Patent Application
20250129346
An emerging cell therapy approach called adoptive cell transfer (ACT) is rapidly changing the scene of human disease treatment. ACT involves collecting cells from a patient (autologous) or healthy donors (allogeneic), engineering these cells (e.g., through gene, mRNA, or protein modifications), and transferring the cells into the patient to fight diseases. One of the complications of allogeneic therapies is that it requires blood type matching between a donor and a recipient. Blood types are determined by the presence or absence of certain antigens on the surface of red blood cells (RBCs) and many other cell types in the body. Since the presence of these antigens can trigger a recipient's immune system to attack the infused cells, safe and effective allogeneic therapies depend on blood type matching.
As of 2019, a total of 41 human blood group systems are recognized by the International Society of Blood Transfusion (ISBT). The two most commonly referenced blood group systems are ABO and Rh. Four major blood types are determined by the presence or absence of the A and B antigens (A, B, AB, and O). In addition to the A and B antigens, the Rh factor can be either present (+) or absent (−), creating the eight most common blood types (A+, A−, B+, B−, AB+, AB−, O+, and O−). Several of the blood type-determining antigens are controlled by a single gene.
The presence of the blood type-determining antigens is usually associated with the absence of antibodies against those antigens in the subject's plasma thereby preventing a potential agglutination reaction. Thus, for allogeneic therapies, blood type compatibility is important to avoid graft rejection and other negative immune reactions. Because type O− individuals do not have A, B, or Rh antigens on the surface of their cells, they are usually referred to as universal donors for any recipient having any blood type.
The present technology provides methods for genetically engineering cells to knock out, knock down or otherwise alter one or more genes associated with blood type, e.g., ABO, FUT1, RHD, to improve the efficacy and safety of allogeneic cell therapies. Also provided herein are cells and compositions derived therefrom, as well as methods of using the same to treat various human diseases.
In some aspects, methods are provided for genetically modifying one or more genes associated with blood type in a cell, the methods comprising introducing into the cell a site-directed nuclease or a nucleotide sequence encoding a site-directed nuclease, wherein the one or more genes associated with blood type are selected from the group consisting of ABO, FUT1, and RHD. In some embodiments, the methods further comprise introducing to the cell a guide RNA (gRNA) targeting the ABO, FUT1, or RHD locus.
In some aspects, gRNAs are provided for use in genetically modifying one or more genes associated with blood type in a cell, wherein the one or more genes associated with blood type is selected from the group consisting of ABO, FUT1, and RHD.
In some embodiments, the site-directed nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a CRISPR-associated transposase, and a CRISPR/Cas nuclease.
In some embodiments, the site-directed nuclease is a CRISPR/Cas nuclease selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, C2c4, C2c8, C2c9, Cmr5, Cse1, Cse2, Csf1, Csm2, Csn2, Csx10, Csx11, Csy1, Csy2, Csy3, and Mad7.
In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) and optionally a transactivating CRISPR RNA (tracrRNA). In some embodiments, the gRNA comprises a crRNA and a tracrRNA as two separate molecules. In some embodiments, the gRNA comprises a crRNA and a tracrRNA as a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a complementary region, a crRNA repeat region, a tetraloop, and a tracrRNA.
In some embodiments, the crRNA repeat region comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:18. In some embodiments, the tetraloop comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO:6 or SEQ ID NO:17. In some embodiments, the tracrRNA comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, or SEQ ID NO:16.
In some embodiments, the crRNA comprises a complementary region specific to a region of the ABO locus, including, for example, a coding sequence (CDS), an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region. In some embodiments, the complementary region comprises, consists of, or consists essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 20-203.
In some embodiments, the crRNA comprises a complementary region specific to a region of the FUT1 locus, including, for example, a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region. In some embodiments, the complementary region comprises, consists of, or consists essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 204-420.
In some embodiments, the crRNA comprises a complementary region specific to a region of the RHD locus, including, for example, a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region. In some embodiments, the complementary region comprises, consists of, or consists essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 421-580.
In some embodiments, the genetic modification occurs via non-homologous end-joining (NHEJ). In some embodiments, the genetic modification occurs via homology-directed repair (HDR). In some embodiments, the genetic modifications include both HDR- and NHEJ-induced modifications.
In some embodiments, provided are compositions comprising the gRNA according to various embodiments of the present technology. In some embodiments, the compositions further comprise a site-directed nuclease or a nucleotide sequence encoding a site-directed nuclease protein as described herein.
In some embodiments, the compositions comprising the gRNA according to various embodiments of the present technology are formulated for delivery into a cell. In some embodiments, the cell further comprises a site-directed nuclease or a nucleotide sequence encoding a site-directed nuclease protein as described herein.
In some aspects, provided are methods of identifying a new genomic locus for genetically modifying one or more genes associated with blood type in a cell, the methods comprising (a) locating a genomic locus based on a known gRNA; and (b) scanning a region of about 500 to 4000 bp on either side of the genomic locus for a PAM sequence, wherein the one or more genes associated with blood type is selected from the group consisting of ABO, FUT1, and RHD. In some embodiments, the known gRNA targets the ABO, FUT1, or RHD locus. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 20-580.
In some aspects, provided are cells having one or more genes associated with blood type genetically modified according to various embodiments of the present technology. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is a pluripotent stem cell, an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). In some embodiments, the cell is differentiated from a pluripotent stem cell (e.g., an ESC or an iPSC). In some embodiments, the cell is the cell is a primary cell. In some embodiments, the cell is a blood cell, e.g., a red blood cell, a platelet cell, a mast cell, a basophil, an eosinophil, a neutrophil, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, a T cell, a B cell, or a plasma cell. In some embodiments, the cell is a T cell, an NK cell, or an NKT cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell is a retinal pigment epithelial cell (RPE). In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is a β islet cell. In some embodiments, the cell is a glial progenitor cell (GPC).
In some embodiments, the cell is modified to have reduced expression of one or more MHC I molecules and/or one or more MHC II molecules, optionally, wherein the one or more MHC I molecules are selected from the group consisting of HLA-A, HLA-B, HLA-C, and optionally, wherein the one or more MHC II molecules are selected from the group consisting of HLA-DR, HLA-DQ, HLA-DP, HLA-DM, and HLA-DO. In some embodiments, the modification is by modulation of the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci. In some embodiments, the modulation of the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci comprises B2M, TAP1, CIITA, MIC-A, and/or MIC-B knockout. In some embodiments, the modulation of the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci comprises knock-in of a transgene at the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci.
In some embodiments, the transgene encodes one or more tolerogenic factors selected from the group consisting of A20/TNFAIP3, CD16, CD16 Fc receptor, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CCL22, CTLA4-Ig, C1 inhibitor, complement receptor (CR1), DUX4, FASL, H2-M3, IDO1, IL15-RF, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, MANF, PD-1, PD-L1, SERPINB9, CCL21, and MFGE8. In some embodiments, the one or more tolerogenic factors comprise CD47, for example, human CD47. In some embodiments, the human CD47 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 583-588. In some embodiments, the human CD47 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:584. In some embodiments, the one or more tolerogenic factors comprise HLA-E. In some embodiments, the one or more tolerogenic factors comprise CD24. In some embodiments, the one or more tolerogenic factors comprise PD-L1. In some embodiments, the one or more tolerogenic factors comprise CD24, CD47, and PD-L1. In some embodiments, the one or more tolerogenic factors comprise CD46. In some embodiments, the one or more tolerogenic factors comprise CD55. In some embodiments, the one or more tolerogenic factors comprise CD59. In some embodiments, the one or more tolerogenic factors comprise C1 inhibitor. In some embodiments, the one or more tolerogenic factors comprise CD46, CD55, CD59, and C1 inhibitor. In some embodiments, the one or more tolerogenic factors comprise HLA-E, CD24, CD47, PD-L1, CD46, CD55, CD59, and C1 inhibitor.
In some embodiments, the cell is modified to have reduced expression of one or more MHC I molecules and/or one or more MHC II molecules; increased expression of CD47, and optionally CD24 and PD-L1; and increased expression of CD46, CD55, CD59, and CR1.
In some embodiments, the cell is modified to have reduced expression of one or more MHC I molecules; reduced expression of TXNIP; increased expression of PD-L1 and HLA-E; and optionally increased expression of A20/TNFAIP3, and/or MANE.
In some embodiments, the cell is modified to have increased expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE8.
In some aspects, provided are pharmaceutical compositions comprising cells having one or more genes associated with blood type genetically modified according to various embodiments of the present technology.
In some aspects, provided are methods of treating a disease in a subject in need thereof, the methods comprising administering the subject cells having one or more genes associated with blood type genetically modified according to various embodiments of the present technology, or pharmaceutical compositions comprising the same.
In some embodiments, the disease is cancer, e.g., a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from the group consisting of myeloid neoplasm, myelodysplastic syndromes (MDS), myeloproliferative/myelodysplastic syndromes, acute lymphoid leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), B cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), T cell lymphoma, and B cell lymphoma.
In some embodiments, the disease is an autoimmune disease, e.g., lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, and celiac disease.
