comprising human cells with modified TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

6. A process for producing a population of cells comprising human cells with modified TYK2 genes, the process comprising the steps:

(a) introducing, into a population of human cells each comprising a TYK2 gene,

7. A process as claimed in any one of the preceding claims, wherein the DNA- targeting polypeptide or the CRISPR polypeptide, and optionally the gRNA, are encoded within a recombinant viral vector, preferably an AAV.

8. A modified human cell, wherein at least one allele of the TYK2 gene in the genome of the cell has a modification which reduces pro-inflammatory cytokine signalling in the cell, or population of such cells.

9. A modified human cell or population of cells as claimed in claim 8, wherein:

(i) the modification is in the coding region of the TYK2 gene, and the modification reduces the level of TYK2 kinase activity obtainable from the encoded TYK2 polypeptide in the cell; or

(ii) the modification is in a regulatory region of the TYK2 gene, and the modification reduces the expressible level of TYK2 polypeptide in the cell.

10. A process or modified human cell as claimed in any one of the preceding claims, wherein the human cell is:

(i) a haematopoietic cell, preferably a haematopoietic stem cell or leukocyte; or

(ii) a stem cell, preferably an induced pluripotent stem cell.

11. A process or modified human cell or population of cells as claimed in any one of the preceding claims, wherein the mutation is a substitution in the coding region of the TYK2 gene corresponding to a substitution selected from the group consisting of Val362Phe, Ne684Ser, Ne684Thr, Asn920Ser, Gly922Ala, Ala931Asp, Ala931Val, Cys936Arg, His940Gln, Arg941 Leu, Gly943Val, Trp944Leu, Lys945Arg, Glu947Lys, Ne948Thr, Asp949Ala, Ne950Ser, Leu951Gln, Thr953Met, His956Arg, His956Gln, Glu957Lys, Tyr962Ser, Lys963Arg, Lys963Met, Gly964Ala, Cys966Gly, Cys966Tyr, Gln969Glu, Glu979Lys, Tyr980Asn, Asp988Val, Tyr989Cys, Arg992Trp, His993Tyr, His993Gln, Ser994Asn, Ne995Val, Gly996Arg, Gln999Glu, Leu1001 Met, Leu1002Phe, Ala1004Gly, Gln1005Arg, Gln1005Leu, Ne1007Met, His1015Gln, Ala1016Ser,

His1018Gln, Ala1026Thr, Asn1028Ser, Asn1033Asp, Arg1035Ser, Arg1035Ser, Lys1038Asn, Pro1049Leu, Glu1050Lys, Glu1050Ala, Glu1050Asp, Pro1064Arg, Tyr1076Cys, Lys1077Gln, Tyr1080His, Tyr1080Cys, Phe1087Cys, Gly1088Arg,

Tyr1092Phe, His1097Tyr, Ser1100Cys, Ser1103Gly, Ser1103Asn, Seri 103Thr and Prd 104Val; or selected from the group consisting of R987Q, Lys930Arg, Tyr1054His, Tyr1055Cys, Tyr1055His and Tyr1055Cys.

12. A process or modified human cell or population of cells as claimed in any one of the preceding claims, wherein the mutation is a substitution in the coding region of the TYK2 gene at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of:

Val911 , preferably Val911 lie Ala928, preferably Ala928Val Lys930, preferably Lys930Arg Asp988, preferably Asp988Asn Asp1041 , preferably Asp1041Asn Pro1104, preferably Pro1104Ala, Pro1104Ser or Pro1104Val.

13. A process or modified human cell or population of cells as claimed in claim 12, wherein the mutation is a substitution in the coding region of the TYK2 gene corresponding to a substitution in SEQ ID NO: 1 selected from the group consisting of Pro1104Ala, Ala928Val or Asp988Asn, preferably Ala928Val.

14. A process or modified human cell or population of cells as claimed in any one of the preceding claims, wherein the mutation is in a regulatory region of the TYK2 gene, preferably wherein the regulatory region is selected from the group consisting of a promoter, an enhancer or a repressor.

