HLA class I MHC cell excision

Abstract

This invention provides compositions and methods for reducing the immunogenicity of cells for transplantation, including cell-based immunotherapy. [Solution] A vector encoding β2-microglobulin (B2M) modified RNA, along with a targeting moiety and other signaling and / or suicide genes, enables the efficient generation of engineered CAR T regulatory cells or other therapeutic cells derived from any source. The present invention provides, for example, vectors encoding β2-microglobulin (B2M) modified RNA and chimeric antigen receptors (CARs).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 014,344, filed on 23 April 2020 (which is incorporated herein by reference in its entirety).

[0002] Sequence List This application was filed via EFS-Web and includes a sequence listing having 11 sequences, which is incorporated herein by reference in its entirety. The above ASCII copy (created April 21, 2021) is named 48216WO_CRF_sequencelisting.txt and is 6,287 bytes in size.

[0003] Field of Invention The present invention provides compositions and methods for the cellular excision of human leukocyte antigens encoding Class I major histocompatibility complex proteins. [Background technology]

[0004] background The emergence of cell-based treatments, including stem cell transplantation and various immunotherapies, has shown remarkable potential in the treatment of diseases, including cancer. In particular, engineered T cells expressing chimeric antigen receptors (CAR-T cells) have come into the spotlight as a new weapon in targeted immunotherapy. Furthermore, as discussed in Patent Application Publication WO 2019 / 190879 (incorporated herein by reference), other immune cells, including T regulatory lymphocytes (Tregs), can be engineered to express chimeric antigen receptors and used to modulate immune responses and inflammation in autoimmune and inflammatory diseases.

[0005] These treatments carry risks associated with their own hurdles and the immunogenicity of the transplanted cells. While the risk of an immune response to the intended treatment is generally problematic, eliciting a further immune response is particularly concerning when the treatment objective is immunosuppression, as is the case with the aforementioned engineered Tregs. Existing methods to mitigate immunogenicity include the use of autologous cells taken from the patient to be treated. However, such methods pose problems associated with treatment delays and hinder large-scale production. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] International Publication No. 2019 / 190879 [Overview of the project] [Means for solving the problem]

[0007] Abstract The compositions and methods of the present invention use β2-microglobulin (B2M) modified RNA to reduce HLA class I MHC expression in various cells. By reducing class I MHC expression, cells are less likely to be recognized as foreign substances or to induce unwanted immune responses.

[0008] Vectors encoding B2M-modified RNA can be used to transduce cells such as Treg cells to express B2M-modified RNA. B2M-modified RNA may include small interfering RNA (siRNA) or small hairpin RNA (shRNA) and can be manipulated to target B2M-coding mRNA, interfere with its translation, reduce class I MHC expression, and consequently produce B2M knockdown cells with reduced immunogenicity.

[0009] The ability to reduce immunogenicity in any cell could open up new cell sources for any cell transplantation-based treatment or technique (e.g., CAR Treg therapy), enabling large-scale production and stockpiling by avoiding the need for autologous starting materials. In certain techniques, such as engineered CAR Tregs, transduction is already required to express CARs or other targeted moieties in Treg cells. Thus, in a preferred embodiment, a single vector encoding both the B2M modified RNA and the targeted moiety (e.g., CAR) can be used to generate immunogenic CAR Tregs resulting from B2M knockdown. Such a simplified, single-vector approach further supports larger-scale production.

[0010] In certain embodiments, further elements may be encoded within the vector (e.g., safety switches, as well as suicide and reporter genes useful for monitoring expression during cell generation and for providing means for monitoring / tracking the expression profiles of therapeutic cells and their expression profiles after administration). Reporter expression may be particularly useful when used in targeted therapies such as glial cell-targeted CAR Tregs to ensure desired CNS concentrations in engineered immunosuppressive cells.

[0011] As noted, some embodiments include B2M-modified, targeted T regulatory lymphocytes (Tregs) that can be used to modulate immune responses and inflammation through specific targeting to select immune cells and / or tissues. Such Tregs may be ligated to chimeric antigen receptors (CARs), antibodies, or their functional components (e.g., single-stranded variable fragments (scFv)) that specifically recognize and bind to various target cells or tissues, and immunosuppressive Tregs or proteins are attracted to inflamed tissues to reduce inflammation, and thus to reduce inflammation-related pain and degeneration. Knockdown of B2M expression in such cells (which can be achieved via transduction in a single vector encoding both the targeting portion and the B2M-modified RNA) may help avoid unwanted immunogenicity, regardless of the source of the manipulated Treg cells. The B2M modified vectors and methods of the present invention are compatible with targeted Treg therapy for treating neurodegenerative disorders as described in Patent Publication WO 2019 / 190879, as well as for treating other immune and inflammatory diseases. Accordingly, the vectors encoding glial cell and other cell / tissue targeting portions described in those applications are intended herein.

[0012] An aspect of the present invention includes vectors encoding β2-microglobulin (B2M) modified RNA and chimeric antigen receptors (CARs), antibodies, or functional components thereof. In certain embodiments, the vector is a lentiviral vector.

[0013] The above B2M modified RNA may include small interfering RNA (siRNA). The above siRNA may include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In certain embodiments, the above B2M modified RNA may include small hairpin RNA (shRNA). The above shRNA may include sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and one or more hairpin loop sequences selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

[0014] In various embodiments, the vector can be operable to knockdown B2M expression in the transfected cells by at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to wild-type.

[0015] In certain embodiments, the CAR can specifically bind to a glial cell marker, an antigen-presenting cell (APC) marker, a T helper 1 cell (Th1) marker, or a T helper 17 cell (Th17) marker. In some embodiments, the CAR can specifically bind to a marker specific for a cell selected from the group consisting of pancreatic islet cells, pancreatic beta cells, cardiomyocytes, monocytes, macrophages, myeloid cells, intestinal cells, liver cells, kidney cells, kidney podocytes, kidney tubule cells, epithelial cells, salivary gland cells, lung cells, fibroblasts, connective tissue cells, Langerhans cells, keratinocytes, melanocytes, skin cells, hair follicle cells, oligodendrocytes, astrocytes, microglial cells, and hair bulb cells.

[0016] The vector of the present invention can further encode a suicide gene and / or a PET reporter gene. In certain embodiments, the vector can encode a TK suicide / PET reporter gene.

[0017] In certain aspects, the present invention can include engineered cells transfected with a vector encoding β2-microglobulin (B2M) modified RNA and a chimeric antigen receptor (CAR). The engineered cells can have B2M expression that is 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of B2M expression of an otherwise equivalent wild-type cell.

[0018] The manipulated cells described above may be stem cells and lymphocytes. In certain embodiments, the cells may be regulatory T cells (Tregs). The cells may be derived from an allogeneic graft donor in the subject. The manipulated Treg cells may be derived from an allogeneic donor without the addition of CAR molecules.