In some embodiments, the disease is diabetes mellitus, e.g., Type I diabetes, Type II diabetes, prediabetes, and gestational diabetes.
In some embodiments, the disease is a neurological disease, e.g., catalepsy, epilepsy, encephalitis, meningitis, migraine, Huntington's, Alzheimer's, Parkinson's, Pelizaeus-Merzbacher disease, and multiple sclerosis.
In some embodiments, the disease is a cardiac disease, e.g., pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, dysfunctional conduction systems, dysfunctional coronary arteries, pulmonary hypertension, cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle degeneration, myocarditis, infective myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, cardiomegaly, mitral insufficiency, and autoimmune endocarditis.
The present technology provides methods for engineering cells, including through genetic editing, to alter the expression of one or more genes associated with blood type, e.g., ABO, FUT1, and/or RHD. Also provided are site-directed nucleases and guide RNAs, as well as compositions and vectors thereof, for use in these methods. Moreover, the present technology provides genetically modified cells and cell populations generated using these gene editing methods as well as methods of using these cells and cell populations to treat various human diseases.
While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention and is not intended to limit the invention to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.
The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
To the extent any materials incorporated by reference herein conflict with the present disclosure, the present disclosure controls.
The term “about,” as used herein when referring to a measurable value, such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
The term “antibody” is used to denote, in addition to natural antibodies, genetically engineered or otherwise modified forms of immunoglobulins or portions thereof, including chimeric antibodies, human antibodies, humanized antibodies, or synthetic antibodies. The antibodies may be monoclonal or polyclonal antibodies. In those embodiments wherein an antibody is an immunogenically active portion of an immunoglobulin molecule, the antibody may include, but is not limited to, a single chain variable fragment antibody (scFv), disulfide linked Fv, single domain antibody (sdAb), VHH antibody, antigen-binding fragment (Fab), Fab′, F(ab′)2 fragment, or diabody. An scFv antibody is derived from an antibody by linking the variable regions of the heavy (VH) and light (VL) chains of the immunoglobulin with a short linker peptide. Similarly, a disulfide linked Fv antibody can be generated by linking the VH and VL using an interdomain disulfide bond. On the other hand, sdAbs consist of only the variable region from either the heavy or light chain and usually are the smallest antigen-binding fragments of antibodies. A VHH antibody is the antigen binding fragment of heavy chain only. A diabody is a dimer of scFv fragment that consists of the VH and VL regions noncovalent connected by a small peptide linker or covalently linked to each other. The antibodies disclosed herein, including those that comprise an immunogenically active portion of an immunoglobulin molecule, retain the ability to bind a specific antigen.
The term “antigen” refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, or both. An antigen may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can also be produced by cells that have been modified or genetically engineered to express an antigen.
The term “autoimmune disease,” “autoimmune disorder,” “inflammatory disease,” or “inflammatory disorder” refers to any disease or disorder in which the subject mounts an immune response against its own tissues and/or cells. Autoimmune disorders can affect almost every organ system in the subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to Hashimoto's thyroiditis, systemic lupus erythematosus, Sjögren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and diabetes.
The term “codon-optimized” or “codon optimization” when referring to a nucleotide sequence is based on the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding nucleotide is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. Codon optimization refers to the process of substituting certain codons in a coding nucleotide sequence with synonymous codons based on the host cell's preference without changing the resulting polypeptide sequence. A variety of codon optimization methods are known in the art, and include, for example, methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.
The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of introducing a specific nucleic acid sequence into a cell or into another nucleic acid sequence, or as a means of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
The term “expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
The term “hypoimmunogenicity,” “hypoimmunogeneic,” “hypoimmunogenic,” “hypoimmunity,” or “hypoimmune” is used interchangeably to describe a cell being less prone to immune rejection by a subject into which such cell is transplanted. For example, relative to an unaltered or unmodified wild-type cell, such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection by a subject into which such cell is transplanted. In some examples described herein, genome editing technologies are used to modulate the expression of MHC I and MHC II genes, and thus, to generate a hypoimmunogenic cell. In other examples described herein, a tolerogenic factor is introduced into a cell and when expressed can modulate or affect the ability of the cell to be recognized by host immune system and thus confer hypoimmunogenicity. Hypoimmunogenicity of a cell can be determined by evaluating the cell's ability to elicit adaptive and innate immune responses. Such immune response can be measured using assays recognized by those skilled in the art, for example, by measuring the effect of a hypoimmunogenic cell on T cell proliferation, T cell activation, T cell killing, NK cell proliferation, NK cell activation, and macrophage activity. Hypoimmunogenic cells may undergo decreased killing by T cells and/or NK cells upon administration to a subject or show decreased macrophage engulfment compared to an unmodified or wildtype cell. In some cases, a hypoimmunogenic cell elicits a reduced or diminished immune response in a recipient subject compared to a corresponding unmodified wild-type cell. In some cases, a hypoimmunogenic cell is nonimmunogenic or fails to elicit an immune response in a recipient subject. Detailed descriptions of hypoimmunogenic cells, methods of producing the same, and methods of using the same are found in WO2016183041 filed May 9, 2015; WO2018132783 filed Jan. 14, 2018; WO2018176390 filed Mar. 20, 2018; WO2020018615 filed Jul. 17, 2019; WO2020018620 filed Jul. 17, 2019; WO2021022223 filed Jul. 31, 2020; WO2021022223 filed Jul. 31, 2020; WO2021041316 filed Aug. 24, 2020; WO2021222285 filed Apr. 27, 2021, 2020; and WO2021222285 filed Apr. 27, 2021, the disclosures including the examples, sequence listings and figures are incorporated herein by reference in their entirety.
The term “nucleic acid” or “polynucleotide” refers to a polymeric compound including covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single- or double-stranded. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.
The term “subject” refers to a mammalian subject, preferably a human. A “subject in need thereof” may refer to a subject who has been diagnosed with a disease or is at an elevated risk of developing a disease. The phrases “subject” and “patient” are used interchangeably herein.
A “therapeutically effective amount” as used herein is an amount that produces a desired effect in a subject for treating a disease. In certain embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited, to the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications), the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of the therapeutic composition and adjusting the dosage accordingly. For additional guidance, see, e.g., Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, 2012, and Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th Edition, McGraw-Hill, New York, NY, 2011, the entire disclosures of which are incorporated by reference herein.
The term “tolerogenic factor” as used herein includes hypoimmunity factors, complement inhibitors, and other factors that modulate or affect the ability of a cell to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment.
The terms “treat,” “treating,” and “treatment” as used herein with regard to a disease refers to alleviating one or more symptoms of the disease partially or entirely; preventing the disease; decreasing the likelihood of occurrence or recurrence of the disease; slowing the progression or development of the disease; eliminating, reducing, or slowing the development of one or more symptoms associated with the disease; or increasing progression-free or overall survival of the disease. For example, “treating” may refer to preventing or slowing the existing disease from progressing and/or slowing the development of certain symptoms of the disease. In some embodiments, the term “treat,” “treating,” or “treatment” means that the subject has a lesser degree of the disease comparing to a subject without being administered with the treatment. In some embodiments, the term “treat,” “treating,” or “treatment” means that one or more symptoms of the disease are alleviated in a subject receiving the treatment as disclosed and described herein comparing to a subject who does not receive such treatment.
A “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
Provided herein in certain embodiments are methods of genetically engineering a cell or population of cells to knock out, knock down, or otherwise alter the expression of one or more genes associated with blood type, including but not limited to ABO, FUT1, and RHD. As used herein, “knock out” includes deleting all or a portion of the target nucleotide sequence in a way that interferes with the function of the target gene. For example, a knock out can be achieved by altering a target nucleotide sequence by inducing an indel in a functional domain of the target nucleotide sequence (e.g., a DNA binding domain) or where base editing and prime editing can be used to change single nucleic acid bases to an alternate base in order to alter the genome sequence. “Knock down” refers to genetic modifications that result in reduced expression of the edited gene. As used herein, “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof, of nucleotide bases in the genome. Thus, an indel typically inserts or deletes nucleotides from a sequence. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. A gene editing system, e.g., the CRISPR/Cas system, of the present disclosure can be used to induce an indel of any length in a target polynucleotide sequence.
In certain embodiments, the methods provided herein utilize gene editing. Gene editing is a type of genetic engineering in which a nucleotide sequence may be inserted, deleted, modified, or replaced in the genome of a living organism. Current gene editing techniques generally utilize the innate mechanism for cells to repair double-strand breaks (DSBs) in DNA.
Eukaryotic cells repair DSBs by two primary repair pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). HDR typically occurs during late S phase or G2 phase, when a sister chromatid is available to serve as a repair template. NHEJ is more common and can occur during any phase of the cell cycle, but it is more error prone. In gene editing, NHEJ is generally used to produce insertion/deletion mutations (indels), which can produce targeted loss of function in a target gene by shifting the open reading frame (ORF) and producing alterations in the coding region or an associated regulatory region. HDR, on the other hand, is a preferred pathway for producing targeted knock-ins, knockouts, or insertions of specific mutations in the presence of a repair template with homologous sequences. Several methods are known to a skilled artisan to improve HDR efficiency, including, for example, chemical modulation (e.g., treating cells with inhibitors of key enzymes in the NHEJ pathway); timed delivery of the gene editing system at S and G2 phases of the cell cycle; cell cycle arrest at S and G2 phases; and introduction of repair templates with homology sequences. The methods provided herein may utilize HDR-mediated repair, NHEJ-mediated repair, or a combination thereof.