15. A process or modified human cell or population of cells as claimed in claim 14, wherein the regulatory region of the TYK2 gene is a region selected from the group consisting of chr19: 10,484,800-10,487,800; chr19: 10,489,994-10,492,729; chr19: 10,499,400-10,500, 150; chr19: 10,500,420-10,501 , 170; chr19: 10,514,530-

10,515,530; chr19: 10,515,830-10,516,830; chr19: 10,518,990-10,519,990; chr19: 10,522,630-10,523,630; chr19: 10,534,500-10,536,500; chr19: 10,538,530-

10,539,530; chr19: 10,542,319-10,543,320; chr19: 10,546,780-10,547,780; chr19: 10,571 ,500-10,572,500; chr19: 10,582,550-10,583,050; and chr19: 10,589,320-

10,590,320.

16. A pharmaceutical composition comprising one or more modified human cells or population of cells as defined in any one of claims 8-15, optionally together with one or more adjuvants, carriers or diluents.

17. A TYK2 gene, wherein the nucleotide sequence of the TYK2 gene comprises:

(ii) a nucleotide sequence having 90-99.9% nucleotide sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3; and wherein (b) the nucleotide sequence of the TYK2 gene encodes an amino acid sequence comprising a non-wild-type amino acid (preferably, the preferred mutant amino acid) at one or more positions corresponding to the positions identified in claim 11 or claim 12, wherein, if the nucleotide sequence of the TYK2 gene encodes an amino acid sequence comprising a substitution at a position corresponding to position 928 or 1104 in SEQ ID NO: 1 , then the amino acid sequence additionally comprises a non-wild-type amino acid (preferably, the preferred mutant amino acid) at one or more other positions corresponding to the positions identified in claim 11 or claim 12.

18. A TYK2 polypeptide, wherein the amino acid sequence of the TYK2 polypeptide comprises:

(a) an amino acid sequence having 95-99.5% amino acid sequence identity to SEQ ID NO: 1 ; and

(b) the amino acid sequence of the TYK2 polypeptide comprises a non-wild-type amino acid (preferably, the preferred mutant amino acid) at one or more of the positions corresponding to the positions identified claim 11 or claim 12, wherein, if the amino acid sequence of the TYK2 polypeptide comprises a substitution at a position corresponding to position 928 or 1104 in SEQ ID NO: 1 , then the amino acid sequence additionally comprises a non-wild-type amino acid (preferably, the preferred mutant amino acid) at one or more other positions (i.e. other than position 928 or 1104) corresponding to the positions identified in claim 11 or claim 12.

19. A modified human cell or population of cells as claimed in any one of claims 8-15 or a composition as claimed in claim 16, for use in treating an immune-mediated disease (IMD).

20. A method of treating an immune-mediated disease (IMD) in a human subject, the method comprising administering an effective amount of modified human cells or a population of cells as claimed in any one of claims 8-15, to a subject in need thereof.

21. A method of treating as claimed in claim 20, wherein the treatment comprises the steps of:

(a) obtaining human cells from the human subject, wherein the human subject is one who is suffering from an IMD;

(b) modifying, ex vivo, at least one allele of the TYK2 gene in the genome of the cells to produce the modified human cells;

(c) treating the subject with chemotherapy and/or radiation therapy; and

(d) transplanting the modified human cells into the human subject.

22. A method of treating as claimed in claim 20, wherein the treatment comprises the steps of:

(c) treating the subject with chemotherapy and/or radiation therapy; and

(d) transplanting the modified human cells into a second human subject, wherein the second human subject is one who is suffering from an IMD.

23. Use of a modified human cell or population of cells as claimed in any one of claims 8-15 in the manufacture of a medicament for treating an immune-mediated disease (IMD).

24. A modified human cell or population of cells for use as claimed in claim 19, a method of treating as claimed in any one of claims 20-22 or a use as claimed in claim 23, wherein the IMD is selected from the group consisting of Addison’s disease, alopecia, ankylosing spondylitis, Crohn’s disease, Graves’ disease, hypothyroidism, juvenile idiopathic arthritis, multiple sclerosis, pernicious anaemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo and COVID-19, preferably multiple sclerosis or scleroderma.

25. A method of preventing or reducing the risk of graft versus host disease, the method comprising administering an effective amount of modified human cells or population of cells as claimed in any one of claims 8-15 to a subject in need thereof.