[0019] An aspect of the present invention is a method for treating an autoimmune disease or inflammatory disease in a subject, comprising administering a therapeutically effective amount of regulatory T cells (Tregs) to the subject, each of which expresses a chimeric antigen receptor (CAR) that specifically binds to β2-microglobulin (B2M) modified RNA and a ligand on the cell surface in a manner that suppresses the immune response in the subject, thereby treating the autoimmune disease or inflammatory disease in the subject. The subject may be human. The Tregs may not be autologous. In various embodiments, the above autoimmune or inflammatory diseases may include: Alzheimer's disease, amyotrophic lateral sclerosis, Batten's disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Dravet syndrome, Krabbe disease, Guillain-Barré syndrome, Huntington's disease, hypoxic-ischemic encephalopathy, West syndrome, Lewy body dementia, metachromatic leukodystrophy (MLD), migraine, multiple system atrophy (MSA), neuromyelitis optica (NMO), Parkinson's disease, Pelizaeus-Merzbacher disease (PMD), post-traumatic stress disorder (PTSD), prion disease, progressive supranuclear palsy, Rett syndrome, spinal muscular atrophy (SMA), Tourette syndrome, traumatic brain injury, tropical spastic paraplegia (TSP), abdominal pain, Absence seizures, acute spinal cord injury, addiction, memory loss, anxiety disorders, eating disorders, arthralgia, ataxia with telangiectasia (AT), attention deficit hyperactivity disorder (ADHD), autism, autoimmune encephalitis, back pain, bipolar disorder, bladder pain, Broca's aphasia, cancer pain, cerebral edema, chemotherapy-induced pain, cluster headache syndrome, cognitive impairment, cognitive dysfunction, complex regional pain syndrome, dementia, toothache, depression, diabetic neuropathic pain, drug-induced dyskinesia, dystonia, encephalomyelitis, epilepsy, epileptic encephalopathy, essential tremor, fatigue, fibromyalgia, hemiplegia, hyperalgesia, inflammatory pain, insomnia, interstitial cystitis, intracerebral hemorrhage, increased intracranial pressure, Kennedy disease, Lennox-Gastaut syndrome, local anesthetic effect, major depressive disorder, MELAS syndrome, meningoencephalitis, nociceptive pain, Morton's metatarsal painmetatarsalgia), motor neuron disease, movement disorder, multifocal motor neuropathy, multiple sclerosis (MS), muscle spasm, musculoskeletal pain, myalgia, fascial pain syndrome, narcolepsy, nerve injury, neuropathic pain, neurotoxic syndrome, obsessive-compulsive disorder, eye pain, opiate withdrawal syndrome, pain of osteoarthritis, panic disorder, paralysis, partial seizure, peripheral nerve injury, pervasive developmental disorder (PDD), polymyalgia rheumatica (PMR), postherpetic neuralgia, postoperative pain, primary progressive multiple sclerosis (PPMS), psychosis, radiculopathy, relapsing multiple sclerosis (RMS), relapsing-remitting multiple sclerosis (RRMR), restless legs syndrome, rheumatoid arthritis pain, schizoaffective disorder, schizophrenia, sciatica, secondary progressive multiple sclerosis (SPMS), sleep disorder, smoking cessation, social anxiety disorder, spasmodic torticollis (cervical dystonia), spinal cord disorder, stiff person syndrome (SPS), tauopathy, pain of tendon and ligament, tonic-clonic (grand mal) seizure, trigeminal neuralgia, upper limb muscle spasm, vascular dementia, vasomotor symptoms (non-menopausal), and visceral pain, type 1 diabetes, transplantation, myocarditis (cardiomyositis), inflammatory bowel disease, ulcerative colitis, Crohn's disease, GVHD, celiac disease, autoimmune hepatitis (AIH), primary sclerosing cholangitis (PSC); primary biliary cirrhosis (PBC), focal segmental glomerulosclerosis (FSGS), systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, systemic sclerosis, membranous glomerular nephropathy (MGN), membranous nephropathy (MN), minimal change disease (MCD); IgA nephropathy, ANCA-associated vasculitis (AAV), Sjögren's syndrome, scleroderma, systemic sclerosis (SSc), vitiligo, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alopecia areata, COVID-19 or other inflammatory diseases resulting from viral infection. anous glomerular nephropathy)(MGN), membranous nephropathy( MN), minimal change disease)(MCD); IgA nephropathy, ANCA-associated vasculitis(AAV), Sjögren's syndrome, scleroderma, systemic sclerosis(SSc), vitiligo, non-alcoholic fatty liver disease(NAFLD), non-alcoholic steatohepatitis(NASH), alopecia areata, COVID-19 or other inflammatory diseases resulting from viral infection.

Brief Description of Drawings

[0020] [Figure 1] Figure 1 illustrates the B2M-modified targeted CAR-Treg. [Figure 2]Figure 2 illustrates the median fluorescence FACS results of HEK-293 cells transduced with various shRNA coding vectors, using anti-B2M staining. [Figure 3] Figure 3 illustrates the median fluorescence FACS results of anti-HLA ABC staining for HEK-293 cells transduced with various shRNA coding vectors. [Figure 4A] Figures 4A-4B show the average fluorescence intensity for HEK-293 cells transduced with various shRNA-coding GFP+ vectors and stained with anti-B2M or anti-HLA ABC. [Figure 4B] Figures 4A-4B show the average fluorescence intensity for HEK-293 cells transduced with various shRNA-coding GFP+ vectors and stained with anti-B2M or anti-HLA ABC. [Figure 5A] Figures 5A–5D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity of anti-B2M and anti-HLA ABC in GFP+ and GFP- cells. [Figure 5B] Figures 5A–5D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity of anti-B2M and anti-HLA ABC in GFP+ and GFP- cells. [Figure 5C] Figures 5A–5D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity of anti-B2M and anti-HLA ABC in GFP+ and GFP- cells. [Figure 5D] Figures 5A–5D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity of anti-B2M and anti-HLA ABC in GFP+ and GFP- cells. [Figure 6A]Figures 6A-6D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity with IgG1 PE / IgG1 APC isotype control antibodies. [Figure 6B] Figures 6A-6D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity with IgG1 PE / IgG1 APC isotype control antibodies. [Figure 6C] Figures 6A-6D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity with IgG1 PE / IgG1 APC isotype control antibodies. [Figure 6D] Figures 6A-6D show the transduction efficiency measured by GFP for HEK-293 cells transduced with various shRNA coding vectors, as well as the staining intensity with IgG1 PE / IgG1 APC isotype control antibodies. [Figure 7] Figure 7 shows a sequence map of shRNA target sites across the B2M gene. [Figure 8]Figure 8 illustrates the generation of universal donor (UD) Tregs with or without CAR or other targeted components. The starting cell type is native (n)Treg (CD4+CD25+CD127-) purified from peripheral blood mononuclear leukocytes (PBMCs) of normal donor blood. nTregs can be transduced with B2M modified RNA and, optionally, lentiviral vectors encoding CAR B2M alone. Both types of engineered nTregs retain endogenous T cell receptor (TCR) expression and have downregulated B2M expression and excision of HLA class I MHC. Only nTregs transduced with CAR / shRNA B2M sequences express CARs specific to protein targets on the defined cell type. Excision of HLA class I MHC protects Tregs from immune attack from alloreactive immune cells after adoptive transfer to MHC mismatch patients. Immunosuppression by UD CAR-Tregs is induced by either the recognition of allo-HLA class II MHC via the endogenous TCR or the recognition of defined cell surface target proteins by CARs. Immunosuppression by UD Tregs is induced solely by the recognition of allo-HLA class II MHC via the endogenous TCR. [Modes for carrying out the invention]

[0021] Detailed explanation The compositions and methods of the present invention relate to the reduction of immunogenicity in various cells via transduction with a vector encoding B2M-modified RNA. In preferred embodiments, the B2M-modified RNA is an siRNA or shRNA that interferes with B2M expression through RNA interference, thereby reducing or preventing the display of MHC class I proteins on the cell surface. Thus, the engineered cells of the present invention can be derived from more readily available non-autologous sources without increasing the risk of inducing an immune response in recipient patients. When combined with cell-based immunotherapy (e.g., CAR Treg immunosuppression), a single vector can be used to induce the expression of B2M-modified RNA and a targeted moiety (e.g., CAR) and other genes (e.g., reporting genes or suicide genes). Figure 1 illustrates engineered Treg cells of the present invention expressing cell-specific CAR and B2M-modified shRNA to reduce MHC class I cell surface display.