In some embodiments, the methods provided herein for genetically modifying a cell or population of cells to knock out, knock down, or otherwise modify one or more genes utilize a site-directed nuclease, including, for example, prime editing, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems.
Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. See, e.g., Anzalone et al., Nature, 576:149-157 (2019); WO2021072328; WO2022067130, all of which are incorporated herein by reference in their entirety. Cas9 and a reverse transcriptase can also be used to insert an integrase site into the genome for insertion of a nucleic acid of interest in a process called Programmable Addition via Site-specific Targeting Elements (PASTE) editing. See, e.g., loannidi et al., bioRxiv 2021.11.01.466786; doi.org/10.1101/2021.11.01.466786, all of which are incorporated herein by reference in their entirety.
ZFNs are fusion proteins comprising an array of site-specific DNA binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme. A ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the DNA binding domains or zinc finger domains. See, e.g., Carroll et al., Genetics Society of America (2011) 188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93:1156-1160. Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and usually recognizes a 3- to 4-bp DNA sequence. Tandem domains can thus potentially bind to an extended nucleotide sequence that is unique within a cell's genome.
Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15, or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Sera et al., Biochemistry (2002) 41:7074-7081; Liu et al., Bioinformatics (2008) 24:1850-1857.
ZFNs containing FokI nuclease domains or other dimeric nuclease domains function as a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. See Bitinaite et al., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575. To cleave a specific site in the genome, a pair of ZFNs are designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand. Upon binding of the ZFNs on either side of the site, the nuclease domains dimerize and cleave the DNA at the site, generating a DSB with 5′ overhangs. HDR can then be utilized to introduce a specific mutation, with the help of a repair template containing the desired mutation flanked by homology arms. The repair template is usually an exogenous double-stranded DNA vector introduced to the cell. See Miller et al., Nat. Biotechnol. (2011) 29:143-148; Hockemeyer et al., Nat. Biotechnol. (2011) 29:731-734.
TALENs are another example of an artificial nuclease which can be used to edit a target gene. TALENs are derived from DNA binding domains termed TALE repeats, which usually comprise tandem arrays with 10 to 30 repeats that bind and recognize extended DNA sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (termed the repeat-variable di-residue, or RVD) conferring specificity for one of the four DNA base pairs. Thus, there is a one-to-one correspondence between the repeats and the base pairs in the target DNA sequences.
TALENs are produced artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for example, a FokI endonuclease domain. See Zhang, Nature Biotech. (2011) 29:149-153. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. See Cermak et al., Nucl. Acids Res. (2011) 39:e82; Miller et al., Nature Biotech. (2011) 29:143-148; Hockemeyer et al., Nature Biotech. (2011) 29:731-734; Wood et al., Science (2011) 333:307; Doyon et al., Nature Methods (2010) 8:74-79; Szczepek et al., Nature Biotech (2007) 25:786-793; Guo et al., J. Mol. Biol. (2010) 200:96. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI nuclease domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al., Nature Biotech. (2011) 29:143-148.
By combining engineered TALE repeats with a nuclease domain, a site-specific nuclease can be produced specific to any desired DNA sequence. Similar to ZFNs, TALENs can be introduced into a cell to generate DSBs at a desired target site in the genome, and so can be used to knock out genes or knock in mutations in similar, HDR-mediated pathways. See Boch, Nature Biotech. (2011) 29:135-136; Boch et al., Science (2009) 326:1509-1512; Moscou et al., Science (2009) 326:3501.
Meganucleases are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and/or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774. On the other hand, the GIY-YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811. The His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. Members of the NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
Because the chance of identifying a natural meganuclease for a particular target DNA sequence is low due to the high specificity requirement, various methods including mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Chevalier et al., Mol. Cell. (2002) 10:895-905; Epinat et al., Nucleic Acids Res (2003) 31:2952-2962; Silva et al., J Mol. Biol. (2006) 361:744-754; Seligman et al., Nucleic Acids Res (2002) 30:3870-3879; Sussman et al., J Mol Biol (2004) 342:31-41; Doyon et al., J Am Chem Soc (2006) 128:2477-2484; Chen et al., Protein Eng Des Sel (2009) 22:249-256; Arnould et al., J Mol Biol. (2006) 355:443-458; Smith et al., Nucleic Acids Res. (2006) 363(2):283-294.
Like ZFNs and TALENs, Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11:11-27.
Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. By linking transposases to other systems such as the CRISPR/Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA. There are two known DNA integration methods using transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons. The transposase-dependent DNA integration does not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.
The CRISPR system was originally discovered in prokaryotic organisms (e.g., bacteria and archaea) as a system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Now it has been adapted and used as a popular gene editing tool in research and clinical applications.
CRISPR/Cas systems generally comprise at least two components: one or more guide RNAs (gRNAs) and a Cas protein. The Cas protein is a nuclease that introduces a DSB into the target site. CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, Ill, and IV; class 2 is divided into types II, V, and VI. Different Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, C2c4, C2c8, C2c9, Cmr5, Cse1, Cse2, Csf1, Csm2, Csn2, Csx10, Csx11, Csy1, Csy2, Csy3, and Mad7. See, e.g., Jinek et al., Science (2012) 337 (6096):816-821; Dang et al., Genome Biology (2015) 16:280; Ran et al., Nature (2015) 520:186-191; Zetsche et al., Cell (2015) 163:759-771; Strecker et al., Nature Comm. (2019) 10:212; Yan et al., Science (2019) 363:88-91. The most widely used Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from S. pyogenes or S. aureus.
In the original microbial genome, the type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs) each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as “protospacer adjacent motifs” (PAMs).
While the foregoing description has focused on Cas9 nuclease, it should be appreciated that other RNA-guided nucleases exist which utilize gRNAs that differ in some ways from those described to this point. For instance, Cpf1 (CRISPR from Prevotella and Franciscella 1; also known as Cas12a) is an RNA-guided nuclease that only requires a crRNA and does not need a tracrRNA to function.
Since its discovery, the CRISPR system has been adapted for inducing sequence specific DSBs and targeted genome editing in a wide range of cells and organisms spanning from bacteria to eukaryotic cells including human cells. In its use in gene editing applications, artificially designed, synthetic gRNAs have replaced the original crRNA:tracrRNA complexes, including in certain embodiments via a single gRNA. For example, the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest. The tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA. One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.
In order for the Cas nuclease to function, there must be a PAM immediately downstream of the target sequence in the genomic DNA. Recognition of the PAM by the Cas protein is thought to destabilize the adjacent genomic sequence, allowing interrogation of the sequence by the gRNA and resulting in gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies depending on the species of the Cas gene. For example, the most commonly used Cas9 nuclease derived from S. pyogenes recognizes a PAM sequence of 5′-NGG-3′ or, at less efficient rates, 5′-NAG-3′, where “N” can be any nucleotide. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing, which are summarized in Table 1 below.
Streptococcus pyogenes
Staphylococcus aureus
Neisseria meningitidis
Campylobacter jejuni
Streptococcus thermophilus
Treponema denticola
Lachnospiraceae bacterium
Acidaminococcus sp.
Alicyclobacillus acidiphilus
Bacillus hisashii
In some embodiments, Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and/or other characteristics. For example, the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, SpCas9-HF1, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9). For another example, the Cas nuclease may have one or more mutations that alter its PAM specificity.
In some embodiments of the methods provided herein, the genomic locus for site-directed knockout, knockdown, or other modification is a gene associated with blood type. In some embodiments, the one or more genes associated with blood types are selected from the group consisting of ABO, FUT1, and RHD. In some embodiments, two or more locations in a gene are modified. In some embodiments, two or more genes are modified.
The specific site for editing within a gene may be located within any suitable region of the gene, including but not limited to a gene coding region (also known as a coding sequence or “CDS”), an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region (e.g., promoter, enhancer). In some embodiments, the gene editing occurs in one allele of the specific genomic locus. In some embodiments, the gene editing occurs in both alleles of the specific genomic locus.
The ABO gene encodes blood group ABO system transferase, which is an enzyme with glycosyltransferase activity, and determines the ABO blood type of an individual by modifying the oligosaccharides on cell surface glycoproteins. The ABO gene locus encodes three alleles. The A allele produces α-1,3-N-acetylgalactosamine transferase (A-transferase), which catalyzes the transfer of GaINAc residues from the UDP-GaINAc donor nucleotide to the Gal residues of the acceptor H antigen, converting the H antigen into A antigen in A and AB individuals. The B allele encodes α-1,3-galactosyl transferase (B-transferase), which catalyzes the transfer of Gal residues from the UDP-Gal donor nucleotide to the Gal residues of the acceptor H antigen, converting the H antigen into B antigen in B and AB individuals. The O allele lacks both enzymatic activities because of a frame shift near the N-terminus resulting in translation of an almost entirely different protein. Thus, neither A nor B antigen is found in O individuals.
The human ABO gene resides on chromosome 9 at the band 9q34.2 (chromosome 9: 133,233,278-133,276,024, reverse strand). The ABO genomic sequence as set forth in Ensembl ID ENSG00000175164.16 is set forth in SEQ ID NO:1.
The FUT1 gene encodes galactoside 2-alpha-L-fucosyltransferase 1, which is involved in the synthesis of the H antigen (determinant of blood type O).
The human FUT1 gene resides on chromosome 19 at the band 19q13.33 (chromosome 19:48,748,011-48,755,390, reverse strand). The FUT1 genomic sequence as set forth in Ensembl ID: ENSG00000174951.12 is set forth in SEQ ID NO:2.
The Rh system is the second most important blood-group system with currently 50 antigens, of which the D antigen is the most significant Rh antigen for its likelihood to provoke an immune system response. The Rh D antigen is encoded by the RHD gene. Other Rh antigen encoding genes include RHCE, RhAG, RhBG, and RhCG.
The human RHD gene resides on chromosome 1 at the band 1p36.11 (chromosome 1: 25,272,393-25,330,445, forward strand). The RHD genomic sequence as set forth in Ensembl ID: ENSG00000187010.21 is set forth in SEQ ID NO:3.
In some embodiments, the modifications to the specific genomic loci may prevent expression of the genes entirely (i.e., knockout). In some embodiments, the modifications to the specific genomic loci may result in reduced expression of the genes (i.e., knockdown). In certain of these embodiments, gene knockout or knockdown may be achieved by any of the site-directed nuclease-based gene editing systems described, including, for example, the CRISPR/Cas system. In some embodiments, the gene knockout or knockdown occurs through insertion-deletion (indel) mutations at the target loci (e.g., through the NHEJ pathway) including, for example, frameshift-inducing indels or indels in a protein-coding region that result in loss-of-function mutations. In some embodiments, the gene knockout or knockdown occurs through deletions of the genes or portions thereof through either the NHEJ or HDR pathway, although HDR is a preferred pathway for introducing specific deletions. In some embodiments, the gene knockout or knockdown occurs through introduction of silencing or loss-of-function mutations at the target loci through the HDR pathway.
In some embodiments, the modifications to the specific genomic loci may involve insertion of exogenous genes to be expressed in place of the genes being edited (i.e., knock-in). In certain of these embodiments, gene knock-in may be achieved by the introduction of a site-directed nuclease and a transgene to be inserted through homologous recombination. The transgene can be flanked by homology arms (e.g., left homology arm (LHA) and right homology arm (RHA), respectively) and delivered to the cell for insertion into specified loci by HDR-based approaches as described. The homology arms are specifically designed for the target loci to serve as a template for HDR. The length of each homology arm is generally dependent on the size of the transgene being inserted, with larger insertions requiring longer homology arms. Any of the gene editing systems described, including, for example, the CRISPR/Cas system, may be employed for gene knock-in. In addition to expression of transgenes, gene knock-in may result in lost or reduced expression of the original genes at the target loci.
In some embodiments, the gene editing occurs at one or more genomic loci associated with blood types including, for example, ABO, FUT1, and RHD, and results in reduced or no expression of one or more of these genes. By modulating (e.g., reducing or deleting) expression of the ABO, FUT1, and/or RHD gene, the blood type of the cells may be modified. For example, the blood type of the cell may be changed from A, B, or AB to O by knocking out the A and/or B alleles of the ABO gene. For another example, the blood type of the cell may be changed from Rh+ to Rh− by knocking out the RHD gene.
C. Guide RNAs (gRNAs) for Gene Editing
In some embodiments, gRNAs are provided for use in targeted gene editing as described herein, especially in association with the CRISPR/Cas system. The gRNAs comprise a crRNA sequence, which in turn comprises a complementary region (also called a spacer) that recognizes and binds a complementary target DNA of interest. The length of the spacer or complementary region is generally between 15 and 30 nucleotides, usually about 20 nucleotides in length, although will vary based on the requirements of the specific CRISPR/Cas system. In certain embodiments, the spacer or complementary region is fully complementary to the target DNA sequence. In other embodiments, the spacer is partially complementary to the target DNA sequence, for example at least 80%, 85%, 90%, 95%, 98%, or 99% complementary.
In certain embodiments, the gRNAs provided herein further comprise a tracrRNA sequence, which comprises a scaffold region for binding to a nuclease. The length and/or sequence of the tracrRNA may vary depending on the specific nuclease being used for editing. In certain embodiments, nuclease binding by the gRNA does not require a tracrRNA sequence. In those embodiments where the gRNA comprises a tracrRNA, the crRNA sequence may further comprise a repeat region for hybridization with complementary sequences of the tracrRNA.
In some embodiments, the gRNAs provided herein comprise two or more gRNA molecules, for example a crRNA and a tracrRNA as two separate molecules. In other embodiments, the gRNAs are single guide RNAs (sgRNAs), including sgRNAs comprising a crRNA and a tracrRNA on a single RNA molecule. In certain of these embodiments, the crRNA and tracrRNA are linked by an intervening tetraloop.
In some embodiments, one gRNA can be used in association with a site-directed nuclease for targeted editing of a gene locus of interest. In other embodiments, two or more gRNAs targeting the same gene locus of interest can be used in association with a site-directed nuclease.
In some embodiments, exemplary gRNAs (e.g., sgRNAs) for use with various common Cas nucleases that require both a crRNA and tracrRNA, including Cas9 and Cas12b (C2c1), are provided in Table 2. See, e.g., Jinek et al., Science (2012) 337 (6096):816-821; Dang et al., Genome Biology (2015) 16:280; Ran et al., Nature (2015) 520:186-191; Strecker et al., Nature Comm. (2019) 10:212. For each exemplary gRNA, sequences for different portions of the gRNA, including the complementary region or spacer, crRNA repeat region, tetraloop, and tracrRNA, are shown. In some embodiments, the gRNA comprises all or a portion of the nucleotide sequences set forth in SEQ ID NOs: 4-7. In some embodiments, the gRNA comprises all or a portion of the nucleotide sequences set forth in SEQ ID NOs: 8-11. In some embodiments, the gRNA comprises all or a portion of the nucleotide sequences set forth in SEQ ID NOs: 12-15. In some embodiments, the gRNA comprises all or a portion of the nucleotide sequences set forth in SEQ ID NOs: 16-19.
In some embodiments, the gRNA comprises a crRNA repeat region comprising, consisting of, or consisting essentially of the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:18. In some embodiments, the gRNA comprises a tetraloop comprising, consisting of, or consisting essentially of the nucleotide sequence set forth in SEQ ID NO:6 or SEQ ID NO:17. In some embodiments, the gRNA comprises a tracrRNA comprising, consisting of, or consisting essentially of the nucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, or SEQ ID NO:16.
In some embodiments, the gRNA comprises a complementary region specific to a blood type gene locus, for example, the ABO locus, the FUT1 locus, or the RHD locus. The complementary region may bind a target sequence in any region of the blood type gene locus, including for example, a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region (e.g., promoter, enhancer). Where the target sequence is a CDS, exon, intron, or sequence spanning portions of an exon and intron, the CDS, exon, intron, or exon/intron boundary may be defined according to any splice variant of the target gene. In some embodiments, the genomic locus targeted by the gRNA is located within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of any of the loci or regions thereof as described.
In some embodiments, the gRNA used herein for targeted gene editing comprises a complementary region that recognizes a target genomic sequence of the ABO gene locus. In certain of these embodiments, the target sequence is located in a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or regulatory regions of the ABO gene. In certain embodiments, the gRNA comprises a complementary region that recognizes a target genomic sequence located entirely within an exon of the ABO gene, for example an exon as identified in Ensembl ENSG00000175164.4, ENST00000611156.4, or ENST00000538324.2, or in NCBI NC_000009.11. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 1-63, 806-863, 13857-13926, 14651-14707, 16159-16206, 17893-17983, 18483-18616, 19669-26168, or 42416-42747 of SEQ ID NO:1. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 824-863, 13857-13926, 14651-14707, 16159-16206, 17893-17928, 18483-18616, or 19669-20849 of SEQ ID NO:1. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 810-863, 13857-13926, 14651-14707, 16159-16206, 17893-17928, 18483-18501, 18504-18616, or 19669-20849 of SEQ ID NO:1. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 810-863, 13857-13926, 14651-14707, 16159-16206, 17893-17928, 18483-18501, 18504-18616, 19669-20350, or 20355-20423 of SEQ ID NO:1.
Exemplary target genomic sequences, the strand in which they are located, their associated PAM sequences, and cut sites of gRNAs targeting the ABO gene are provided in Table 4. In some embodiments, the gRNA targeting the ABO gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 20-203. In some embodiments, the gRNA targeting the ABO gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to the reverse complement of any of SEQ ID NOs: 20-203.