[0022] Major histocompatibility complex antigens (MHC, also known as human leukocyte antigens or HLA) are protein molecules found on the surface of cells and play a crucial role in cell-based immune responses, specifically in the display of foreign proteins to cytotoxic T cells, to induce immune attack. HLA antigens are divided into two major classes: MHC class I and MHC class II. HLAs corresponding to MHC class I (HLA-A, HLA-B, and HLA-C) enable cells to be recognized as self, while HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR) are involved in the reaction between lymphocytes and antigen-presenting cells. Both are involved in the rejection of transplanted organs. See U.S. Patent No. 9,997,807 (referenced herein).

[0023] B2M, along with α1, α2, and α3 proteins, is a component of MHC class I proteins and is essential for MHC class I peptide presentation. B2M is located on the α3 chain side on the cell surface and lacks a transmembrane domain. Studies have shown that B2M is necessary for the cell surface expression of MHC class I and the stability of peptide bond grooves, and consequently, the amount of detectable MHC class I on the cell surface is severely limited in the absence of B2M. Therefore, reducing or eliminating B2M expression in cells may help avoid recognition of the cell as an exogenous substance and reduce immunogenicity. Thus, class I MHC excision via interference with B2M expression may open up a new source of non-autologous cell supply for use in manipulating cells for transplantation.

[0024] The nucleotide sequence of the human B2M gene (SEQ ID NO: 8) and the peptide sequence of the B2M protein (SEQ ID NO: 9; UniProtKB - P61769(B2MG_HUMAN)) are shown in Figure 7. Human B2M, including adjacent sequences, is shown in SEQ ID NO: 10 below (NCBI Reference Sequence: NM_004048): [ka] [ka]

[0025] The B2M protein (SEQ ID NO: 9) is the signal transduction portion. MSRSVALAVLALLSLSGLEA (Sequence ID 11) Includes.

[0026] In a preferred embodiment, siRNA or shRNA is used for RNA interference of B2M expression in transduced cells. Selected siRNA and shRNA targets within the B2M gene were chosen as mapped in Figure 7. Examples of genetic excision of B2M to reduce immunogenicity have been shown in previous studies. See: Chang et al., 2014, Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells. PLoS ONE 9(5); Aldrich et al., 1994, Positive selection of self- and alloreactive CD8+ T cells in TAP1 mutant mice, PNAS, 91:6525-6528; Wang et al., 2015, Targeted Disruption of the B2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells, Stem Cells Translational Medicine, 4:1234-1245; Zijlstra et al., 1990, β2-Microglobulin Deficient Mice Lack CD4 - 8 + Cytolytic T Cells, Nature, 344:742-746; U.S. Patent Publication 2019 / 0233797; the contents of each of these are incorporated herein by reference.

[0027] The present invention provides novel B2M modified RNA sequences and vectors encoding B2M modified RNA, along with further CARs, reporting genes, and suicide genes, for efficiently generating targeted cells for cell-based immunotherapy and other treatments from any source with suppressed immunogenicity. The B2M modified vectors of the present invention can knock down B2M expression in transduced cells by at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to wild-type.

[0028] In various embodiments, the B2M modified RNA may be siRNA, shRNA, microRNA, or single-stranded interfering RNA. In preferred embodiments, the B2M modified RNA is siRNA or shRNA. siRNA is a double-stranded non-coding RNA, generally 20–25 base pairs in length. It interferes with the expression of specific genes having complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation. In certain embodiments, the vector sequence encoding the B2M modified siRNA includes one or more of the following sequences: [ka]

[0029] B2M modified vectors encoding the above-mentioned siRNA or shRNA sequences, including those sequences, may be referred herein by corresponding ID numbers (e.g., 703, 704, 705, 706, and 707). Vector 728 (discussed in the examples) is included as a control and contains a scrambled siRNA sequence that is not complementary to any target sequence in the B2M mRNA. The human B2M gene sequence, along with the target region for each of the siRNA sequences referenced above, is shown in Figure 7.

[0030] ShRNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference. ShRNA can be incorporated into plasmid vectors and integrated into genomic DNA for stable expression, producing a longer knockdown effect on target mRNA. In application, the shRNA molecule is processed intracellularly to form siRNA, which subsequently knocks down gene expression. In certain embodiments, a hairpin loop such as one of the following may be added to the siRNA sequence to generate the B2M modified shRNA of the present invention: [ka]

[0031] In the examples discussed below, vectors 703, 704a, 705, 706, and 707 contain their respective siRNA sequences along with the hairpin sequence in SEQ ID NO: 6. Vector 704b (discussed in the examples) contains the siRNA in SEQ ID NO: 2 along with the hairpin loop in SEQ ID NO: 7. The present invention recognizes that certain variations of the sequences provided herein may still exhibit the desired level of B2M expression interference. Therefore, in various embodiments, the siRNA or shRNA may contain sequences having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity with SEQ ID NOs: 1-7.

[0032] In various embodiments, the vector may include a reporter gene. The reporter gene encodes an identifiable marker such that their detection in cells confirms the expression of that gene. The reporter gene may provide a measurable signal, when expressed, which can be further used to quantify the expression of that gene and to infer the transduction efficiency of the vector containing them. Commonly used reporter genes include those that provide a visual signal, such as green fluorescent protein (GFP), which can be readily observed and measured. In certain embodiments, the vector of the present invention may include a positron emission tomography (PET) reporter gene. See: Yaghoubi et al., 2012, Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications, Teranostics, 2(4):374-391 (as incorporated herein by reference). The use of a PET reporter gene enables non-invasive imaging using PET scans to determine expression and, therefore, transduction efficiency. Furthermore, when used in targeted cell-based therapies (e.g., CAR-Treg treatment), PET reporter expression can be used to non-invasively monitor the localization of the administered cells and verify that the cells are properly enriched in the target tissue. For example, when expressed in CNS-glial cell-targeted CAR-Tregs, the PET reporter can be used in conjunction with a PET scan of the brain to verify that the CAR-Tregs have crossed the blood-brain barrier and reached the target glial cells.

[0033] In certain embodiments, the vector may further contain a suicide gene. The suicide gene can cause the cell to kill itself through apoptosis upon activation. The suicide gene may be incorporated as a safety switch to selectively kill the genetically modified cell in case of adverse reactions. Therefore, they may be useful in cell-based immunotherapies such as CAR-Treg immunosuppression. In a preferred embodiment, a TK suicide / PET reporter gene may be included in the vector to provide a dual function of PET reporting and suicide safety switch. (Gschweng et al., 2014, HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized) See mice, Cancer Res., 74(18):5173-5183 (as incorporated herein by reference).

[0034] B2M-modified Treg cells and other cells can be engineered by known methods for preparing CAR-T cells or other engineered cells. Treg cells can be isolated from any source by subsequent excision of class I MHC cells. The genes of the Treg cells can then be modified through known techniques (e.g., electroporation, viral vectors, or other forms of transfection with nucleic acids encoding one or more of the following: B2M-modified RNA, optimally engineered chimeric antigen receptors, reporter genes, and suicide genes). The engineered cells can then be experimentally validated before being introduced into a patient's system for treatment. In preferred embodiments, lentiviral vectors can be used to transduce cells with B2M-modified RNA as well as various targeting, signaling, and suicide genes. (Elegheert et al., 2018, Lentiviral transduction of mammalian cells for fast, scalable, and high-level production) See *of soluble and membrane proteins*, Nature Protocols, 13, 2991-3017 (referenced herein).