In some embodiments, the gRNA used herein for targeted gene editing comprises a complementary region that recognizes a target genomic sequence of the FUT1 gene locus. In certain of these embodiments, the target sequence is located in a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or regulatory region of the FUT1 gene. In certain embodiments, the gRNA comprises a complementary region that recognizes a target genomic sequence located entirely within an exon of the FUT1 gene, for example an exon as identified in Ensembl ENSG00000174951.12 or ENST00000645652.2, or in NCBI NG_007510. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 1-101, 1731-2066, 2269-2901, or 4108-7380 of SEQ ID NO:2. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 2750-2901 or 4108-7380 of SEQ ID NO:2. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 33-101, 1731-1743, 1828-2066, 2269-2901, or 4108-7380 of SEQ ID NO:2. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 33-101, 1731-1743, 1828-2066, 2269-2499, or 4108-7380 of SEQ ID NO:2. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 33-101, 1731-1743, 1828-2066, 2666-2901, or 4108-7380 of SEQ ID NO:2. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 33-101, 1731-1743, 1828-2066, 2750-2901, or 4108-7380 of SEQ ID NO:2.
Exemplary target genomic sequences, the strand in which they are located, their associated PAM sequences, and cut sites of gRNAs targeting the FUT1 gene are provided in Table 4. In some embodiments, the gRNA targeting the FUT1 gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 204-420. In some embodiments, the gRNA targeting the FUT1 gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to the reverse complement of any of SEQ ID NOs: 204-420.
In some embodiments, the gRNA used herein for targeted gene editing comprises a complementary region that recognizes a target genomic sequence of the RHD gene locus. In certain of these embodiments, the target sequence is located in a CDS, an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or regulatory regions of the RHD gene. In certain embodiments, the gRNA comprises a complementary region that recognizes a target genomic sequence located entirely within an exon of the RHD gene, for example an exon as identified in Ensembl ENSG00000187010.21, ENST00000328664.9, ENST00000622561.4, ENST00000423810.6, ENST00000342055.9, ENST00000568195.5, ENST00000357542.8, ENST00000417538.6, ENST00000454452.6, or ENST00000648012.1, or in NCBI NG_007494.1. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 1-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34550, 35255-35424, 44608-44687, 49497-49570, or 56506-58053 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 117-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 44608-44687, 49497-49570, or 56506-58053 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 94-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 35255-35424, 44608-44687, 49497-49570, or 56506-58053 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 156-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 35255-35424, 44608-44687, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 156-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 35255-35424, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 156-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 44608-44687, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 156-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 156-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 44608-44687, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 106-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, or 56506-56819 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 47-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 44608-44687, 49497-49570, or 56506-56769 of SEQ ID NO:3. In certain embodiments, the gRNA comprises a complementary region that recognizes a 15-30 nucleotide target sequence located within nucleotides 117-303, 12181-12367, 18249-18399, 28554-28701, 29128-29294, 30930-31067, 34204-34337, 44608-44687, 49497-49570, or 56506-58053 of SEQ ID NO:3.
Exemplary target genomic sequences, the strand in which they are located, their associated PAM sequences, and cut sites of gRNAs targeting the RHD gene are provided in Table 4. In some embodiments, the gRNA targeting the RHD gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 421-580. In some embodiments, the gRNA targeting the RHD gene comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence complementary to the reverse complement of any of SEQ ID NOs: 421-580.
In some embodiments, provided are methods of identifying new loci and/or gRNA sequences for use in the gene editing systems as described. For example, for CRISPR/Cas systems, when an existing gRNA for a particular locus (e.g., any of the exemplary gRNAs provided) is known, an “inch worming” approach can be used to identify additional loci by scanning the flanking regions on either side of the known locus for PAM sequences, which usually occurs about every 100 base pairs (bp) across the genome. The PAM sequence will depend on the particular Cas nuclease used because different nucleases usually have different corresponding PAM sequences. The flanking regions on either side of the locus can be between about 500 to 4000 bp long, for example, about 500 bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, or about 4000 bp long. When a PAM sequence is identified within the search range, a new guide can be designed according to the sequence of that locus for use in association with any of the gene editing system described. In certain embodiments, the new gRNAs identified using this approach can target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, within 500 bp, within 400 bp, within 300 bp, within 200 bp, within 100 bp, or within 50 bp of any of the genomic cut sites provided in Table 4. In certain embodiments, the gRNA is configured to produce a cut site at a position within 5, 10, 15, 20, 30, 40, or 50 nucleotides of any of the genomic cut sites provided in Table 4.
In some embodiments, the activity, stability, and/or other characteristics of gRNAs can be altered through the incorporation of chemical and/or sequential modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not being bound by a particular theory, it is believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into a population of cells, particularly the cells of the present technology. As used herein, the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. Other common chemical modifications of gRNAs to improve stabilities, increase nuclease resistance, and/or reduce immune response include 2′-O-methyl modification, 2′-fluoro modification, 2′-O-methyl phosphorothioate linkage modification, and 2′-O-methyl 3′ thioPACE modification.
One common 3′ end modification is the addition of a poly A tract comprising one or more (and typically 5-200) adenine (A) residues. The poly A tract can be contained in the nucleic acid sequence encoding the gRNA or can be added to the gRNA during chemical synthesis, or following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase). In vivo, poly-A tracts can be added to sequences transcribed from DNA vectors through the use of polyadenylation signals. Other suitable gRNA modifications include, without limitations, those described in U.S. Patent Application No. US 2017/0073674 A1 and International Publication No. WO 2017/165862 A1, the entire contents of each of which are incorporated by reference herein.
D. Delivery of Gene Editing Systems into a Cell
In some embodiments, provided are compositions comprising one or more components of a gene editing system described herein, including one or more gRNAs, a site-directed nuclease (e.g., a Cas nuclease) or a nucleotide sequence encoding a site-directed nuclease protein, and optionally a transgene for targeted insertion. In some embodiments, the compositions are formulated for delivery into a cell.
In some embodiments, components of a gene editing system provided herein, including one or more gRNAs, a site-directed nuclease (e.g., a Cas nuclease) or a nucleotide sequence encoding a site-directed nuclease protein, and optionally a transgene for targeted insertion, may be delivered into a cell in the form of a vector. The delivery vector can be any type of vector suitable for introduction of nucleotide sequences into a cell, including, for example, plasmids, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, phages, and HDR-based donor vectors. The different components may be introduced into a cell together or separately, and may be delivered in a single vector or multiple vectors.
In some embodiments, the vector may be introduced into a cell by any known method in the field, including, for example, viral transformation, calcium phosphate transfection, lipid-mediated transfection, DEAE-dextran, electroporation, microinjection, nucleoporation, liposomes, nanoparticles, or other methods.
In some embodiments, the present technology provides compositions comprising a vector according to various embodiments disclosed herein. In some embodiments, the compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or a combination thereof. A “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier or excipient may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier or excipient must be “pharmaceutically acceptable,” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In some embodiments, compositions comprising cells as disclosed herein further comprise a suitable infusion media.
In some embodiments, provided are cells or compositions thereof comprising one or more components of a gene editing system described herein, including one or more gRNAs, a site-directed nuclease (e.g., a Cas nuclease) or a nucleotide sequence encoding a site-directed nuclease protein, and optionally a transgene for targeted insertion.
In some embodiments, in addition to the gene editing methods as described to knock out, knock down, or otherwise alter the expression of one or more genes associated with blood type (e.g., ABO, FUT1, and RHD) in a cell, the cell, for example, in cases of allogeneic cells, may have additional genetic modifications, to further reduce potential graft-versus-host risks after infusion into the recipient or risks of being eliminated by the recipient's innate immune system. These additional modifications may include, for example, reducing or eliminating the expression of major histocompatibility complex (MHC) class I and/or MHC class II (MHC I and/or MHC II) genes, which encode cell surface molecules specialized to present antigenic peptides to immune cells. Reduced expression of MHC I and/or MHC II molecules in allogeneic cells may prevent recognition of these cells by the immune cells of the recipient and thus rejection of the graft. The step of modifying (e.g., reducing or eliminating) MHC I and/or MHC II molecules may occur before, at the same time as, or after the step of modifying the expression of one or more blood type genes. The MHC in humans is called human leukocyte antigen (HLA). Class I HLA (HLA I) corresponding to MHC I include the HLA-A, HLA-B, and HLA-C genes, and Class II HLA (HLA I) corresponding to MHC II include the HLA-DR, HLA-DQ, HLA-DP, HLA-DM, and HLA-DO genes.
In some embodiments, the additional modifications to a cell to reduce the immunogenicity of the cell comprise genetically modifying the cell to reduce the expression of one or more immune factors, including, for example, class II transactivator (CIITA), P2 microglobulin (B2M), NLRC5, CTLA-4, PD-1, HLA-A, HLA-BM, HLA-C, RFX-ANK, NFY-A, RFX5, RFX-AP, NFY-B, NFY-C, IRF1, MIC-A, MIC-B, TXNIP, CD142, CD38, PCDH11Y, NLGN4Y, and TAP1.
In some embodiments, the cell may be modified to have reduced expression of MHC I genes by targeting and modulating one or more of the HLA loci individually, such as HLA-A, HLA-B, and/or HLA-C, or collectively with HLA-Razor. In some embodiments, the modulation occurs through insertion-deletion (indel) modifications of one of more of the HLA loci, including HLA-A, HLA-B, and/or HLA-C, for example, by using the CRISPR/Cas system as described. By modulating (e.g., reducing or deleting) expression of any of the HLA genes, the cell can be rendered hypoimmunogenic and have a reduced ability to induce an immune response in a recipient subject. In some embodiments, reduced expression of any of the HLA loci reduces or eliminates expression of one or more of the HLA-A, HLA-B, and HLA-C genes. In some embodiments, the cell has HLA-A, HLA-B, and/or HLA-C knockout. In some embodiments, the genetic modification targeting any of the HLA loci comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as described herein at the HLA locus. In certain of these embodiments, insertion of the transgene into any of the HLA loci results in HLA-A, HLA-B, and/or HLA-C knockout.
In some embodiments, the cell may be modified to have reduced expression of MHC I genes by targeting and modulating the B2M locus. The B2M gene encodes a component of MHC I molecules. In some embodiments, the modulation occurs through insertion-deletion (indel) modifications or targeted mutations of the B2M locus, for example, by using the CRISPR/Cas system as described. By modulating (e.g., reducing or deleting) expression of B2M, surface trafficking of MHC I molecules is blocked, and the cell is thus rendered hypoimmunogenic. In some embodiments, the allogeneic cell modified to have reduced expression of MHC I genes has a reduced ability to induce an immune response in a recipient subject. In some embodiments, reduced expression of B2M reduces or eliminates expression of one or more of the HLA-A, HLA-B, and HLA-C genes. In some embodiments, the cell has B2M knockout. In some embodiments, the genetic modification targeting the B2M locus comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as described herein at the B2M locus. In certain of these embodiments, insertion of the transgene into the B2M locus results in B2M knockout.
In some embodiments, the cell may be modified to have reduced expression of MHC I genes by targeting and modulating the TAP1 locus. TAP1 encoded by the TAP1 gene assembles with TAP2 encoded by the TAP2 gene to form the transporter associated with antigen processing (TAP) complex, which is found in the endoplasmic reticulum (ER) and transports peptides of foreign origin into the ER to be attached to MHC class I proteins for presentation on the cell surface to the immune system. In some embodiments, the modulation occurs through insertion-deletion (indel) modifications of the TAP1 locus, for example, by using the CRISPR/Cas system as described. By modulating (e.g., reducing or deleting) expression of TAP1, surface trafficking of MHC I molecules is blocked, and the cell is thus rendered hypoimmunogenic. In some embodiments, reduced expression of TAP1 reduces or eliminates expression of one or more of the HLA-A, HLA-B, and HLA-C genes. In some embodiments, the cell has TAP1 knockout. In some embodiments, the genetic modification targeting the TAP1 locus comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as disclosed herein at the TAP1 locus. In certain of these embodiments, insertion of the transgene into the TAP1 locus results in TAP1 knockout.
In some embodiments, the cell may be modified to have reduced expression of MHC II genes by overexpression of CD74.
In some embodiments, the cell may be modified to have reduced expression of MHC II genes by targeting and modulating the CIITA locus. CIITA is a member of the nucleotide binding domain (NBD) leucine-rich repeat (LRR) family of proteins and regulates the transcription of MHC II by associating with the MHC enhanceosome. In some embodiments, the modulation occurs through insertion-deletion (indel) modifications of the CIITA locus, for example, by using the CRISPR/Cas system as described. In some embodiments, reduced expression of CIITA reduces or eliminates expression of one or more of the HLA-DR, HLA-DQ, HLA-DP, HLA-DM, and HLA-DO genes. In some embodiments, the cell has CIITA knockout. In some embodiments, the genetic modification targeting the CIITA locus comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as disclosed herein at the CIITA locus. In certain of these embodiments, insertion of the transgene into the CIITA locus results in CIITA knockout.
In certain embodiments, the cell comprises a modification, such as a genetic modification, targeting the MIC-A gene. MIC-A is a protein having known isoforms and variants (see, e.g., UniProt Q29983, accessed Jul. 18, 2022); all such forms of MIC-A are encompassed by the disclosure provided herein. In some embodiments, the genetic modification targeting the MIC-A gene is by using a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the MIC-A gene. In some embodiments, the genetic modification occurs using a CRISPR/Cas system as described. For example, in some embodiments, a gRNA with a targeting sequence GATGACCCTGGCTCATATCA (SEQ ID NO:581) can be used. In some embodiments, methods of gene editing with a CRISPR/Cas system and gRNA targeting MIC-A, such as with a targeting sequence GATGACCCTGGCTCATATCA (SEQ ID NO:581), knocks out all alleles of MIC-A in a cell. In some embodiments, the cell has MIC-A knockout. In some embodiments, the genetic modification targeting the MIC-A locus comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as disclosed herein at the MIC-A locus. In certain of these embodiments, insertion of the transgene into the MIC-A locus results in MIC-A knockout.
In certain embodiments, the cell comprises a modification, such as a genetic modification, targeting the MIC-B gene. MIC-B is a protein having known isoforms and variants (see, e.g., UniProt Q29980, accessed Jul. 18, 2022); all such forms of MIC-B are encompassed by the disclosure provided herein. In some embodiments, the genetic modification targeting the MIC-B gene is by using a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the MIC-B gene. In some embodiments, the genetic modification occurs using a CRISPR/Cas system as described. For example, in some embodiments, a gRNA with a targeting sequence GTTTCTGCCTGTCATAGCGC (SEQ ID NO:582) can be used. In some embodiments, methods of gene editing with a CRISPR/Cas system and gRNA targeting MIC-B, such as with a targeting sequence GTTTCTGCCTGTCATAGCGC (SEQ ID NO:582) knocks out all alleles of MIC-B in a cell. In some embodiments, the cell has MIC-B knockout. In some embodiments, the genetic modification targeting the MIC-B locus comprises inserting an exogenous nucleic acid or transgene encoding a polypeptide (e.g., a tolerogenic factor) as disclosed herein at the MIC-B locus. In certain of these embodiments, insertion of the transgene into the MIC-B locus results in MIC-B knockout.
In some embodiments, the cell has genetic modifications at the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci, have B2M, TAP1, CIITA, MIC-A, and/or MIC-B knockout, or have CD74 overexpression. The B2M, TAP1, CIITA, MIC-A, and/or MIC-B knockout can occur at one allele, or both alleles, of the respective gene locus. In some embodiments, the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci are modified so that the cell has reduced or no expression of B2M, TAP1, CIITA, MIC-A, and/or MIC-B, respectively. In these embodiments, the cell has reduced expression of MHC I and/or MHC II genes (HLA I and/or HLA II in humans) as a result of B2M, TAP1, CIITA, MIC-A, and/or MIC-B deletion or knockout, or overexpression of CD74.
In some embodiments, the transgene for targeted insertion (i.e., knock-in) at a genomic locus for genetic modification as described (e.g., the ABO, FUT1, RHD, B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci) may encode a tolerogenic factor that can improve the hypoimmunogenicity of the resulting cells so that they will not be subject to immune rejection when transplanted into a recipient and thus increasing the effectiveness of cell-based therapies. Examples of a tolerogenic factor include, but are not limited to, A20/TNFAIP3, CD16, CD16 Fc receptor, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CCL22, CTLA4-Ig, C1 inhibitor, complement receptor (CR1), DUX4, FASL, H2-M3, IDO1, IL15-RF, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, MANF, PD-1, PD-L1, SERPINB9, CCL21, MFGE8, and truncations, modifications, or fusions of any of the above.
In some embodiments, the tolerogenic factor is CD47, which is a leukocyte surface antigen and has a role in cell adhesion and modulation of integrins. It is expressed on the surface of a cell (e.g., a T cell) and signals to circulating macrophages not to phagocytize the cell. Overexpression of CD47 thus can reduce the immunogenicity of the cell when grafted and improve immune protection in allogeneic recipients.
CD47 is a transmembrane protein that, in humans, is encoded by the CD47 gene. It is a member of the immunoglobulin (Ig) superfamily. CD47 has a molecular weight of about −50 kDa. It is glycosylated and ubiquitously expressed by virtually all cells in the human body. It has a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments, and an alternatively spliced cytoplasmic tail at its C-terminus. In addition, it has two extracellular regions and two intracellular regions between neighboring membrane-spanning segments. A signal peptide, when it exists on a CD47 isoform, is located at the N-terminus of the IgV-like domain.
CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 interacts with multiple extracellular ligands, such as TSP-1, integrins, other CD47 proteins, and SIRPa. The CD47/SIRPa interaction regulates a multitude of intercellular interactions in many body systems, such as the immune system where it regulates lymphocyte homeostasis, dendritic cell (DC) maturation and activation, proper localization of certain DC subsets in secondary lymphoid organs, and cellular transmigration. CD47 on cells, including on donor cells in the context of transplantation or cell therapy applications, can function as a “marker of self” and regulate phagocytosis by binding to SIRPα on the surface of circulating immune cells to deliver an inhibitory “don't kill me” signal. CD47-SIRPα binding results in phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs) on SIRPα, which triggers recruitment of the SHP1 and SHP2 Src homology phosphatases. These phosphatases, in turn, inhibit accumulation of myosin II at the phagocytic synapse, preventing phagocytosis (Fujioka et al., Mol. Cell. Biol., 16:6887-6899 (1996)). Phagocytosis of target cells by macrophages is ultimately regulated by a balance of activating signals (e.g., FcγR, CRT, LRP-1) and inhibitory signals (e.g., SIRPα-CD47). Elevated expression of CD47 can help the cell evade immune surveillance, subsequent destruction, and innate immune cell killing. Thus, CD47 can be used as a tolerogenic factor to induce immune tolerance, for example, when there is pathological or undesirable activation of an otherwise normal immune response. This can occur, for example, when a patient develops an immune reaction to donor antigens after receiving an allogeneic transplantation or an allogeneic cell therapy, or when the body responds inappropriately to self-antigens implicated in autoimmune diseases.
The human CD47 gene has six naturally occurring transcripts, five of which each encode a protein isoform of CD47 (Ensembl, Gene: CD47, ENSG00000196776). The six transcripts are named CD47-201, CD47-202, CD47-203, CD47-204, CD47-205, and CD47-206. The coding DNA sequence (CDS) of the six transcripts are as set forth in SEQ ID NOs: 589-594, respectively. The amino acid sequences of the five protein isoforms are as set forth in SEQ ID NOs: 583-588 respectively (see Table 3).
Transcript CD47-201 (SEQ ID NO:589) encodes isoform CD47-201 (SEQ ID NO:583), which has 305 amino acids. Isoform CD47-201 has a C-terminal truncation of 18 amino acids from isoform CD47-202. All splice junctions of the CD47-201 transcript are supported by at least one non-suspect mRNA.
Transcript CD47-202 (SEQ ID NO:590) encodes isoform CD47-202 (SEQ ID NO:584), which has 323 amino acids. CD47-202 is the longest transcript of the human CD47 gene. It is designated as the representative transcript in the Ensembl database. In identifying the representative transcript, Ensembl aims to identity the transcript that, on balance, has the highest coverage of conserved exons, highest expression, longest coding sequence and is represented in other key resources, such as NCBI and UniProt. All splice junctions of the CD47-202 transcript are supported by at least one non-suspect mRNA. Amino acids 1-18 are the signal peptide. The amino acid sequence of CD47-202 without the signal peptide is set forth in SEQ ID NO:585.
Transcript CD47-203 (SEQ ID NO:591) encodes isoform CD47-203 (SEQ ID NO:586), which has 86 amino acids. The only support for the transcript model is from a single expressed sequence tag (EST).
Transcript CD47-204 (SEQ ID NO:592) does not encode any protein. All splice junctions of this transcript are supported by at least one non-suspect mRNA
Transcript CD47-205 (SEQ ID NO:593) encodes isoform CD47-205 (SEQ ID NO:587), which has 109 amino acids. Isoform 205 comprises 3 transmembrane domains and a truncated intracellular domain from isoform CD47-202. The best supporting mRNA for the transcript model is flagged as suspect or the support is from multiple ESTs.
Transcript CD47-206 (SEQ ID NO:594) encodes isoform CD47-206 (SEQ ID NO:588), which has 183 amino acids. Isoform 206 comprises a truncated extracellular domain and 5 transmembrane domains from isoform CD47-202.
In some embodiments, the transgene for targeted insertion (i.e., knock-in) at a genomic locus for genetic modification as described (e.g., the B2M, TAP1, CIITA, MIC-A, and/or MIC-B loci) may encode CD47, for example, human CD47. In certain of these embodiments, the human CD47 comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 583-588, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in any one of SEQ ID NOs: 583-588. In some embodiments, the human CD47 comprises or consists of an amino acid sequence set forth in SEQ ID NO:584 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:584. In some embodiments, the nucleotide sequence encoding CD47 corresponds to an mRNA sequence of human CD47. In some embodiments, the nucleotide sequence encoding CD47 is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in any one of SEQ ID NOs: 589-594. In some embodiments, the nucleotide sequence encoding CD47 is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO:590.
In some embodiments, the nucleotide sequence encoding CD47 is codon-optimized for expression in a mammalian cell, for example, a human cell. In some embodiments, the codon-optimized nucleotide sequence encoding CD47 is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO:595.
Provided herein in some aspects are cells that have been genetically edited according to various embodiments disclosed herein. In some embodiments, the cells have genetic modifications at one or more genomic loci associated with blood type, including, for example, the ABO, FUT1, and/or RHD loci. In certain of these embodiments, the cells have modified expression (e.g., reduced or no expression) of one or more of these genes. As a result of the modified expression of the ABO, FUT1, and/or RHD gene, the cells may have modified blood type.
In some embodiments, the cell is an autologous cell, i.e., obtained from the subject who will receive the cell after genetic modification. In some embodiments, the cell is an allogeneic cell, i.e., obtained from someone other than the subject who will receive the cell after genetic modification.
In some embodiments, the cell is a mesenchymal stem cell or a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic cell or a blood cell, for example, a red blood cell (erythrocyte), a platelet cell (thrombocyte), a mast cell, a basophil, an eosinophil, a neutrophil, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, a lymphocyte (e.g., a T cell, a B cell), or a plasma cell.
In some embodiments, the cell is a T cell, for example, a naïve T cell, a helper T cell (CD4+), a cytotoxic T cell (CD8+), a regulatory T cell (Treg), a central memory T cell (TCM), an effector memory T cell (TEM), a stem cell memory T cell (TSCM), or any combination thereof. More specifically, the T cell can be naïve (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (antigen-experienced and long-lived), or effector cells (antigen-experienced, cytotoxic). Memory T cells can be further divided into subsets of TCM (increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naïve T cells) and TEM (decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naïve T cells or TCM). Effector T cells refer to antigen-experienced CD8+ cytotoxic T cells that has decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Helper T cells are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate or suppress an adaptive immune response, and which of those two functions is induced will depend on the presence of other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. In some embodiments, the T cell can be a primary T cell obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In other embodiments, the T cell can be derived or differentiated from embryonic stem cells (ESCs) or induced pluripotent cells (iPSCs). In some embodiments, T cells may be modified to delete T cell receptors, e.g., by deletion of the TRAC or TRB locus, and may also be modified to express a chimeric antigen receptor.
In some embodiments, the cell is an NK cell. NK cells (also defined as large granular lymphocytes) represent a cell lineage differentiated from the common lymphoid progenitor (which also gives rise to B lymphocytes and T lymphocytes). Unlike T-cells, NK cells do not naturally express CD3 at the plasma membrane. Importantly, NK cells do not express a TCR and typically also lack other antigen-specific cell surface receptors. NK cells' cytotoxic activity does not require sensitization but is enhanced by activation with a variety of cytokines including IL-2. NK cells are generally thought to lack appropriate or complete signaling pathways necessary for antigen-receptor-mediated signaling, and thus are not thought to be capable of antigen receptor-dependent signaling, activation and expansion. NK cells are cytotoxic, and they balance activating and inhibitory receptor signaling to modulate their cytotoxic activity. For instance, NK cells expressing CD16 may bind to the Fc domain of antibodies bound to an infected cell, resulting in NK cell activation. By contrast, activity is reduced against cells expressing high levels of MHC class I proteins. On contact with a target cell, NK cells release proteins such as perforin, and enzymes such as proteases (granzymes). Perforin can form pores in the cell membrane of a target cell, inducing apoptosis or cell lysis. In some embodiments, the NK cells can be primary NK cells obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the NK cells can be derived or differentiated from ESCs or iPSCs. There are a number of techniques that can be used to generate NK cells from pluripotent stem cells (e.g., iPSCs). See, for example, Zhu et al., Methods Mol Biol. 2019; 2048:107-119; Knorr et al., Stem Cells Transl Med. 2013 2(4):274-83. doi: 10.5966/sctm.2012-0084; Zeng et al., Stem Cell Reports. 2017 Dec. 12; 9(6):1796-1812; Ni et al., Methods Mol Biol. 2013; 1029:33-41; Bernareggi et al., Exp Hematol. 2019 71:13-23; Shankar et al., Stem Cell Res Ther. 2020; 11(1):234, all of which are incorporated herein by reference in their entirety and specifically for the methodologies and reagents for differentiation. Differentiation can be assayed as is known in the art, generally by evaluating the presence of NK cell associated and/or specific markers, including, but not limited to, CD56, KIRs, CD16, NKp44, NKp46, NKG2D, TRAIL, CD122, CD27, CD244, NK1.1, NKG2A/C, NCR1, Ly49, CD49b, CD11b, KLRG1, CD43, CD62L, and/or CD226.
In some embodiments, the cell is an NKT cell. NKT cells are a heterogeneous group of T cells that share properties of both T cells and NK cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 1% of all peripheral blood T cells. In some embodiments, the NKT cells can be primary NKT cells obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the NKT cells can be derived or differentiated from ESCs or iPSCs.