[0035] In a preferred embodiment, the transduced cells may be stem cells or lymphocytes (e.g., Tregs). The engineered Tregs of the present invention can be used to treat a variety of autoimmune and / or inflammatory diseases, including various neurodegenerative diseases. The engineered cells can target cell-specific markers, directing Tregs to target immune cells or tissues, thereby disrupting autoimmune attacks and inflammation that contribute to the symptoms of a wide variety of diseases.

[0036] As noted above, in addition to the B2M modified RNA, reporter, and / or suicide gene, the vector may encode a chimeric antigen receptor (CAR), antibody, or single-stranded variable fragment (scFv) that specifically binds to a target cell marker. The target cell may be an immune cell such as an APC or T helper cell (Th1 or Th17), or a specific tissue in which suppression of the immune response is desired. Exemplary target cells, ligands, and targeting moieties for treating neurodegenerative diseases, autoimmune diseases, and / or inflammatory diseases are described in Patent Publication WO 2019 / 190879, and the present invention intends to incorporate such targeting moieties and methods of treatment in the B2M modified vector described herein.

[0037] In various embodiments, vectors may encode CARs, scFvs, or antibodies to target specific cells or tissues. CARs are engineered receptors that can provide specificity to immune effector cells (T cells). CARs have been used to confer tumor cell specificity to cytotoxic T lymphocytes for use in cancer immunotherapy. See: Couzin-Frankel, 2013, Cancer immunotherapy, Science, 342(6165):1432-33; Smith et al., 2016, Chimeric antigen receptor (CAR) T cell therapy for malignant cancers: Summary and perspective, Journal of Cellular Immunotherapy, 2(2):59-68; the contents of each of these are incorporated herein by reference. Using a similar principle, the compounds and methods of the present invention involve manipulating CARs that are specific to markers found on the above-mentioned immune cells or other specific cell types, but instead of grafting cell-specific CARs onto cytotoxic T cells, they are grafted onto engineered immunosuppressive Tregs. The CAR-Tregs of the present invention may express multiple chimeric antigen receptors that target the same, two, or more different cellular markers.

[0038] ScFv is the heavy chain of immunoglobulin (V H ) and light chain (V L ScFv is a fusion protein containing the variable region of ) in mice or other animals immunized with the desired target molecule. H and V L It can be produced by cloning genes. Then, the above V H and V L The gene can be expressed in multiple orientations and with various linkers to form various scFvs, which can then be experimentally validated to provide desired stability, expression levels, and binding affinity to specific markers or cells.

[0039] Antibodies that target cell markers can be generated by methods known in the art, including, for example, commercially available services from Pacific Immunology (San Diego, CA) or ABclonal (Woburn, MA) to generate custom antibodies.

[0040] To generate engineered Tregs, the starting cell type is natural (n) Tregs (CD4 + CD25 + CD127 - ), which can be used. The nTregs can be purified from peripheral blood mononuclear leukocytes (PBMCs) derived from normal donor blood. The CAR gene can be introduced into nTregs by transduction with a lentiviral vector in cis with shRNA against B2M. In certain embodiments, the targeted CAR gene can be excluded, such that the nTregs are simply engineered with shRNA against B2M on the lentiviral vector. With or without CAR expression, the engineered nTregs should retain expression of the endogenous T cell receptor (TCR) and show downregulation of B2M and excision of HLA class I MHC. Only nTregs transduced with the CAR / shRNA B2M sequence express a CAR specific for a protein target on a defined cell type. Excision of HLA-class I MHC can protect Tregs from immune attack by alloreactive immune cells after adoptive transfer into MHC-mismatched patients. Immunosuppression by UD CAR-Tregs can be induced by either recognition of allogeneic HLA class II MHC via the endogenous TCR or recognition of a defined cell surface target protein by the CAR. Alternatively, immunosuppression by UD Tregs may not be induced by CAR recognition, because the CAR is not expressed and instead is induced by recognition of allogeneic class II MHC by the endogenous TCR.

[0041] Regulatory T cells, or Tregs, modulate the immune system and generally downregulate the induction and proliferation of effector T cells. Tregs prevent autoimmune responses and assist the immune system in distinguishing between self and non-self. Regulatory T cells produce inhibitory cytokines, including transforming growth factor β, interleukin 35, and interleukin 10, and can induce other cell types to express interleukin-10. Tregs can also produce granzyme B, which can subsequently induce apoptosis in effector cells. Tregs also function via reverse signaling through direct interaction with dendritic cells and induction of immunosuppressive indoleamine 2,3-dioxygenase. Tregs can also downregulate immune responses mediated by the exenzymes CD39 and CD73, along with the production of immunosuppressive adenosine. Tregs also suppress immune responses mediated by direct interaction with dendritic cells, both by LAG3 and TIGIT. Another regulatory mechanism involves the IL-2 feedback loop. Another mechanism of immunosuppression by Treg cells involves the activation of the molecule CTLA-4, which prevents CD28-mediated co-stimulation on effector T cells.

[0042] The B2M-modified CAR-Treg of the present invention may be incorporated into a carrier system comprising one or more of the therapeutic compounds described herein. In certain embodiments, the carrier system may be nanoparticles comprising disulfide-crosslinked polyethyleneimine (CLPEI) and lipids. The lipids may be bile acids (e.g., cholic acid, deoxycholic acid, and lithocholic acid). Such carrier systems are further described in the following examples. Other exemplary carrier systems are described, for example, by Wittrup et al. (Nature Reviews / Genetics, 16:543-552, 2015) (their contents are incorporated herein by reference in their entirety).

[0043] Figure 8 illustrates the generation of universal donor (UD) CAR-Treg or Treg. The starting cell type is native (n)Treg (CD4) purified from peripheral blood mononuclear leukocytes (PBMCs) of normal donor blood. + CD25 + CD127 - nTregs can be transduced with lentiviral vectors encoding shRNA for CAR and B2M, or the shRNA alone. Both types of engineered nTregs should retain the expression of endogenous T cell receptors (TCRs), and therefore endogenous Treg functionality. Both types of engineered nTregs should also exhibit B2M downregulation and excision of HLA-class I MHCs. Furthermore, the nTregs transduced with CAR / shRNA B2M sequences may express CARs specific to protein targets on a given cell type. Excision of HLA-class I MHCs protects the engineered Tregs from immune attack from alloreactive immune cells after adoptive transfer to MHC mismatch patients. Immunosuppression by UD CAR-Tregs can be induced by either the recognition of allo-HLA-class II MHCs via the endogenous TCR or the recognition of a defined cell surface target protein by the CAR. Immunosuppression by UD Tregs can only be induced by the recognition of alloclasm II MHC by endogenous TCRs.

[0044] The terms “parenteral administration” and “administered parenterally,” as used herein, typically mean modes of administration other than enteral and local administration by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0045] The terms “systemic administration,” “administered systemically,” “peripheral administration,” and “peripherally administered,” as used herein, mean the administration of a compound, drug, or other substance other than that administered directly to the central nervous system, so as to enter the patient’s system and thus undergo metabolism and other similar processes (e.g., subcutaneous administration).