In some embodiments, the cell is a pancreatic islet cell, including, for example, a β cell (also referred to as beta cell or β islet cell). Exemplary pancreatic islet cell types include, but are not limited to, pancreatic islet progenitor cells, immature pancreatic islet cells, mature pancreatic islet cells, and the like. In some embodiments, the β islet cells can be primary β islet cells. In some embodiments, the β islet cells can be derived or differentiated from ESCs or iPSCs. Useful method for differentiating pluripotent stem cells into pancreatic islet cells are disclosed, for example, in U.S. Pat. Nos. 9,683,215; 9,157,062; and 8,927,280. In some embodiments, the β islet cells genetically engineered by the methods as disclosed herein secretes insulin. In some embodiments, a β islet cell exhibits at least two characteristics of an endogenous pancreatic islet cell, for example, but not limited to, secretion of insulin in response to an increase in glucose, and expression of beta cell markers. In some embodiments, the β islet cells disclosed herein are administered to a subject to treat diabetes. Exemplary p cell markers or p cell progenitor markers include, but are not limited to, c-peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinase (GCK), prohormone convertase (PC 1/3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2, Pax4, Pax6, Ptfla, Isll, Sox9, Soxl7, and FoxA2. In some embodiments, the PSCs are differentiated into beta-like cells or islet organoids for transplantation to address type I diabetes mellitus (T1DM). Cell systems are a promising way to address T1DM, see, e.g., Ellis et al., Nat Rev Gastroenterol Hepatol. 2017 October; 14(10):612-628, incorporated herein by reference. Additionally, Pagliuca et al. (Cell, 2014, 159(2):428-39) reports on the successful differentiation of β cells from hiPSCs, the contents incorporated herein by reference in its entirety and in particular for the methods and reagents disclosed there for the large-scale production of functional human β cells from human pluripotent stem cells). Furthermore, Vegas et al. shows the production of human β cells from human pluripotent stem cells followed by encapsulation to avoid immune rejection by the recipient; Vegas et al., Nat Med, 2016, 22(3):306-11, incorporated herein by reference in its entirety and in particular for the methods and reagents disclosed there for the large-scale production of functional human B cells from human pluripotent stem cells. Additional disclosure of pancreatic islet cells including pancreatic β islet cells for use in the present technology are found in WO2020/018615, the disclosure is herein incorporated by reference in its entirety.
In some embodiments, the cell is a pluripotent stem cell, for example, an ESC or iPSC. In some embodiments, the cell is a cell differentiated from pluripotent stem cells, e.g., ESCs or iPSCs. ESCs and iPSCs have the ability to differentiated into any cell type of the body, including, for example, neurons, astrocytes, oligodendrocytes, retinal epithelial cells, epidermal cells, hair cells, keratinocytes, hepatocytes, pancreatic β islet cells, intestinal epithelial cells, lung alveolar cells, hematopoietic cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, renal cells, adipocytes, chondrocytes, thyroid cell, NK cells, NKT cells, macrophages, T cells, B cells, and osteocytes.
In some embodiments, the cell is a primary cell, including, for example, neurons, astrocytes, oligodendrocytes, retinal epithelial cells, epidermal cells, hair cells, keratinocytes, hepatocytes, pancreatic β islet cells, intestinal epithelial cells, lung alveolar cells, hematopoietic cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, renal cells, adipocytes, chondrocytes, thyroid cells, NK cells, NKT cells, macrophages, T cells, B cells, and osteocytes. In some embodiments, the cell is a cardiomyocyte, a retinal pigment epithelial cell (RPE), an endothelial cell, a β islet cell, or a glial progenitor cell (GPC).
In some aspects, the present technology provides pharmaceutical compositions comprising a cell according to various embodiments disclosed herein.
In some embodiments, the compositions can have various formulations, for example, injectable formulations, lyophilized formulations, liquid formulations, oral formulations, etc., depending on the suitable routes of administration.
In some embodiments, the compositions can be co-formulated in the same dosage unit or can be individually formulated in separate dosage units. The terms “dose unit” and “dosage unit” herein refer to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (i.e., 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.
In some embodiments, a single dosage unit includes at least about 1×102, 5×102, 1×103, 5×103, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, or 5×1010 cells.
In some aspects, provided are methods for treating and/or preventing a disease in a subject in need thereof. The method entails obtaining a cell from the subject (autologous) or from a donor (allogeneic); genetically modifying the cell, including targeted gene editing at one or more genomic loci associated with blood types, according to various embodiments disclosed herein; and administering to the subject a therapeutically effective amount of the genetically modified cell or a pharmaceutical composition containing the same.
In some embodiments, the disease is cancer. In some embodiments, the cancer is a hematologic malignancy. Non-limiting examples of hematologic malignancies include myeloid neoplasm, myelodysplastic syndromes (MDS), myeloproliferative/myelodysplastic syndromes, acute lymphoid leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), B cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), T cell lymphoma, and B cell lymphoma.
In some embodiments, the disease is an autoimmune disease, including, for example, lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, and celiac disease.
In some embodiments, the disease is diabetes mellitus, including, for example, Type I diabetes, Type II diabetes, prediabetes, and gestational diabetes.
In some embodiments, the disease is a neurological disease, including, for example, catalepsy, epilepsy, encephalitis, meningitis, migraine, Huntington's, Alzheimer's, Parkinson's, Pelizaeus-Merzbacher disease, and multiple sclerosis.
In some embodiments, the disease is a cardiac disease or disorder, i.e., a condition and/or disorder relating to the heart, including the valves, endothelium, infarcted zones, or other components or structures of the heart. Cardiac diseases or disorders include, for example, pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, dysfunctional conduction systems, dysfunctional coronary arteries, pulmonary hypertension, cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle degeneration, myocarditis, infective myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, cardiomegaly, mitral insufficiency, and autoimmune endocarditis.
In some embodiments, the genetically modified cell or pharmaceutical composition containing the same according to the present technology may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art. In any of the above embodiments, the genetically modified cell may be administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid, so as to encounter the target antigen or cells. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the undesired type or level or activity of the tagged cells, the particular form of the active ingredient; and the method of administration.
In some embodiments, the amount of genetically modified cells of the present technology in a pharmaceutical composition is typically greater than 102 cells, for example, about 1×102, 5×102, 1×103, 5×103, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010 cells, or more.
In some embodiments, the methods comprise administering to the subject the genetically modified cells or pharmaceutical composition containing the same once a day, twice a day, three times a day, or four times a day for a period of about 3 days, about 5 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.25 years, about 1.5 years, about 1.75 years, about 2 years, about 2.25 years, about 2.5 years, about 2.75 years, about 3 years, about 3.25 years, about 3.5 years, about 3.75 years, about 4 years, about 4.25 years, about 4.5 years, about 4.75 years, about 5 years, or more than about 5 years. In some embodiments, the genetically modified cells or pharmaceutical composition containing the same can be administered every day, every other day, every third day, weekly, biweekly (i.e., every other week), every third week, monthly, every other month, or every third month.
In some embodiments, the genetically modified cells or pharmaceutical composition containing the same may be administered over a pre-determined time period. Alternatively, the genetically modified cell or pharmaceutical composition containing the same may be administered until a particular therapeutic benchmark is reached. In some embodiments, the methods provided herein include a step of evaluating one or more therapeutic benchmarks in a biological sample, such as, but not limited to, the level of a disease related biomarker, to determine whether to continue administration of the genetically modified cell or pharmaceutical composition containing the same.
In some embodiments, the methods further comprise administering the subject a pharmaceutically effective amount of one or more additional therapeutic agents to obtain improved or synergistic therapeutic effects. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of an immunotherapy agent, a chemotherapy agent, and a biologic agent. In some embodiments, the subject was administered the one or more additional therapeutic agents before administration of the genetically modified cells or pharmaceutical composition containing the same. In some embodiments, the subject is co-administered the one or more additional therapeutic agents and the genetically modified cells or pharmaceutical composition containing the same. In some embodiments, the subject was administered the one or more additional therapeutic agents after administration of the genetically modified cells or pharmaceutical composition containing the same.
As one of ordinary skill in the art would understand, the one or more additional therapeutic agents and the genetically modified cells or pharmaceutical composition containing the same can be administered to a subject in need thereof one or more times at the same or different doses, depending on the diagnosis and prognosis of the subject. One skilled in the art would be able to combine one or more of these therapies in different orders to achieve the desired therapeutic results. In some embodiments, the combinational therapy achieves improved or synergistic effects in comparison to any of the treatments administered alone.
The above detailed description of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise forms disclosed above. Although specific embodiments of, and examples for, the technology are described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology as those skilled in the relevant art will recognize. For example, although steps are presented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments.
From the foregoing, it will be appreciated that specific embodiments of the technology have been described herein for purposes of illustration, but well-known components and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the technology. Where the context permits, singular or plural terms may also include the plural or singular term, respectively. Further, while advantages associated with some embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and associated technology can encompass other embodiments not expressly shown or described herein.
This application claims the benefit of U.S. Provisional Patent Application No. 63/232,142, filed on Aug. 11, 2021, the contents of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/074846 | 8/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63232142 | Aug 2021 | US |