[0046] When the compounds of the present invention are administered to humans and mammals as pharmaceuticals, they may be given either by themselves or as pharmaceutical compositions containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of an active ingredient, i.e., at least one therapeutic compound of the present invention and / or a derivative thereof, in combination with a pharmaceutically acceptable carrier.

[0047] The effective dose of each drug can be readily determined by those skilled in the art, taking into account representative factors such as the patient's age, weight, sex, and clinical history. Generally, the appropriate daily dose of the compounds of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose generally depends on the factors mentioned above.

[0048] If desired, the effective daily dose of the active compound may be administered in unit dose form as needed, in two, three, four, five, six or more partial doses, administered separately at appropriate intervals throughout the day.

[0049] The pharmaceutical compositions of the present invention comprise a "therapeutically effective amount" or a "preventively effective amount" of one or more of the compounds of the present invention or their functional derivatives. "Effective amount" is the amount as defined herein in the Definitions section, and refers to the amount effective for achieving the desired therapeutic outcome, e.g., the dose and duration required to reduce or prevent effects associated with neuropathic and / or inflammatory pain. The therapeutically effective amount of the compounds of the present invention or their functional derivatives may vary depending on factors such as the subject's disease state, age, sex, and weight, as well as the ability of the therapeutic compound to elicit the desired response in the subject. The therapeutically effective amount is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the therapeutic agent.

[0050] "Prophylactically effective dose" refers to the effective amount of a drug in the dosage and duration necessary to achieve the desired prophylactic effect. Typically, since prophylactic doses are administered to subjects before the onset of disease or at an earlier stage of the disease, the prophylactically effective dose may be less than the therapeutically effective dose. The prophylactic or therapeutically effective dose is also the amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the compound.

[0051] The administration regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, or several divided doses may be administered over time, and the doses may be proportionally reduced or increased as indicated by the urgency of the treatment situation. Formulating parenteral compositions in dosing unit form is particularly advantageous for ease of administration and uniformity of dosage. The actual dose levels of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the patient.

[0052] The term “dosage unit,” as used herein, refers to a physically distinct unit suitable as a unit dose for a mammalian subject being treated; each unit contains a predetermined amount of the active compound calculated to produce its desired therapeutic effect in association with the required pharmaceutical carrier. The specifications relating to the dosage unit forms of the present invention are defined and directly depend on (a) the specific characteristics of the compound and (b) the inherent limitations of the technique for formulating such active compounds for the treatment of hypersensitivity in an individual.

[0053] In some embodiments, a therapeutically effective dose can be initially estimated in either a cell culture assay or an animal model (typically a mouse, rabbit, dog, or pig). The animal model may also be used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. Generally, the therapeutically effective dose is sufficient to reduce or inhibit neuropathic and / or inflammatory pain in the subject. In some embodiments, the therapeutically effective dose is sufficient to eliminate neuropathic and / or inflammatory pain in the subject.

[0054] Dosages for specific patients can be determined by those skilled in the art using conventional considerations (e.g., by appropriate conventional pharmacological protocols). A physician may, for example, prescribe a relatively low dose initially, and then increase the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to produce a beneficial therapeutic response over time in the patient, or to reduce, for example, symptoms or other appropriate activities, as appropriate for the application. The dose is determined by the efficacy of the particular formulation, the activity, stability, or serum half-life of the compound or functional derivative of the present invention, as well as the patient's condition and the patient's body weight or surface area to be treated. The magnitude of the dose is also determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular vector, formulation, etc., in a particular subject. Therapeutic compositions comprising one or more compounds or functional derivatives of the present invention may be tested as necessary in one or more appropriate in vitro and / or in vivo animal models of the disease (e.g., models of neuropathic and / or inflammatory pain) to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art. In particular, the dosage may be first determined in the relevant assay by the activity, stability, or other appropriate means (e.g., comparison of treated versus untreated cells or animal models). The formulation is administered in a proportion determined by the LD50 of the relevant formulation and / or observation of any adverse effects at various concentrations of the compound or functional derivative of the present invention, for example, in relation to the patient's body weight and overall health. Dosage may be achieved via a single dose or divided doses.

[0055] Administration typically includes administering a pharmaceutically acceptable dosage form, where the dosage form means the dosage forms of the compounds described herein, including, for example, tablets, sugar-coated tablets, powders, elixirs, syrups, liquid preparations (including suspensions), sprays, inhalation tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid injectable preparations (including liposomal preparations). The techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition (which is incorporated herein by reference in its entirety). Administration may be performed orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or intranasally. The compounds may be administered alone or with a suitable pharmaceutically acceptable carrier, and may be in solid or liquid form (e.g., tablets, capsules, powders, solutions, suspensions, or emulsions).

[0056] Pharmaceutical compositions containing the above-mentioned active ingredients may exist in forms suitable for oral use (e.g., as tablets, lozenges, watery or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs). Compositions intended for oral use may be prepared according to any method known in the art with respect to the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents and preservatives to provide a pharmaceutically refined and palatable preparation. Tablets may contain the above-mentioned active ingredients in mixture with non-toxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients may be, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate); granulating and disintegrating agents (e.g., corn starch or alginic acid); binders (e.g., starch, gelatin or acacia) and lubricants (e.g., magnesium stearate, stearic acid or talc). The above tablets may be uncoated or coated by known techniques that slow disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a long period. For example, time-delaying substances (e.g., glyceryl monostearate or glyceryl distearate) may be used. They may also be osmotic therapeutic tablets for release control. To form a therapeutic tablet, it may be coated by the techniques described in U.S. Patents 4,256,108, 4,166,452 and 4,265,874 (the contents of each of these are incorporated herein by reference in whole).

[0057] Formulations for oral use may also be presented as rigid gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0058] The formulation may also include a complex of the parent (non-ionized) compound with a derivative of β-cyclodextrin, particularly with hydroxypropyl-β-cyclodextrin.

[0059] An alternative oral formulation may be achieved using a controlled-release formulation, in which the compound is encapsulated in an enteric coating.

[0060] The aqueous suspension contains the active substance in mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum); dispersants or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), or condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitan monooleate), such as condensation products of polyoxyethylenes and partial esters derived from fatty acids and hexitol anhydrides. The aqueous suspension described above may also contain one or more preservatives (e.g., ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate), one or more colorants, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0061] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or mineral oil (e.g., liquid paraffin). These oily suspensions may contain thickening agents (e.g., beeswax, solid paraffin, or cetyl alcohol). Sweeteners (e.g., those listed above) and flavoring agents may be added to provide an oral preparation that is palatable. These compositions may be preserved by the addition of antioxidants (e.g., ascorbic acid).

[0062] Dispersible powders and granules suitable for the preparation of aqueous suspensions by the addition of water provide the above-mentioned active ingredients in a mixture with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified, and sweeteners, flavoring agents, and coloring agents may also be present, for example.

[0063] The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil (e.g., olive oil or peanut oil) or a mineral oil (e.g., liquid paraffin) or a mixture thereof. Suitable emulsifiers include naturally occurring gums (e.g., acacia gum or tragacanth gum), naturally occurring phosphatides (e.g., soybean lecithin), and those derived from fatty acids and hexitol anhydrides. The emulsion may be an ester or partial ester (e.g., sorbitan monooleate) and a condensation product of the partial ester and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion may also contain sweeteners and flavoring / odorizing agents.

[0064] Syrups and elixirs may be formulated with sweeteners (e.g., glycerol, propylene glycol, sorbitol, or sucrose). Such formulations may also contain lubricants, preservatives, and flavoring and coloring agents. The above pharmaceutical compositions may be in the form of sterile aqueous or oily suspensions for injection. These suspensions may be formulated according to known techniques using the appropriate dispersants or wetting agents and suspending agents mentioned above. The above sterile injectable preparations may also be in the form of sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents (e.g., as solutions in 1,3-butanediol). Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils have been conventionally used as solvents or suspension media. For this purpose, any non-irritating non-volatile oil may be used (including synthetic monoglycerides or diglycerides). Furthermore, fatty acids (e.g., oleic acid) have found applications in the preparation of injectable drugs.

[0065] Each active agent may also be administered in the form of a suppository for rectal administration of the drug. These compositions may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at normal temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0066] For topical use, creams, ointments, gels, liquids, or suspensions are suitable. Topical application includes the use of mouthwashes and gargles.

[0067] The term "pharmaceutical composition" means a composition comprising the compounds described herein, as well as at least one component, depending on the nature of the mode and form of administration, including pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles (e.g., preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispersants).

[0068] The term “pharmaceutically acceptable carrier” is used to mean any carrier, diluent, adjuvant, excipient, or vehicle as described herein. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxyoxide, bentonite, agar, and tragacanth, or mixtures thereof. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Inclusion of isotonic agents, e.g., sugars, sodium chloride, etc., may be desirable. Long-term absorption of pharmaceutical forms for injection can be achieved by the use of absorption-delaying agents, e.g., aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Examples of excipients include lactose, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrants include starch, alginate, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.

[0069] The term "pharmaceutically acceptable" means that, within reasonable medical judgment, it is suitable for use in contact with human and lower animal cells without excessive toxicity, irritation, or allergic reactions, and the benefits / risks are balanced in a reasonable way. [Examples]

[0070] Examples Example 1 - B2M knockdown in HEK293 cells HEK293 cells were transduced with a lentiviral vector encoding shRNA sequences 703–707 and 728 (including siRNA sequence numbers 1–5 and hairpin loops of sequence number 6 or 7) as described above, and a GFP reporter gene to monitor transduction efficiency. The constructs exhibited transduction of 90% or better, as measured by GFP.

[0071] Cells were stained, and the knockdown effects of various shRNA vectors were measured using fluorescence-activated cell sorting, compared to a control without transduction (NV) and a scrambled shRNA-coding vector (728). Figure 2 shows the median fluorescence FACS results with anti-B2M staining, and Figure 3 shows the median fluorescence FACS results with anti-HLA ABC staining. As shown, SEQ ID NOs. 1–5 (703–707) each had a significant knockdown effect on both surface B2M and HLA-A / B / C class I MHC expression compared to the two controls mentioned above. Vectors 703 (encoding SEQ ID NO 1) and 707 (encoding SEQ ID NO 5) showed the most significant knockdown effects. Furthermore, the B2M knockdown levels were consistent with the HLA knockdown levels across the various vectors.

[0072] Figures 4A-B show the mean fluorescence intensity of HEK-293 cells transduced with various shRNA-coding GFP+ vectors and stained with anti-B2M or anti-HLA ABC staining. Figures 5A-5D show the transduction efficiency measured by GFP of HEK-293 cells transduced with various shRNA-coding vectors and stained with anti-B2M and anti-HLA staining. Figures 6A-6D show the transduction efficiency measured by GFP of HEK-293 cells transduced with various shRNA-coding vectors and stained with IgG1 PE IgG1 APC isotype control. The transduction efficiency and B2M knockdown effect of vectors containing siRNA (703-707) including SEQ ID NOs: 1-5 are further supported by the results shown in Figures 4A-B, 5A-D, and 6A-D.

[0073] Equivalents and references All references cited herein are invoked by reference to the same extent as each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is specifically and individually indicated as being invoked by reference for all purposes in whole. This statement of referencing by reference is intended by the applicant to relate to each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent (each of which is clearly identified in accordance with 37 CFR §1.57(b)(2), even if such reference is not immediately adjacent to a dedicated statement of referencing by reference). The inclusion of dedicated statements of referencing by reference within this specification, if any, does not diminish this general statement of referencing by reference. The references made herein are not intended to constitute an endorsement that such references are the relevant prior art, nor do they constitute any endorsement of the content or date of such publications or documents.

[0074] While the present invention has been described and illustrated in detail with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made within the present invention without departing from the spirit and scope of the invention. In certain embodiments, for example, the following items are provided: (Item 1) β2 microglobulin (B2M) modified RNA; and Chimeric antigen receptor (CAR), A vector that codes. (Item 2) The vector is a lentiviral vector, as described in item 1. (Item 3) The aforementioned B2M modified RNA is a vector containing small interfering RNA (siRNA), as described in item 1. (Item 4) The vector described in item 3, wherein the siRNA is encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 5) The aforementioned B2M modified RNA is the vector described in item 1, which contains small hairpin RNA (shRNA). (Item 6) The vector according to item 5, wherein the shRNA is encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and one or more hairpin loop sequences are encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. (Item 7) The vector described in item 1, which can be manipulated to knock down B2M expression in transduced cells by at least 75% compared to the wild type. (Item 8) The vector described in item 1, which can be manipulated to knock down B2M expression in transduced cells by at least 80% compared to the wild type. (Item 9) The vector described in item 1, which can be manipulated to knock down B2M expression in transduced cells by at least 85% compared to the wild type. (Item 10) The vector described in item 1, which can be manipulated to knock down B2M expression in transduced cells by at least 90% compared to the wild type. (Item 11) The vector described in item 1, which can be manipulated to knock down B2M expression in transduced cells by at least 95% compared to the wild type. (Item 12) The CAR is the vector described in item 1, which specifically binds to glial cell markers. (Item 13) The CAR is a vector described in item 1 that specifically binds to an antigen-presenting cell (APC) marker. (Item 14) The aforementioned CAR is a vector described in item 1 that specifically binds to a T helper 1 cell (Th1) marker. (Item 15) The aforementioned CAR is the vector described in item 1, which specifically binds to the T helper 17 cell (Th17) marker. (Item 16) The CAR is a vector as described in item 1, which specifically binds to a marker specific to cells selected from the group consisting of pancreatic islet cells, pancreatic β-cells, cardiomyocytes, monocytes, macrophages, myeloid cells, intestinal cells, hepatocytes, kidney cells, renal podocytes, renal tubular cells, epithelial cells, salivary gland cells, lung cells, fibroblasts, connective tissue cells, Langerhans cells, keratinocytes, melanocytes, skin cells, hair follicle cells, and hair bulb cells. (Item 17) The vector described in item 1 further codes for the suicide gene. (Item 18) A vector, as described in item 1, that further encodes the PET reporter gene. (Item 19) A vector, as described in item 1, that further encodes the TK suicide / PET reporter gene. (Item 20) Engineered cells transduced with vectors encoding β2-microglobulin (B2M) modified RNA and chimeric antigen receptors (CARs). (Item 21) The B2M modified RNA is a modified cell as described in item 20, containing small interfering RNA (siRNA). (Item 22) The manipulated cell described in item 21, wherein the siRNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 23) The aforementioned B2M modified RNA is the engineered cell described in item 20, which contains small hairpin RNA (shRNA). (Item 24) The manipulated cell described in item 23, wherein the shRNA is encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and one or more hairpin loop sequences are encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. (Item 25) Manipulated cells as described in item 20, having B2M expression of 25% or less than that of equivalent wild-type cells. (Item 26) Manipulated cells as described in item 20, having B2M expression of 20% or less than that of equivalent wild-type cells. (Item 27) Manipulated cells as described in item 20, having B2M expression of 15% or less of that of equivalent wild-type cells. (Item 28) Manipulated cells as described in item 20, having B2M expression of 10% or less of that of equivalent wild-type cells. (Item 29) Manipulated cells as described in item 20, having B2M expression of 5% or less than that of equivalent wild-type cells. (Item 30) The CAR is an engineered cell as described in item 20, which specifically binds a CNS glial cell marker. (Item 31) The CAR is an engineered cell as described in item 20, which specifically binds an antigen-presenting cell (APC) marker. (Item 32) The CAR is a manipulated cell as described in item 20, which specifically binds to a T helper 1 cell (Th1) marker. (Item 33) The CAR is a manipulated cell as described in item 20, which specifically binds to the T helper 17 cell (Th17) marker. (Item 34) The CARs are modified cells as described in item 20, which specifically bind to markers specific to cells selected from the group consisting of pancreatic islet cells, pancreatic β-cells, cardiomyocytes, monocytes, macrophages, myeloid cells, intestinal cells, hepatocytes, kidney cells, renal podocytes, renal tubular cells, epithelial cells, salivary gland cells, lung cells, fibroblasts, connective tissue cells, Langerhans cells, keratinocytes, melanocytes, skin cells, hair follicle cells, and hair bulb cells, oligodendrocytes, astrocytic cells, and microglia cells. (Item 35) The cells are the manipulated cells described in item 20, selected from the group consisting of stem cells and lymphocytes. (Item 36) The aforementioned cells are regulatory T cells (Tregs), which are the manipulated cells described in item 20. (Item 37) The cells are the manipulated cells described in item 20, derived from the donor of the allogeneic graft in the subject. (Item 38) A method for treating an autoimmune disease or inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of regulatory T cells (Tregs) to the subject, each expressing β2-microglobulin (B2M) modified RNA and a chimeric antigen receptor (CAR) that specifically binds to a ligand on the cell surface in a manner that suppresses the immune response in the subject, thereby treating the autoimmune disease or inflammatory disease in the subject. (Item 39) The B2M modified RNA is the method described in item 38, which includes small interfering RNA (siRNA). (Item 40) The method according to item 39, wherein the siRNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 41) The B2M modified RNA is the method described in item 38, which includes small hairpin RNA (shRNA). (Item 42) The method according to item 41, wherein the shRNA is encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and one or more hairpin loop sequences are encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. (Item 43) The method according to item 38, wherein the Treg has a B2M expression of 25% or less than that of a wild-type Treg. (Item 44) The subject is a human, as described in item 38. (Item 45) The aforementioned Treg is not autologous, as described in item 38. (Item 46) The cell is selected from the group consisting of glial cells, antigen-presenting cells (APCs), T helper 1 cells (Th1), and T helper 17 cells (Th17), as described in item 38. (Item 47) A vector encoding β2-microglobulin (B2M) modified RNA, wherein the vector's sequence includes a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 48) The vector is a lentiviral vector, as described in item 47. (Item 49) The aforementioned B2M modified RNA is a vector containing small interfering RNA (siRNA), as described in item 47. (Item 50) The aforementioned B2M modified RNA is a vector containing small hairpin RNA (shRNA), as described in item 47. (Item 51) The vector according to item 50, wherein the shRNA comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence numbers 6 and 7. (Item 52) The vector described in item 47, which can be manipulated to knock down B2M expression in transduced cells by at least 75% compared to the wild type. (Item 53) The vector described in item 47, which can be manipulated to knock down B2M expression in transduced cells by at least 80% compared to the wild type. (Item 54) The vector described in item 47, which can be manipulated to knock down B2M expression in transduced cells by at least 85% compared to the wild type. (Item 55) The vector described in item 47, which can be manipulated to knock down B2M expression in transduced cells by at least 90% compared to the wild type. (Item 56) The vector described in item 47, which can be manipulated to knock down B2M expression in transduced cells by at least 95% compared to the wild type. (Item 57) A vector, as described in item 47, that further codes for the suicide gene. (Item 58) A vector, as described in item 47, that further encodes the PET reporter gene. (Item 59) A vector, as described in item 47, that further encodes the TK suicide / PET reporter gene. (Item 60) Engineered cells transduced with a vector encoding β2-microglobulin (B2M) modified RNA, wherein the vector sequence includes a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 61) The B2M modified RNA is a modified cell as described in item 60, containing small interfering RNA (siRNA). (Item 62) The aforementioned B2M modified RNA is the engineered cell described in item 60, which contains small hairpin RNA (shRNA). (Item 63) The manipulated cell described in item 62, wherein the shRNA comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence numbers 6 and 7. (Item 64) Manipulated cells as described in item 60, having B2M expression of 25% or less than that of equivalent wild-type cells. (Item 65) Manipulated cells as described in item 60, having B2M expression of 20% or less than that of equivalent wild-type cells. (Item 66) Manipulated cells as described in item 60, having B2M expression of 15% or less of that of equivalent wild-type cells. (Item 67) Manipulated cells as described in item 60, having B2M expression of 10% or less of that of equivalent wild-type cells. (Item 68) Manipulated cells as described in item 60, having B2M expression of 5% or less than that of equivalent wild-type cells. (Item 69) The cells are the manipulated cells described in item 60, selected from the group consisting of stem cells and lymphocytes. (Item 70) The aforementioned cells are regulatory T cells (Treg), which are the manipulated cells described in item 60. (Item 71) The cells are the manipulated cells described in item 60, derived from the donor of the allogeneic graft in the subject. (Item 72) A method for treating an autoimmune disease or inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of regulatory T cells (Tregs) to the subject, each of which expresses β2-microglobulin (B2M) modified RNA in a manner that suppresses the immune response in the subject, thereby treating the autoimmune disease or inflammatory disease in the subject. (Item 73) The B2M modified RNA is the method described in item 72, which includes small interfering RNA (siRNA). (Item 74) The method according to item 73, wherein the siRNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 75) The B2M modified RNA is the method described in item 72, which includes small hairpin RNA (shRNA). (Item 76) The method according to item 75, wherein the shRNA is encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and one or more hairpin loop sequences are encoded by a sequence containing a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. (Item 77) The method according to item 72, wherein the Treg has a B2M expression of 25% or less than that of a wild-type Treg. (Item 78) The subject is a human, as described in item 72. (Item 79) The aforementioned Treg is not autologous, as described in item 72.

Claims

1. β2-microglobulin (B2M) modified RNA; and Chimeric antigen receptor (CAR), A vector that codes.

2. The vector according to claim 1, wherein the vector is a lentiviral vector.

3. The vector according to claim 2, wherein the B2M modified RNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

4. The vector according to claim 1, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

5. The vector according to claim 4, wherein the shRNA can be processed into small interfering RNA (siRNA).

6. The vector according to claim 4, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

7. The vector according to claim 1, which can be manipulated to knock down B2M expression in transduced cells by at least 75% compared to the wild type.

8. The vector according to claim 1, which can be manipulated to knock down B2M expression in transduced cells by at least 80% compared to the wild type.

9. The vector according to claim 1, which can be manipulated to knock down B2M expression in transduced cells by at least 85% compared to the wild type.

10. The vector according to claim 1, which can be manipulated to knock down B2M expression in transduced cells by at least 90% compared to the wild type.

11. The vector according to claim 1, which can be manipulated to knock down B2M expression in transduced cells by at least 95% compared to the wild type.

12. The vector according to claim 1, wherein the CAR specifically binds a glial cell marker.

13. The vector according to claim 1, wherein the CAR specifically binds to an antigen-presenting cell (APC) marker.

14. The vector according to claim 1, wherein the CAR specifically binds to a T helper 1 cell (Th1) marker.

15. The vector according to claim 1, wherein the CAR specifically binds to a T helper 17 cell (Th17) marker.

16. The vector according to claim 1, wherein the CAR specifically binds to a marker specific to cells selected from the group consisting of pancreatic islet cells, pancreatic β-cells, cardiomyocytes, monocytes, macrophages, myeloid cells, intestinal cells, hepatocytes, kidney cells, renal podocytes, renal tubular cells, epithelial cells, salivary gland cells, lung cells, fibroblasts, connective tissue cells, Langerhans cells, keratinocytes, melanocytes, skin cells, hair follicle cells, and hair bulb cells.

17. The vector according to claim 1, further encoding a suicide gene.

18. The vector according to claim 1, further encoding a PET reporter gene.

19. The vector according to claim 1, further encoding the TK suicide / PET reporter gene.

20. Engineered cells transduced with a vector encoding β2-microglobulin (B2M) modified RNA and a chimeric antigen receptor (CAR).

21. The manipulated cell according to claim 20, wherein the B2M modified RNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

22. The manipulated cell according to claim 20, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

23. The manipulated cell according to claim 22, wherein the shRNA can be processed into small interfering RNA (siRNA).

24. The manipulated cell according to claim 22, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

25. The manipulated cell according to claim 20, having B2M expression of 25% or less of that of equivalent wild-type cells.

26. The engineered cell according to claim 20, having B2M expression of 20% or less of that of an equivalent wild-type cell.

27. ​​The manipulated cell according to claim 20, having a B2M expression of 15% or less of that of an equivalent wild-type cell.

28. The manipulated cell according to claim 20, having B2M expression of 10% or less of that of equivalent wild-type cells.

29. The manipulated cell according to claim 20, having a B2M expression of 5% or less of that of an equivalent wild-type cell.

30. The manipulated cell according to claim 20, wherein the CAR specifically binds to a CNS glial cell marker.

31. The manipulated cell according to claim 20, wherein the CAR specifically binds to an antigen-presenting cell (APC) marker.

32. The manipulated cell according to claim 20, wherein the CAR specifically binds to a T helper 1 cell (Th1) marker.

33. The manipulated cell according to claim 20, wherein the CAR specifically binds to a T helper 17 cell (Th17) marker.

34. The manipulated cell according to claim 20, wherein the CAR specifically binds to a marker specific to cells selected from the group consisting of islet cells, pancreatic β-cells, cardiomyocytes, monocytes, macrophages, myeloid cells, intestinal cells, hepatocytes, kidney cells, renal podocytes, renal tubular cells, epithelial cells, salivary gland cells, lung cells, fibroblasts, connective tissue cells, Langerhans cells, keratinocytes, melanocytes, skin cells, hair follicle cells, and hair bulb cells, oligodendrocytes, astrocytic cells, and microglia cells.

35. The manipulated cells according to claim 20, wherein the cells are selected from the group consisting of stem cells and lymphocytes.

36. The manipulated cell according to claim 20, wherein the cell is a regulatory T cell (Treg).

37. The manipulated cells according to claim 20, wherein the cells are derived from an allogeneic graft donor in the subject.

38. A composition for treating an autoimmune disease or inflammatory disease in a subject, wherein the composition comprises regulatory T cells (Treg), each expressing a chimeric antigen receptor (CAR) that specifically binds β2-microglobulin (B2M) modified RNA and a ligand on the cell surface in a manner that suppresses the immune response in the subject.

39. The composition according to claim 38, wherein the B2M modified RNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

40. The composition according to claim 38, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

41. The composition according to claim 40, wherein the shRNA can be processed into small interfering RNA (siRNA).

42. The composition according to claim 40, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

43. The composition according to claim 38, wherein the Treg has a B2M expression of 25% or less of that of wild-type Treg.

44. The composition according to claim 38, wherein the subject is a human.

45. The composition according to claim 38, wherein the Treg is not in-house.

46. The composition according to claim 38, wherein the cells are selected from the group consisting of glial cells, antigen-presenting cells (APCs), T helper 1 cells (Th1), and T helper 17 cells (Th17).

47. A vector encoding β2-microglobulin (B2M) modified RNA, wherein the sequence of the vector includes a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

48. The vector according to claim 47, wherein the vector is a lentiviral vector.

49. The vector according to claim 47, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

50. The vector according to claim 49, wherein the shRNA can be processed into small interfering RNA (siRNA).

51. The vector according to claim 49, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

52. The vector according to claim 47, which can be manipulated to knock down B2M expression in transduced cells by at least 75% compared to the wild type.

53. The vector according to claim 47, which can be manipulated to knock down B2M expression in transduced cells by at least 80% compared to the wild type.

54. The vector according to claim 47, which can be manipulated to knock down B2M expression in transduced cells by at least 85% compared to the wild type.

55. The vector according to claim 47, which can be manipulated to knock down B2M expression in transduced cells by at least 90% compared to the wild type.

56. The vector according to claim 47, which can be manipulated to knock down B2M expression in transduced cells by at least 95% compared to the wild type.

57. The vector according to claim 47, further encoding a suicide gene.

58. The vector according to claim 47, further encoding a PET reporter gene.

59. The vector according to claim 47, further encoding the TK suicide / PET reporter gene.

60. An engineered cell transduced with a vector encoding β2-microglobulin (B2M) modified RNA, wherein the sequence of the vector includes a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

61. The manipulated cell according to claim 60, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

62. The manipulated cell according to claim 61, wherein the shRNA can be processed into small interfering RNA (siRNA).

63. The manipulated cell according to claim 61, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

64. The manipulated cell according to claim 60, having B2M expression of 25% or less of that of equivalent wild-type cells.

65. The manipulated cell according to claim 60, having B2M expression of 20% or less of that of equivalent wild-type cells.

66. The manipulated cell according to claim 60, having B2M expression of 15% or less of that of an equivalent wild-type cell.

67. The manipulated cell according to claim 60, having B2M expression of 10% or less of that of equivalent wild-type cells.

68. The manipulated cell according to claim 60, having a B2M expression of 5% or less of that of an equivalent wild-type cell.

69. The manipulated cells according to claim 60, wherein the cells are selected from the group consisting of stem cells and lymphocytes.

70. The manipulated cell according to claim 60, wherein the cell is a regulatory T cell (Treg).

71. The manipulated cells according to claim 60, wherein the cells are derived from an allogeneic graft donor in the subject.

72. A composition for treating an autoimmune disease or inflammatory disease in a subject, wherein the composition comprises regulatory T cells (Treg), each of which expresses β2-microglobulin (B2M) modified RNA in a manner that suppresses the immune response in the subject.

73. The composition according to claim 72, wherein the B2M modified RNA is encoded by a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, and SEQ ID NO:

2.

74. The composition according to claim 72, wherein the B2M modified RNA comprises small hairpin RNA (shRNA).

75. The composition according to claim 74, wherein the shRNA can be processed into small interfering RNA (siRNA).

76. The composition according to claim 74, wherein the B2M modified RNA further comprises one or more hairpin loop sequences encoded by a sequence selected from the group consisting of sequence number 6 and sequence number 7.

77. The composition according to claim 72, wherein the Treg has a B2M expression of 25% or less of that of wild-type Treg.

78. The composition according to claim 72, wherein the subject is a human.

79. The composition according to claim 72, wherein the Treg is not in-house.

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